US20080261833A1 - Methods for generating polynucleotides having desired characteristics by iterative selection and recombination - Google Patents

Methods for generating polynucleotides having desired characteristics by iterative selection and recombination Download PDF

Info

Publication number
US20080261833A1
US20080261833A1 US11/843,351 US84335107A US2008261833A1 US 20080261833 A1 US20080261833 A1 US 20080261833A1 US 84335107 A US84335107 A US 84335107A US 2008261833 A1 US2008261833 A1 US 2008261833A1
Authority
US
United States
Prior art keywords
fragments
stranded
double
sequence
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/843,351
Inventor
Willem P.C. Stemmer
Andreas Crameri
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Maxygen Inc
Codexis Inc
Original Assignee
Maxygen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=22733354&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20080261833(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Maxygen Inc filed Critical Maxygen Inc
Priority to US11/843,351 priority Critical patent/US20080261833A1/en
Publication of US20080261833A1 publication Critical patent/US20080261833A1/en
Priority to US12/557,519 priority patent/US7981614B2/en
Assigned to CODEXIS, INC. reassignment CODEXIS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CODEXIS MAYFLOWER HOLDINGS, LLC
Abandoned legal-status Critical Current

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/02Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/545IL-1
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102Mutagenizing nucleic acids
    • C12N15/1027Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034Isolating an individual clone by screening libraries
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034Isolating an individual clone by screening libraries
    • C12N15/1037Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/78Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811Selection methods for production or design of target specific oligonucleotides or binding molecules
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y302/00Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y305/00Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/02Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
    • C12Y305/02006Beta-lactamase (3.5.2.6)
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/06Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565Complementarity determining region [CDR]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/60Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622Single chain antibody (scFv)

Definitions

  • the present invention relates to a method for the production of polynucleotides conferring a desired phenotype and/or encoding a protein having an advantageous predetermined property which is selectable.
  • the method is used for generating and selecting nucleic acid fragments encoding mutant proteins.
  • the information content of a protein has been defined as the resistance of the active protein to amino acid sequence variation, calculated from the minimum number of invariable amino acids (bits) required to describe a family of related sequences with the same function (9, 10). Proteins that are sensitive to random mutagenesis have a high information content. In 1974, when this definition was coined, protein diversity existed only as taxonomic diversity.
  • Information density is the Information Content/unit length of a sequence. Active sites of enzymes tend to have a high information density. By contrast, flexible linkers in enzymes have a low information density (8).
  • Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. Error prone PCR can be used to mutagenize a mixture of fragments of unknown sequence. However, computer simulations have suggested that point mutagenesis alone may often be too gradual to allow the block changes that are required for continued sequence evolution. The published error-prone PCR protocols do not allow amplification of DNA fragments greater than 0.5 to 1.0 kb, limiting their practical application. Further, repeated cycles of error-prone PCR lead to an accumulation of neutral mutations, which, for example, may make a protein immunogenic.
  • oligonucleotide-directed mutagenesis a short sequence is replaced with a synthetically mutagenized oligonucleotide. This approach does not generate combinations of distant mutations and is thus not combinatorial.
  • the limited library size relative to the vast sequence length means that many rounds of selection are unavoidable for protein optimization.
  • Mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round followed by grouping into families, arbitrarily choosing a single family, and reducing it to a consensus motif, which is resynthesized and reinserted into a single gene followed by additional selection. This process constitutes a statistical bottleneck, it is labor intensive and not practical for many rounds of mutagenesis.
  • Error-prone PCR and oligonucleotide-directed mutagenesis are thus useful for single cycles of sequence fine tuning but rapidly become limiting when applied for multiple cycles.
  • Error-prone PCR can be used to mutagenize a mixture of fragments of unknown sequence (11, 12).
  • the published error-prone PCR protocols (11, 12) suffer from a low processivity of the polymerase. Therefore, the protocol is unable to result in the random mutagenesis of an average-sized gene. This inability limits the practical application of error-prone PCR.
  • cassette mutagenesis a sequence block of a single template is typically replaced by a (partially) randomized sequence. Therefore, the maximum information content that can be obtained is statistically limited by the number of random sequences (i.e., library size). This constitutes a statistical bottleneck, eliminating other sequence families which are not currently best, but which may have greater long term potential.
  • mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round (20). Therefore, this approach is tedious and is not practical for many rounds of mutagenesis.
  • Error-prone PCR and cassette mutagenesis are thus best suited and have been widely used for fine-tuning areas of comparatively low information content.
  • One apparent exception is the selection of an RNA ligase ribozyme from a random library using many rounds of amplification by error-prone PCR and selection (13).
  • Marton et al. (27) describes the use of PCR in vitro to monitor recombination in a plasmid having directly repeated sequences. Marton et al. discloses that recombination will occur during PCR as a result of breaking or nicking of the DNA. This will give rise to recombinant molecules. Meyerhans et al. (23) also disclose the existence of DNA recombination during in vitro PCR.
  • AME Applied Molecular Evolution
  • Caren et al. (45) describe a method for generating a large population of multiple mutants using random in vivo recombination. However, their method requires the recombination of two different libraries of plasmids, each library having a different selectable marker. Thus the method is limited to a finite number of recombinations equal to the number of selectable markers existing, and produces a concomitant linear increase in the number of marker genes linked to the selected sequence(s).
  • Calogero et al. (46) and Galizzi et al. (47) report that in vivo recombination between two homologous but truncated insect-toxin genes on a plasmid can produce a hybrid gene.
  • Radman et al. (49) report in vivo recombination of substantially mismatched DNA sequences in a host cell having defective mismatch repair enzymes, resulting in hybrid molecule formation.
  • the invention described herein is directed to the use of repeated cycles of point mutagenesis, nucleic acid shuffling and selection which allow for the directed molecular evolution in vitro of highly complex linear sequences, such as proteins through random recombination.
  • the invention described herein is directed to the use of repeated cycles of mutagenesis, in vivo recombination and selection which allow for the directed molecular evolution in vivo of highly complex linear sequences, such as DNA, RNA or proteins through recombination.
  • the present invention is directed to a method for generating a selected polynucleotide sequence or population of selected polynucleotide sequences, typically in the form of amplified and/or cloned polynucleotides, whereby the selected polynucleotide sequence(s) possess a desired phenotypic characteristic (e.g., encode a polypeptide, promote transcription of linked polynucleotides, bind a protein, and the like) which can be selected for.
  • a desired phenotypic characteristic e.g., encode a polypeptide, promote transcription of linked polynucleotides, bind a protein, and the like
  • One method of identifying polypeptides that possess a desired structure or functional property involves the screening of a large library of polypeptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the polypeptide.
  • the present invention provides a method for generating libraries of displayed polypeptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening.
  • the method comprises (1) obtaining a first plurality of selected library members comprising a displayed polypeptide or displayed antibody and an associated polynucleotide encoding said displayed polypeptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequence, optionally introducing mutations into said polynucleotides or copies, and (2) pooling and fragmenting, typically randomly, said associated polynucleotides or copies to form fragments thereof under conditions suitable for PCR amplification, performing PCR amplification and optionally mutagenesis, and thereby homologously recombining said fragments to form a shuffled pool of recombined polynucleotides, whereby a substantial fraction (e.g., greater than 10 percent) of the recombined polynucleotides of
  • the method comprises the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind or otherwise interact (e.g., such as catalytic antibodies) with a predetermined macromolecule, such as for example a proteinaceous receptor, peptide, oligosaccharide, virion, or other predetermined compound or structure.
  • a predetermined macromolecule such as for example a proteinaceous receptor, peptide, oligosaccharide, virion, or other predetermined compound or structure.
  • the displayed polypeptides, antibodies, peptidomimetic antibodies, and variable region sequences that are identified from such libraries can be used for therapeutic, diagnostic, research, and related purposes (e.g., catalysts, solutes for increasing osmolarity of an aqueous solution, and the like), and/or can be subjected to one or more additional cycles of shuffling and/or affinity selection.
  • the method can be modified such that the step of selecting is for a phenotypic characteristic other than binding affinity for a predetermined molecule (e.g., for catalytic activity, stability, oxidation resistance, drug resistance, or detectable phenotype conferred on a host cell).
  • a phenotypic characteristic other than binding affinity for a predetermined molecule (e.g., for catalytic activity, stability, oxidation resistance, drug resistance, or detectable phenotype conferred on a host cell).
  • the first plurality of selected library members is fragmented and homologously recombined by PCR in vitro.
  • the first plurality of selected library members is fragmented in vitro, the resultant fragments transferred into a host cell or organism and homologously recombined to form shuffled library members in vivo.
  • the first plurality of selected library members is cloned or amplified on episomally replicable vectors, a multiplicity of said vectors is transferred into a cell and homologously recombined to form shuffled library members in vivo.
  • the first plurality of selected library members is not fragmented, but is cloned or amplified on an episomally replicable vector as a direct repeat, which each repeat comprising a distinct species of selected library member sequence, said vector is transferred into a cell and homologously recombined by intra-vector recombination to form shuffled library members in vivo.
  • combinations of in vitro and in vivo shuffling are provided to enhance combinatorial diversity.
  • the present invention provides a method for generating libraries of displayed antibodies suitable for affinity interaction screening.
  • the method comprises (1) obtaining a first plurality of selected library members comprising a displayed antibody and an associated polynucleotide encoding said displayed antibody, and obtaining said associated polynucleotides or copies thereof, wherein said associated polynucleotides comprise a region of substantially identical variable region framework sequence, and (2) pooling and fragmenting said associated polynucleotides or copies to form fragments thereof under conditions suitable for PCR amplification and thereby homologously recombining said fragments to form a shuffled pool of recombined polynucleotides comprising novel combinations of CDRs, whereby a substantial fraction (e.g., greater than 10 percent) of the recombined polynucleotides of said shuffled pool comprise CDR combinations are not present in the first plurality of selected library members, said shuffled pool composing a library of displayed antibodies comprising CDR permutations and suitable for affinity
  • the shuffled pool is subjected to affinity screening to select shuffled library members which bind to a predetermined epitope (antigen) and thereby selecting a plurality of selected shuffled library members.
  • the plurality of selected shuffled library members can be shuffled and screened iteratively, from 1 to about 1000 cycles or as desired until library members having a desired binding affinity are obtained.
  • one aspect of the present invention provides a method for introducing one or more mutations into a template double-stranded polynucleotide, wherein the template double-stranded polynucleotide has been cleaved into random fragments of a desired size, by adding to the resultant population of double-stranded fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the template polynucleotide; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at regions of identity between the single-stranded fragments and formation of a mutagenized double-stranded polynucleotide; and repeating the above steps as desired.
  • the present invention is directed to a method of producing recombinant proteins having biological activity by treating a sample comprising double-stranded template polynucleotides encoding a wild-type protein under conditions which provide for the cleavage of said template polynucleotides into random double-stranded fragments having a desired size; adding to the resultant population of random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise areas of identity and areas of heterology to the template polynucleotide; denaturing the resultant mixture of double-stranded fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at the areas of identity and formation of a mutagenized double-stranded polynucleotide; repeating the above steps as desired; and
  • a third aspect of the present invention is directed to a method for obtaining a chimeric polynucleotide by treating a sample comprising different double-stranded template polynucleotides wherein said different template polynucleotides contain areas of identity and areas of heterology under conditions which provide for the cleavage of said template polynucleotides into random double-stranded fragments of a desired size; denaturing the resultant random double-stranded fragments contained in the treated sample into single-stranded fragments; incubating the resultant single-stranded fragments with polymerase under conditions which provide for the annealing of the single-stranded fragments at the areas of identity and the formation of a chimeric double-stranded polynucleotide sequence comprising template polynucleotide sequences; and repeating the above steps as desired.
  • a fourth aspect of the present invention is directed to a method of replicating a template polynucleotide by combining in vitro single-stranded template polynucleotides with small random single-stranded fragments resulting from the cleavage and denaturation of the template polynucleotide, and incubating said mixture of nucleic acid fragments in the presence of a nucleic acid polymerase under conditions wherein a population of double-stranded template polynucleotides is formed.
  • the invention also provides the use of polynucleotide shuffling, in vitro and/or in vivo to shuffle polynucleotides encoding polypeptides and/or polynucleotides comprising transcriptional regulatory sequences.
  • the invention also provides the use of polynucleotide shuffling to shuffle a population of viral genes (e.g., capsid proteins, spike glycoproteins, polymerases, proteases, etc.) or viral genomes (e.g., paramyxoviridae, orthomyxoviridae, herpesviruses, retroviruses, reoviruses, rhinoviruses, etc.).
  • viral genes e.g., capsid proteins, spike glycoproteins, polymerases, proteases, etc.
  • viral genomes e.g., paramyxoviridae, orthomyxoviridae, herpesviruses, retroviruses, reoviruses, rhinoviruses, etc.
  • the invention provides a method for shuffling sequences encoding all or portions of immunogenic viral proteins to generate novel combinations of epitopes as well as novel epitopes created by recombination; such shuffled viral proteins may comprise epitopes or combinations of epitopes which are likely to arise in the natural environment as a consequence of viral evolution (e.g., such as recombination of influenza virus strains).
  • the invention also provides a method suitable for shuffling polynucleotide sequences for generating gene therapy vectors and replication-defective gene therapy constructs, such as may be used for human gene therapy, including but not limited to vaccination vectors for DNA-based vaccination, as well as anti-neoplastic gene therapy and other gene therapy formats.
  • FIG. 1 is a schematic diagram comparing mutagenic shuffling over error-prone PCR; (a) the initial library; (b) pool of selected sequences in first round of affinity selection; (d) in vitro recombination of the selected sequences (‘shuffling’); (f) pool of selected sequences in second round of affinity selection after shuffling; (c) error-prone PCR; (e) pool of selected sequences in second round of affinity selection after error-prone PCR.
  • FIG. 2 illustrates the reassembly of a 1.0 kb LacZ alpha gene fragment from 10-50 bp random fragments.
  • FIG. 3 is a schematic illustration of the LacZ alpha gene stop codon mutants and their DNA sequences.
  • the boxed regions are heterologous areas, serving as markers.
  • the stop codons are located in smaller boxes or underlined. “+” indicates a wild-type gene and “ ⁇ ” indicates a mutated area in the gene.
  • FIG. 4 is a schematic illustration of the introduction or spiking of a synthetic oligonucleotide into the reassembly process of the LacZ alpha gene.
  • FIG. 5 illustrates the regions of homology between a murine IL1-B gene (M) and a human IL1-B gene (H) with E. coli codon usage. Regions of heterology are boxed. The “
  • FIG. 6 is a schematic diagram of the antibody CDR shut fling model system using the scFv of anti-rabbit IgG antibody (A10B).
  • FIG. 7 illustrates the observed frequency of occurrence of certain combinations of CDRs in the shuffled DNA of the scFv of anti-rabbit IgG antibody (A10B).
  • FIG. 8 illustrates the improved avidity of the scFv anti-rabbit antibody after DNA shuffling and each cycle of selection.
  • FIG. 9 schematically portrays pBR322-Sfi-BL-LA-Sfi and in vivo intraplasmidic recombination via direct repeats, as well as the rate of generation of ampicillin-resistant colonies by intraplasmidic recombination reconstituting a functional beta-lactamase gene.
  • FIG. 10 schematically portrays pBR322-Sfi-2Bla-Sfi and in vivo intraplasmidic recombination via direct repeats, as well as the rate of generation of ampicillin-resistant colonies by intraplasmidic recombination reconstituting a functional beta-lactamase gene.
  • FIG. 11 illustrates the method for testing the efficiency of multiple rounds of homologous recombination after the introduction of polynucleotide fragments into cells for the generation of recombinant proteins.
  • FIG. 12 schematically portrays generation of a library of vectors by shuffling cassettes at the following loci: promoter, leader peptide, terminator, selectable drug resistance gene, and origin of replication.
  • the multiple parallel lines at each locus represents the multiplicity of cassettes for that cassette.
  • FIG. 13 schematically shows some examples of cassettes suitable at various loci for constructing prokaryotic vector libraries by shuffling.
  • the present invention relates to a method for nucleic acid molecule reassembly after random fragmentation and its application to mutagenesis of DNA sequences. Also described is a method for the production of nucleic acid fragments encoding mutant proteins having enhanced biological activity. In particular, the present invention also relates to a method of repeated cycles of mutagenesis, nucleic acid shuffling and selection which allow for the creation of mutant proteins having enhanced biological activity.
  • the present invention is directed to a method for generating a very large library of DNA, RNA or protein mutants. This method has particular advantages in the generation of related DNA fragments from which the desired nucleic acid fragment(s) may be selected.
  • the present invention also relates to a method of repeated cycles of mutagenesis, homologous recombination and selection which allow for the creation of mutant proteins having enhanced biological activity.
  • DNA reassembly is used when recombination occurs between identical sequences.
  • DNA shuffling is used herein to indicate recombination between substantially homologous but non-identical sequences, in some embodiments DNA shuffling may involve crossover via nonhomologous recombination, such as via cre/lox and/or flp/frt systems and the like.
  • amplification means that the number of copies of a nucleic acid fragment is increased.
  • nucleic acid sequences have the same sequence or a complementary sequence.
  • areas of identity means that regions or areas of a nucleic acid fragment or polynucleotide are identical or complementary to another polynucleotide or nucleic acid fragment.
  • a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA”.
  • reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, such as a polynucleotide sequence of FIG. 1 or FIG. 2( b ), or may comprise a complete cDNA or gene sequence.
  • a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • homologous or “homologous” means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence.
  • the degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later.
  • the region of identity is greater than about 5 bp, more preferably the region of identity is greater than 10 bp.
  • heterologous means that one single-stranded nucleic acid sequence is unable to hybridize to another single-stranded nucleic acid sequence or its complement.
  • areas of heterology means that nucleic acid fragments or polynucleotides have areas or regions in the sequence which are unable to hybridize to another nucleic acid or polynucleotide. Such regions or areas are, for example, areas of mutations.
  • cognate refers to a gene sequence that is evolutionarily and functionally related between species.
  • the human CD4 gene is the cognate gene to the mouse CD4 gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode a protein which functions in signaling T cell activation through MHC class II-restricted antigen recognition.
  • wild-type means that the nucleic acid fragment does not comprise any mutations.
  • a “wild-type” protein means that the protein will be active at a level of activity found in nature and will comprise the amino acid sequence found in nature.
  • related polynucleotides means that regions or areas of the polynucleotides are identical and regions or areas of the polynucleotides are heterologous.
  • chimeric polynucleotide means that the polynucleotide comprises regions which are wild-type and regions which are mutated. It may also mean that the polynucleotide comprises wild-type regions from one polynucleotide and wild-type regions from another related polynucleotide.
  • cleaving means digesting the polynucleotide with enzymes or breaking the polynucleotide.
  • population means a collection of components such as polynucleotides, nucleic acid fragments or proteins.
  • a “mixed population” means a collection of components which belong to the same family of nucleic acids or proteins (i.e. are related) but which differ in their sequence (i.e. are not identical) and hence in their biological activity.
  • specific nucleic acid fragment means a nucleic acid fragment having certain end points and having a certain nucleic acid sequence.
  • Two nucleic acid fragments wherein one nucleic acid fragment has the identical sequence as a portion of the second nucleic acid fragment but different ends comprise two different specific nucleic acid fragments.
  • mutants means changes in the sequence of a wild-type nucleic acid sequence or changes in the sequence of a peptide. Such mutations may be point mutations such as transitions or transversions. The mutations may be deletions, insertions or duplications.
  • the lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • the lefthand end of single-stranded polynucleotide sequences is the 5′ end; the lefthand direction of double-stranded polynucleotide sequences is referred to as the 5′ direction.
  • RNA transcripts The direction of 5′ to 3′ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5′ to the 5′ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA and which are 3′ to the 3′ end of the coding RNA transcript are referred to as “downstream sequences”.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • the term naturally-occurring refers to an object as present in a non-pathological (undiseased) individual, such as would be typical for the species.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, an array of spatially localized compounds (e.g., a VLSIPS peptide array, polynucleotide array, and/or combinatorial small molecule array), a biological macromolecule, a bacteriophage peptide display library, a bacteriophage antibody (e.g., scFv) display library, a polysome peptide display library, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • Agents are evaluated for potential activity as antineoplastics, anti-inflammatories, or apoptosis modulators by inclusion in screening assays described hereinbelow.
  • Agents are evaluated for potential activity as specific protein interaction inhibitors (i.e., an agent which selectively inhibits a binding interaction between two predetermined polypeptides but which does not substantially interfere with cell viability) by inclusion in screening assays described hereinbelow.
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual macromolecular species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
  • physiological conditions refers to temperature, pH, ionic strength, viscosity, and like biochemical parameters which are compatible with a viable organism, and/or which typically exist intracellularly in a viable cultured yeast cell or mammalian cell.
  • the intracellular conditions in a yeast cell grown under typical laboratory culture conditions are physiological conditions.
  • Suitable in vitro reaction conditions for in vitro transcription cocktails are generally physiological conditions.
  • in vitro physiological conditions comprise 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45° C.
  • aqueous conditions may be selected by the practitioner according to conventional methods.
  • buffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition of divalent cation(s) and/or metal chelators and/or nonionic detergents and/or membrane fractions and/or antifoam agents and/or scintillants.
  • hybridization is defined herein as the formation of hybrids between a first polynucleotide and a second polynucleotide (e.g., a polynucleotide having a distinct but substantially identical sequence to the first polynucleotide), wherein the first polynucleotide preferentially hybridizes to the second polynucleotide under stringent hybridization conditions wherein substantially unrelated polynucleotide sequences do not form hybrids in the mixture.
  • a first polynucleotide and a second polynucleotide e.g., a polynucleotide having a distinct but substantially identical sequence to the first polynucleotide
  • single-chain antibody refers to a polypeptide comprising a V H domain and a V L domain in polypeptide linkage, generally linked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser] x ), and which may comprise additional amino acid sequences at the amino- and/or carboxy-termini.
  • a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.
  • a scFv is a single-chain antibody.
  • Single-chain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 contiguous amino acids substantially encoded by genes of the immunoglobulin superfamily (e.g., see The Immunoglobulin Gene Superfamily , A. F. Williams and A. N. Barclay, in Immunoglobulin Genes , T. Honjo, F. W. Alt, and T. H. Rabbitts, eds., (1989) Academic Press: San Diego, Calif., pp. 361-387, which is incorporated herein by reference), most frequently encoded by a rodent, non-human primate, avian, porcine, bovine, ovine, goat, or human heavy chain or light chain gene sequence.
  • a functional single-chain antibody generally contains a sufficient portion of an immunoglobulin superfamily gene product so as to retain the property of binding to a specific target molecule, typically a receptor or antigen (epitope).
  • CDR complementarity-determining region
  • Variable region domains typically comprise the amino-terminal approximately 105-115 amino acids of a naturally-occurring immunoglobulin chain (e.g., amino acids 1-110), although variable domains somewhat shorter or longer are also suitable for forming single-chain antibodies.
  • An immunoglobulin light or heavy chain variable region consists of a “framework” region interrupted by three hypervariable regions, also called CDR's.
  • the extent of the framework region and CDR's have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., 4th Ed., U.S. Department of Health and Human Services, Bethesda, Md. (1987)).
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • a “human framework region” is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's.
  • the CDR's are primarily responsible for binding to an epitope of an antigen.
  • variable segment refers to a portion of a nascent peptide which comprises a random, pseudorandom, or defined kernal sequence.
  • a variable segment can comprise both variant and invariant residue positions, and the degree of residue variation at a variant residue position may be limited; both options are selected at the discretion of the practitioner.
  • variable segments are about 5 to 20 amino acid residues in length (e.g., 8 to 10), although variable segments may be longer and may comprise antibody portions or receptor proteins, such as an antibody fragment, a nucleic acid binding protein, a receptor protein, and the like.
  • random peptide sequence refers to an amino acid sequence composed of two or more amino acid monomers and constructed by a stochastic or random process.
  • a random peptide can include framework or scaffolding motifs, which may comprise invariant sequences.
  • random peptide library refers to a set of polynucleotide sequences that encodes a set of random peptides, and to the set of random peptides encoded by those polynucleotide sequences, as well as the fusion proteins containing those random peptides.
  • the term “pseudorandom” refers to a set of sequences that have limited variability, so that for example the degree of residue variability at one position is different than the degree of residue variability at another position, but any pseudorandom position is allowed some degree of residue variation, however circumscribed.
  • defined sequence framework refers to a set of defined sequences that are selected on a nonrandom basis, generally on the basis of experimental data or structural data; for example, a defined sequence framework may comprise a set of amino acid sequences that are predicted to form a ⁇ -sheet structure or may comprise a leucine zipper heptad repeat motif, a zinc-finger domain, among other variations.
  • a “defined sequence kernal” is a set of sequences which encompass a limited scope of variability.
  • a completely random 10-mer sequence of the 20 conventional amino acids can be any of (20) 10 sequences
  • a pseudorandom 10-mer sequence of the conventional amino acids can be any of (20) 10 sequences but will exhibit a bias for certain residues at certain positions and/or overall
  • a defined sequence kernal is a subset of sequences which is less that the maximum number of potential sequences if each residue position was allowed to be any of the allowable 20 conventional amino acids (and/or allowable unconventional amino/imino acids).
  • a defined sequence kernal generally comprises variant and invariant residue positions and/or comprises variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), and the like, either segmentally or over the entire length of the individual selected library member sequence.
  • sequence kernals can refer to either amino acid sequences or polynucleotide sequences.
  • sequences (NNK) 10 and (NNM) 10 where N represents A, T, G, or C; K represents G or T; and M represents A or C, are defined sequence kernals.
  • epitope refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of an antibody. Typically, such binding interaction is manifested as an intermolecular contact with one or more amino acid residues of a CDR.
  • receptor refers to a molecule that has an affinity for a given ligand. Receptors can be naturally occurring or synthetic molecules. Receptors can be employed in an unaltered state or as aggregates with other species. Receptors can be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells, or other materials), cell membrane receptors, complex carbohydrates and glycoproteins, enzymes, and hormone receptors.
  • ligand refers to a molecule, such as a random peptide or variable segment sequence, that is recognized by a particular receptor.
  • a molecule or macromolecular complex
  • the binding partner having a smaller molecular weight is referred to as the ligand and the binding partner having a greater molecular weight is referred to as a receptor.
  • linker refers to a molecule or group of molecules that connects two molecules, such as a DNA binding protein and a random peptide, and serves to place the two molecules in a preferred configuration, e.g., so that the random peptide can bind to a receptor with minimal steric hindrance from the DNA binding protein.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • Nucleic acid shuffling is a method for in vitro or in vivo homologous recombination of pools of nucleic acid fragments or polynucleotides. Mixtures of related nucleic acid sequences or polynucleotides are randomly fragmented, and reassembled to yield a library or mixed population of recombinant nucleic acid molecules or polynucleotides.
  • FIG. 1 a schematic diagram of DNA shuffling as described herein.
  • the initial library can consist of related sequences of diverse origin (i.e. antibodies from naive mRNA) or can be derived by any type of mutagenesis (including shuffling) of a single antibody gene.
  • a collection of selected complementarity determining regions (“CDRs”) is obtained after the first round of affinity selection ( FIG. 1 ).
  • CDRs complementarity determining regions
  • FIG. 1 the thick CDRs confer onto the antibody molecule increased affinity for the antigen.
  • Shuffling allows the free combinatorial association of all of the CDR1s with all of the CDR2s with all of the CDR3s, etc. ( FIG. 1 ).
  • This method differs from PCR, in that it is an inverse chain reaction.
  • PCR the number of polymerase start sites and the number of molecules grows exponentially.
  • sequence of the polymerase start sites and the sequence of the molecules remains essentially the same.
  • nucleic acid reassembly or shuffling of random fragments the number of start sites and the number (but not size) of the random fragments decreases over time.
  • the theoretical endpoint is a single, large concatemeric molecule.
  • Rare shufflants will contain a large number of the best (eg. highest affinity) CDRs and these rare shufflants may be selected based on their superior affinity ( FIG. 1 ).
  • CDRs from a pool of 100 different selected antibody sequences can be permutated in up to 100 6 different ways. This large number of permutations cannot be represented in a single library of DNA sequences. Accordingly, it is contemplated that multiple cycles of DNA shuffling and selection may be required depending on the length of the sequence and the sequence diversity desired.
  • Error-prone PCR in contrast, keeps all the selected CDRs in the same relative sequence ( FIG. 1 ), generating a much smaller mutant cloud.
  • the template polynucleotide which may be used in the methods of this invention may be DNA or RNA. It may be of various lengths depending on the size of the gene or DNA fragment to be recombined or reassembled. Preferably the template polynucleotide is from 50 bp to 50 kb. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest can be used in the methods of this invention, and in fact have been successfully used.
  • the template polynucleotide may be obtained by amplification using the PCR reaction (U.S. Pat. Nos. 4,683,202 and 4,683,195) or other amplification or cloning methods.
  • PCR reaction U.S. Pat. Nos. 4,683,202 and 4,683,195
  • the removal of free primers from the PCR product before fragmentation provides a more efficient result. Failure to adequately remove the primers can lead to a low frequency of crossover clones.
  • the template polynucleotide often should be double-stranded.
  • a double-stranded nucleic acid molecule is required to ensure that regions of the resulting single-stranded nucleic acid fragments are complementary to each other and thus can hybridize to form a double-stranded molecule.
  • single-stranded or double-stranded nucleic acid fragments having regions of identity to the template polynucleotide and regions of heterology to the template polynucleotide may be added to the template polynucleotide at this step. It is also contemplated that two different but related polynucleotide templates can be mixed at this step.
  • the double-stranded polynucleotide template and any added double- or single-stranded fragments are randomly digested into fragments of from about 5 bp to 5 kb or more.
  • the size of the random fragments is from about 10 bp to 1000 bp, more preferably the size of the DNA fragments is from about 20 bp to 500 bp.
  • double-stranded nucleic acid having multiple nicks may be used in the methods of this invention.
  • a nick is a break in one strand of the double-stranded nucleic acid.
  • the distance between such nicks is preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp.
  • the nucleic acid fragment may be digested by a number of different methods.
  • the nucleic acid fragment may be digested with a nuclease, such as DNAseI or RNAse.
  • the nucleic acid may be randomly sheared by the method of sonication or by passage through a tube having a small orifice.
  • nucleic acid may also be partially digested with one or more restriction enzymes, such that certain points of cross-over may be retained statistically.
  • the concentration of any one specific nucleic acid fragment will not be greater than 1% by weight of the total nucleic acid, more preferably the concentration of any one specific nucleic acid sequence will not be greater than 0.1% by weight of the total nucleic acid.
  • the number of different specific nucleic acid fragments in the mixture will be at least about 100, preferably at least about 500, and more preferably at least about 1000.
  • single-stranded or double-stranded nucleic acid fragments may be added to the random double-stranded nucleic acid fragments in order to increase the heterogeneity of the mixture of nucleic acid fragments.
  • populations of double-stranded randomly broken nucleic acid fragments may be mixed or combined at this step.
  • single-stranded or double-stranded nucleic acid fragments having a region of identity to the template polynucleotide and a region of heterology to the template polynucleotide may be added in a 20 fold excess by weight as compared to the total nucleic acid, more preferably the single-stranded nucleic acid fragments may be added in a 10 fold excess by weight as compared to the total nucleic acid.
  • populations of nucleic acid fragments from each of the templates may be combined at a ratio of less than about 1:100, more preferably the ratio is less than about 1:40.
  • a backcross of the wild-type polynucleotide with a population of mutated polynucleotide may be desired to eliminate neutral mutations (e.g., mutations yielding an insubstantial alteration in the phenotypic property being selected for).
  • the ratio of randomly digested wild-type polynucleotide fragments which may be added to the randomly digested mutant polynucleotide fragments is approximately 1:1 to about 100:1, and more preferably from 1:1 to 40:1.
  • the mixed population of random nucleic acid fragments are denatured to form single-stranded nucleic acid fragments and then reannealed. Only those single-stranded nucleic acid fragments having regions of homology with other single-stranded nucleic acid fragments will reanneal.
  • the random nucleic acid fragments may be denatured by heating.
  • One skilled in the art could determine the conditions necessary to completely denature the double stranded nucleic acid.
  • the temperature is from 80° C. to 100° C., more preferably the temperature is from 90° C. to 96° C.
  • Other methods which may be used to denature the nucleic acid fragments include pressure (36) and pH.
  • the nucleic acid fragments may be reannealed by cooling.
  • the temperature is from 20° C. to 75° C., more preferably the temperature is from 40° C. to 65° C. If a high frequency of crossovers is needed based on an average of only 4 consecutive bases of homology, recombination can be forced by using a low annealing temperature, although the process becomes more difficult.
  • the degree of renaturation which occurs will depend on the degree of homology between the population of single-stranded nucleic acid fragments.
  • Renaturation can be accelerated by the addition of polyethylene glycol (“PEG”) or salt.
  • the salt concentration is preferably from 0 mM to 200 mM, more preferably the salt concentration is from 10 mM to 100 mM.
  • the salt may be KCl or NaCl.
  • the concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%.
  • the annealed nucleic acid fragments are next incubated in the presence of a nucleic acid polymerase and dNTP's (i.e. dATP, dCTP, dGTP and dTTP).
  • a nucleic acid polymerase and dNTP's i.e. dATP, dCTP, dGTP and dTTP.
  • the nucleic acid polymerase may be the Klenow fragment, the Taq polymerase or any other DNA polymerase known in the art.
  • the approach to be used for the assembly depends on the minimum degree of homology that should still yield crossovers. If the areas of identity are large, Taq polymerase can be used with an annealing temperature of between 45-65° C. If the areas of identity are small, Klenow polymerase can be used with an annealing temperature of between 20-30° C. One skilled in the art could vary the temperature of annealing to increase the number of cross-overs achieved.
  • the polymerase may be added to the random nucleic acid fragments prior to annealing, simultaneously with annealing or after annealing.
  • the cycle of denaturation, renaturation and incubation in the presence of polymerase is referred to herein as shuffling or reassembly of the nucleic acid.
  • This cycle is repeated for a desired number of times.
  • the cycle is repeated from 2 to 50 times, more preferably the sequence is repeated from 10 to 40 times.
  • the resulting nucleic acid is a larger double-stranded polynucleotide of from about 50 bp to about 100 kb, preferably the larger polynucleotide is from 500 bp to 50 kb.
  • This larger polynucleotide fragment may contain a number of copies of a nucleic acid fragment having the same size as the template polynucleotide in tandem.
  • This concatemeric fragment is then digested into single copies of the template polynucleotide.
  • the result will be a population of nucleic acid fragments of approximately the same size as the template polynucleotide.
  • the population will be a mixed population where single or double-stranded nucleic acid fragments having an area of identity and an area of heterology have been added to the template polynucleotide prior to shuffling.
  • the single nucleic acid fragments may be obtained from the larger concatemeric nucleic acid fragment by amplification of the single nucleic acid fragments prior to cloning by a variety of methods including PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202) rather than by digestion of the concatemer.
  • the vector used for cloning is not critical provided that it will accept a DNA fragment of the desired size. If expression of the DNA fragment is desired, the cloning vehicle should further comprise transcription and translation signals next to the site of insertion of the DNA fragment to allow expression of the DNA fragment in the host cell.
  • Preferred vectors include the pUC series and the pBR series of plasmids.
  • the resulting bacterial population will include a number of recombinant DNA fragments having random mutations. This mixed population may be tested to identify the desired recombinant nucleic acid fragment. The method of selection will depend on the DNA fragment desired.
  • the proteins expressed by each of the DNA fragments in the population or library may be tested for their ability to bind to the ligand by methods known in the art (i.e. panning, affinity chromatography). If a DNA fragment which encodes for a protein with increased drug resistance is desired, the proteins expressed by each of the DNA fragments in the population or library may be tested for their ability to confer drug resistance to the host organism.
  • methods known in the art i.e. panning, affinity chromatography
  • the proteins expressed by each of the DNA fragments in the population or library may be tested for their ability to confer drug resistance to the host organism.
  • One skilled in the art given knowledge of the desired protein, could readily test the population to identify DNA fragments which confer the desired properties onto the protein.
  • a phage display system in which fragments of the protein are expressed as fusion proteins on the phage surface (Pharmacia, Milwaukee Wis.).
  • the recombinant DNA molecules are cloned into the phage DNA at a site which results in the transcription of a fusion protein a portion of which is encoded by the recombinant DNA molecule.
  • the phage containing the recombinant nucleic acid molecule undergoes replication and transcription in the cell.
  • the leader sequence of the fusion protein directs the transport of the fusion protein to the tip of the phage particle.
  • the fusion protein which is partially encoded by the recombinant DNA molecule is displayed on the phage particle for detection and selection by the methods described above.
  • nucleic acid shuffling may be conducted with nucleic acid fragments from a subpopulation of the first population, which subpopulation contains DNA encoding the desired recombinant protein. In this manner, proteins with even higher binding affinities or enzymatic activity could be achieved.
  • a number of cycles of nucleic acid shuffling may be conducted with a mixture of wild-type nucleic acid fragments and a subpopulation of nucleic acid from the first or subsequent rounds of nucleic acid shuffling in order to remove any silent mutations from the subpopulation.
  • nucleic acid in purified form can be utilized as the starting nucleic acid.
  • the process may employ DNA or RNA including messenger RNA, which DNA or RNA may be single or double stranded.
  • a DNA-RNA hybrid which contains one strand of each may be utilized.
  • the nucleic acid sequence may be of various lengths depending on the size of the nucleic acid sequence to be mutated. Preferably the specific nucleic acid sequence is from 50 to 50000 base pairs. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest may be used in the methods of this invention.
  • the nucleic acid may be obtained from any source, for example, from plasmids such a pBR322, from cloned DNA or RNA or from natural DNA or RNA from any source including bacteria, yeast, viruses and higher organisms such as plants or animals.
  • DNA or RNA may be extracted from blood or tissue material.
  • the template polynucleotide may be obtained by amplification using the polynucleotide chain reaction (PCR) (U.S. Pat. Nos. 4,683,202 and 4,683,195).
  • PCR polynucleotide chain reaction
  • the polynucleotide may be present in a vector present in a cell and sufficient nucleic acid may be obtained by culturing the cell and extracting the nucleic acid from the cell by methods known in the art.
  • Any specific nucleic acid sequence can be used to produce the population of mutants by the present process. It is only necessary that a small population of mutant sequences of the specific nucleic acid sequence exist or be created prior to the present process.
  • the initial small population of the specific nucleic acid sequences having mutations may be created by a number of different methods. Mutations may be created by error-prone PCR. Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. Alternatively, mutations can be introduced into the template polynucleotide by oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, a short sequence of the polynucleotide is removed from the polynucleotide using restriction enzyme digestion and is replaced with a synthetic polynucleotide in which various bases have been altered from the original sequence.
  • the polynucleotide sequence can also be altered by chemical mutagenesis.
  • Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
  • Other agents which are analogues of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, these agents are added to the PCR reaction in place of the nucleotide precursor thereby mutating the sequence.
  • Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.
  • Random mutagenesis of the polynucleotide sequence can also be achieved by irradiation with X-rays or ultraviolet light.
  • plasmid DNA or DNA fragments so mutagenized are introduced into E. coli and propagated as a pool or library of mutant plasmids.
  • the small mixed population of specific nucleic acids may be found in nature in that they may consist of different alleles of the same gene or the same gene from different related species (i.e., cognate genes). Alternatively, they may be related DNA sequences found within one species, for example, the immunoglobulin genes.
  • the polynucleotides can be used directly or inserted into an appropriate cloning vector, using techniques well-known in the art.
  • the choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of this invention.
  • the templates of this invention may be plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof (e.g., coat protein, spike glycoprotein, capsid protein).
  • viruses e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like
  • selected portions thereof e.g., coat protein, spike glycoprotein, capsid protein.
  • cosmids and phagemids are preferred where the specific nucleic acid sequence to be mutated is larger because these vectors are able to stably propagate large nucleic acid fragments.
  • the mixed population of the specific nucleic acid sequence is cloned into a vector it can be clonally amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector. This is referred to as clonal amplification because while the absolute number of nucleic acid sequences increases, the number of mutants does not increase.
  • the DNA shuffling method of this invention can be performed blindly on a pool of unknown sequences.
  • any sequence mixture can be incorporated at any specific position into another sequence mixture.
  • mixtures of synthetic oligonucleotides, PCR fragments or even whole genes can be mixed into another sequence library at defined positions.
  • the insertion of one sequence (mixture) is independent from the insertion of a sequence in another part of the template.
  • the degree of recombination, the homology required, and the diversity of the library can be independently and simultaneously varied along the length of the reassembled DNA.
  • This approach of mixing two genes may be useful for the humanization of antibodies from murine hybridomas.
  • the approach of mixing two genes or inserting mutant sequences into genes may be useful for any therapeutically used protein, for example, interleukin I, antibodies, tPA, growth hormone, etc.
  • the approach may also be useful in any nucleic acid for example, promoters or introns or 3′ untranslated region or 5′ untranslated regions of genes to increase expression or alter specificity of expression of proteins.
  • the approach may also be used to mutate ribozymes or aptamers.
  • Shuffling requires the presence of homologous regions separating regions of diversity. Scaffold-like protein structures may be particularly suitable for shuffling.
  • the conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding. Examples of such scaffolds are the immunoglobulin beta-barrel, and the four-helix bundle (24). This shuffling can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.
  • the equivalents of some standard genetic matings may also be performed by shuffling in vitro.
  • a ‘molecular backcross’ can be performed by repeated mixing of the mutant's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest.
  • this approach can be used to combine phenotypes from different sources into a background of choice. It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (i.e. immunogenicity).
  • it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not, an advantage which cannot be achieved by error-prone mutagenesis or cassette mutagenesis methods.
  • the method of this invention can be used for the in vitro amplification of a whole genome from a single cell as is needed for a variety of research and diagnostic applications.
  • DNA amplification by PCR is in practice limited to a length of about 40 kb.
  • Amplification of a whole genome such as that of E. coli (5,000 kb) by PCR would require about 250 primers yielding 125 forty kb fragments. This approach is not practical due to the unavailability of sufficient sequence data.
  • random digestion of the genome with DNAseI, followed by gel purification of small fragments will provide a multitude of possible primers. Use of this mix of random small fragments as primers in a PCR reaction alone or with the whole genome as the template should result in an inverse chain reaction with the theoretical endpoint of a single concatemer containing many copies of the genome.
  • 100 fold amplification in the copy number and an average fragment size of greater than 50 kb may be obtained when only random fragments are used (see Example 2). It is thought that the larger concatemer is generated by overlap of many smaller fragments. The quality of specific PCR products obtained using synthetic primers will be indistinguishable from the product obtained from unamplified DNA. It is expected that this approach will be useful for the mapping of genomes.
  • the polynucleotide to be shuffled can be fragmented randomly or non-randomly, at the discretion of the practitioner.
  • the mixed population of the specific nucleic acid sequence is introduced into bacterial or eukaryotic cells under conditions such that at least two different nucleic acid sequences are present in each host cell.
  • the fragments can be introduced into the host cells by a variety of different methods.
  • the host cells can be transformed with the fragments using methods known in the art, for example treatment with calcium chloride. If the fragments are inserted into a phage genome, the host cell can be transfected with the recombinant phage genome having the specific nucleic acid sequences.
  • the nucleic acid sequences can be introduced into the host cell using electroporation, transfection, lipofection, biolistics, conjugation, and the like.
  • the specific nucleic acids sequences will be present in vectors which are capable of stably replicating the sequence in the host cell.
  • the vectors will encode a marker gene such that host cells having the vector can be selected. This ensures that the mutated specific nucleic acid sequence can be recovered after introduction into the host cell.
  • the entire mixed population of the specific nucleic acid sequences need not be present on a vector sequence. Rather only a sufficient number of sequences need be cloned into vectors to ensure that after introduction of the fragments into the host cells each host cell contains one vector having at least one specific nucleic acid sequence present therein. It is also contemplated that rather than having a subset of the population of the specific nucleic acids sequences cloned into vectors, this subset may be already stably integrated into the host cell.
  • the host cell transformants are placed under selection to identify those host cell transformants which contain mutated specific nucleic acid sequences having the qualities desired. For example, if increased resistance to a particular drug is desired then the transformed host cells may be subjected to increased concentrations of the particular drug and those transformants producing mutated proteins able to confer increased drug resistance will be selected. If the enhanced ability of a particular protein to bind to a receptor is desired, then expression of the protein can be induced from the transformants and the resulting protein assayed in a ligand binding assay by methods known in the art to identify that subset of the mutated population which shows enhanced binding to the ligand. Alternatively, the protein can be expressed in another system to ensure proper processing.
  • the recombined specific nucleic acid sequences may be mixed with the original mutated specific nucleic acid sequences (parent sequences) and the cycle repeated as described above. In this way a set of second recombined specific nucleic acids sequences can be identified which have enhanced characteristics or encode for proteins having enhanced properties. This cycle can be repeated a number of times as desired.
  • a backcross can be performed in the second or subsequent recombination cycle.
  • a molecular backcross can be performed by mixing the desired specific nucleic acid sequences with a large number of the wild-type sequence, such that at least one wild-type nucleic acid sequence and a mutated nucleic acid sequence are present in the same host cell after transformation. Recombination with the wild-type specific nucleic acid sequence will eliminate those neutral mutations that may affect unselected characteristics such as immunogenicity but not the selected characteristics.
  • a subset of the specific nucleic acid sequences can be fragmented prior to introduction into the host cell.
  • the size of the fragments must be large enough to contain some regions of identity with the other sequences so as to homologously recombine with the other sequences.
  • the size of the fragments will range from 0.03 kb to 100 kb more preferably from 0.2 kb to 10 kb. It is also contemplated that in subsequent rounds, all of the specific nucleic acid sequences other than the sequences selected from the previous round may be cleaved into fragments prior to introduction into the host cells.
  • Sequences can be accomplished by a variety of method known in the art.
  • the sequences can be randomly fragmented or fragmented at specific sites in the nucleic acid sequence. Random fragments can be obtained by breaking the nucleic acid or exposing it to harsh physical treatment (e.g., shearing or irradiation) or harsh chemical agents (e.g., by free radicals; metal ions; acid treatment to depurinate and cleave). Random fragments can also be obtained, in the case of DNA by the use of DNase or like nuclease.
  • the sequences can be cleaved at specific sites by the use of restriction enzymes.
  • the fragmented sequences can be single-stranded or double-stranded. If the sequences were originally single-stranded they can be denatured with heat, chemicals or enzymes prior to insertion into the host cell.
  • the reaction conditions suitable for separating the strands of nucleic acid are well known in the art.
  • steps of this process can be repeated indefinitely, being limited only by the number of possible mutants which can be achieved. After a certain number of cycles, all possible mutants will have been achieved and further cycles are redundant.
  • the same mutated template nucleic acid is repeatedly recombined and the resulting recombinants selected for the desired characteristic.
  • the initial pool or population of mutated template nucleic acid is cloned into a vector capable of replicating in a bacteria such as E. coli .
  • the particular vector is not essential, so long as it is capable of autonomous replication in E. coli .
  • the vector is designed to allow the expression and production of any protein encoded by the mutated specific nucleic acid linked to the vector. It is also preferred that the vector contain a gene encoding for a selectable marker.
  • the population of vectors containing the pool of mutated nucleic acid sequences is introduced into the E. coli host cells.
  • the vector nucleic acid sequences may be introduced by transformation, transfection or infection in the case of phage.
  • the concentration of vectors used to transform the bacteria is such that a number of vectors is introduced into each cell. Once present in the cell, the efficiency of homologous recombination is such that homologous recombination occurs between the various vectors. This results in the generation of mutants (daughters) having a combination of mutations which differ from the original parent mutated sequences.
  • the host cells are then clonally replicated and selected for the marker gene present on the vector. Only those cells having a plasmid will grow under the selection.
  • the host cells which contain a vector are then tested for the presence of favorable mutations. Such testing may consist of placing the cells under selective pressure, for example, if the gene to be selected is an improved drug resistance gene. If the vector allows expression of the protein encoded by the mutated nucleic acid sequence, then such selection may include allowing expression of the protein so encoded, isolation of the protein and testing of the protein to determine whether, for example, it binds with increased efficiency to the ligand of interest.
  • nucleic acid is isolated either already linked to the vector or separated from the vector. This nucleic acid is then mixed with the first or parent population of nucleic acids and the cycle is repeated.
  • nucleic acid sequences having enhanced desired properties can be selected.
  • the first generation of mutants are retained in the cells and the parental mutated sequences are added again to the cells. Accordingly, the first cycle of Embodiment I is conducted as described above. However, after the daughter nucleic acid sequences are identified, the host cells containing these sequences are retained.
  • the parent mutated specific nucleic acid population is introduced into the host cells already containing the daughter nucleic acids. Recombination is allowed to occur in the cells and the next generation of recombinants, or granddaughters are selected by the methods described above.
  • This cycle can be repeated a number of times until the nucleic acid or peptide having the desired characteristics is obtained. It is contemplated that in subsequent cycles, the population of mutated sequences which are added to the preferred mutants may come from the parental mutants or any subsequent generation.
  • the invention provides a method of conducting a “molecular” backcross of the obtained recombinant specific nucleic acid in order to eliminate any neutral mutations.
  • Neutral mutations are those mutations which do not confer onto the nucleic acid or peptide the desired properties. Such mutations may however confer on the nucleic acid or peptide undesirable characteristics. Accordingly, it is desirable to eliminate such neutral mutations.
  • the method of this invention provide a means of doing so.
  • the nucleic acid after the mutant nucleic acid, having the desired characteristics, is obtained by the methods of the embodiments, the nucleic acid, the vector having the nucleic acid or the host cell containing the vector and nucleic acid is isolated.
  • the nucleic acid or vector is then introduced into the host cell with a large excess of the wild-type nucleic acid.
  • the nucleic acid of the mutant and the nucleic acid of the wild-type sequence are allowed to recombine.
  • the resulting recombinants are placed under the same selection as the mutant nucleic acid. Only those recombinants which retained the desired characteristics will be selected. Any silent mutations which do not provide the desired characteristics will be lost through recombination with the wild-type DNA. This cycle can be repeated a number of times until all of the silent mutations are eliminated.
  • the in vivo recombination method of this invention can be performed blindly on a pool of unknown mutants or alleles of a specific nucleic acid fragment or sequence. However, it is not necessary to know the actual DNA or RNA sequence of the specific nucleic acid fragment.
  • the approach of using recombination within a mixed population of genes can be useful for the generation of any useful proteins, for example, interleukin I, antibodies, tPA, growth hormone, etc.
  • This approach may be used to generate proteins having altered specificity or activity.
  • the approach may also be useful for the generation of mutant nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 3′ untranslated regions or 5′ untranslated regions of genes.
  • This approach may be used to generate genes having increased rates of expression.
  • This approach may also be useful in the study of repetitive DNA sequences.
  • this approach may be useful to mutate ribozymes or aptamers.
  • Scaffold-like regions separating regions of diversity in proteins may be particularly suitable for the methods of this invention.
  • the conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding.
  • Examples of such scaffolds are the immunoglobulin beta barrel, and the four-helix bundle.
  • the methods of this invention can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.
  • a “molecular” backcross can be performed by repeated mixing of the mutant's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest.
  • this approach can be used to combine phenotypes from different sources into a background of choice. It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (i.e. immunogenicity). Thus it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not.
  • the present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by peptide display methods, wherein an associated polynucleotide encodes a displayed peptide which is screened for a phenotype (e.g., for affinity for a predetermined receptor (ligand).
  • a phenotype e.g., for affinity for a predetermined receptor (ligand).
  • peptide structures including the primary amino acid sequences, of peptides or peptidomimetics that interact with biological macromolecules.
  • One method of identifying peptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule (e.g., a receptor) involves the screening of a large library or peptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the peptide.
  • each bacteriophage particle or cell serves as an individual library member displaying a single species of displayed peptide in addition to the natural bacteriophage or cell protein sequences.
  • Each bacteriophage or cell contains the nucleotide sequence information encoding the particular displayed peptide sequence; thus, the displayed peptide sequence can be ascertained by nucleotide sequence determination of an isolated library member.
  • a well-known peptide display method involves the presentation of a peptide sequence on the surface of a filamentous bacteriophage, typically as a fusion with a bacteriophage coat protein.
  • the bacteriophage library can be incubated with an immobilized, predetermined macromolecule or small molecule (e.g., a receptor) so that bacteriophage particles which present a peptide sequence that binds to the immobilized macromolecule can be differentially partitioned from those that do not present peptide sequences that bind to the predetermined macromolecule.
  • the bacteriophage particles i.e., library members
  • the bacteriophage particles which are bound to the immobilized macromolecule are then recovered and replicated to amplify the selected bacteriophage subpopulation for a subsequent round of affinity enrichment and phage replication.
  • the bacteriophage library members that are thus selected are isolated and the nucleotide sequence encoding the displayed peptide sequence is determined, thereby identifying the sequence(s) of peptides that bind to the predetermined macromolecule (e.g., receptor).
  • the predetermined macromolecule e.g., receptor
  • the fusion protein/vector DNA complexes can be screened against a predetermined macromolecule in much the same way as bacteriophage particles are screened in the phage-based display system, with the replication and sequencing of the DNA vectors in the selected fusion protein/vector DNA complexes serving as the basis for identification of the selected library peptide sequence(s).
  • RNA molecules with the ability to bind a predetermined protein or a predetermined dye molecule were selected by alternate rounds of selection and PCR amplification (Tuerk and Gold (1990) Science 249: 505; Ellington and Szostak (1990) Nature 346: 818).
  • a similar technique was used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res.
  • library members comprise a fusion protein having a first polypeptide portion with DNA binding activity and a second polypeptide portion having the library member unique peptide sequence; such methods are suitable for use in cell-free in vitro selection formats, among others.
  • the displayed peptide sequences can be of varying lengths, typically from 3-5000 amino acids long or longer, frequently from 5-100 amino acids long, and often from about 8-15 amino acids long.
  • a library can comprise library members having varying lengths of displayed peptide sequence, or may comprise library members having a fixed length of displayed peptide sequence. Portions or all of the displayed peptide sequence(s) can be random, pseudorandom, defined set kernal, fixed, or the like.
  • the present display methods include methods for in vitro and in vivo display of single-chain antibodies, such as nascent scFv on polysomes or scFv displayed on phage, which enable large-scale screening of scFv libraries having broad diversity of variable region sequences and binding specificities.
  • the present invention also provides random, pseudorandom, and defined sequence framework peptide libraries and methods for generating and screening those libraries to identify useful compounds (e.g., peptides, including single-chain antibodies) that bind to receptor molecules or epitopes of interest or gene products that modify peptides or RNA in a desired fashion.
  • useful compounds e.g., peptides, including single-chain antibodies
  • the random, pseudorandom, and defined sequence framework peptides are produced from libraries of peptide library members that comprise displayed peptides or displayed single-chain antibodies attached to a polynucleotide template from which the displayed peptide was synthesized.
  • the mode of attachment may vary according to the specific embodiment of the invention selected, and can include encapsidation in a phage particle or incorporation in a cell.
  • a method of affinity enrichment allows a very large library of peptides and single-chain antibodies to be screened and the polynucleotide sequence encoding the desired peptide(s) or single-chain antibodies to be selected.
  • the polynucleotide can then be isolated and shuffled to recombine combinatorially the amino acid sequence of the selected peptide(s) (or predetermined portions thereof) or single-chain antibodies (or just V H , V L , or CDR portions thereof).
  • Using these methods one can identify a peptide or single-chain antibody as having a desired binding affinity for a molecule and can exploit the process of shuffling to converge rapidly to a desired high-affinity peptide or scFv.
  • the peptide or antibody can then be synthesized in bulk by conventional means for any suitable use (e.g., as a therapeutic or diagnostic agent).
  • a significant advantage of the present invention is that no prior information regarding an expected ligand structure is required to isolate peptide ligands or antibodies of interest.
  • the peptide identified can have biological activity, which is meant to include at least specific binding affinity for a selected receptor molecule and, in some instances, will further include the ability to block the binding of other compounds, to stimulate or inhibit metabolic pathways, to act as a signal or messenger, to stimulate or inhibit cellular activity, and the like.
  • the present invention also provides a method for shuffling a pool of polynucleotide sequences selected by affinity screening a library of polysomes displaying nascent peptides (including single-chain antibodies) for library members which bind to a predetermined receptor (e.g., a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as heterodimers and the like) or epitope (e.g., an immobilized protein, glycoprotein, oligosaccharide, and the like).
  • a predetermined receptor e.g., a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as heterodimers and the like
  • epitope e.g., an im
  • Polynucleotide sequences selected in a first selection round are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.
  • the recombined selected polynucleotide sequences are subjected to at least one subsequent selection round.
  • the polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection.
  • Selected sequences can also be backcrossed with polynucleotide sequences encoding neutral sequences (i.e., having insubstantial functional effect on binding), such as for example by backcrossing with a wild-type or naturally-occurring sequence substantially identical to a selected sequence to produce native-like functional peptides, which may be less immunogenic. Generally, during backcrossing subsequent selection is applied to retain the property of binding to the predetermined receptor (ligand).
  • the sequences Prior to or concomitant with the shuffling of selected sequences, the sequences can be mutagenized.
  • selected library members are cloned in a prokaryotic vector (e.g., plasmid, phagemid, or bacteriophage) wherein a collection of individual colonies (or plaques) representing discrete library members are produced.
  • Individual selected library members can then be manipulated (e.g., by site-directed mutagenesis, cassette mutagenesis, chemical mutagenesis, PCR mutagenesis, and the like) to generate a collection of library members representing a kernal of sequence diversity based on the sequence of the selected library member.
  • sequence of an individual selected library member or pool can be manipulated to incorporate random mutation, pseudorandom mutation, defined kernal mutation (i.e., comprising variant and invariant residue positions and/or comprising variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), codon-based mutation, and the like, either segmentally or over the entire length of the individual selected library member sequence.
  • the mutagenized selected library members are then shuffled by in vitro and/or in vivo recombinatorial shuffling as disclosed herein.
  • the invention also provides peptide libraries comprising a plurality of individual library members of the invention, wherein (1) each individual library member of said plurality comprises a sequence produced by shuffling of a pool of selected sequences, and (2) each individual library member comprises a variable peptide segment sequence or single-chain antibody segment sequence which is distinct from the variable peptide segment sequences or single-chain antibody sequences of other individual library members in said plurality (although some library members may be present in more than one copy per library due to uneven amplification, stochastic probability, or the like).
  • the invention also provides a product-by-process, wherein selected polynucleotide sequences having (or encoding a peptide having) a predetermined binding specificity are formed by the process of: (1) screening a displayed peptide or displayed single-chain antibody library against a predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching library members which bind to the predetermined receptor or epitope to produce a pool of selected library members, (2) shuffling by recombination the selected library members (or amplified or cloned copies thereof) which binds the predetermined epitope and has been thereby isolated and/or enriched from the library to generate a shuffled library, and (3) screening the shuffled library against the predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching shuffled library members which bind to the predetermined receptor or epi
  • the present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by antibody display methods, wherein an associated polynucleotide encodes a displayed antibody which is screened for a phenotype (e.g., for affinity for binding a predetermined antigen (ligand).
  • a phenotype e.g., for affinity for binding a predetermined antigen (ligand).
  • V variable
  • D tandem array of diversity
  • J tandem array of joining
  • V-D-J rearrangement occurs wherein a heavy chain variable region gene (V H ) is formed by rearrangement to form a fused D-J segment followed by rearrangement with a V segment to form a V-D-J joined product gene which, if productively rearranged, encodes a functional variable region (V H ) of a heavy chain.
  • V H heavy chain variable region gene
  • V L variable region gene
  • variable regions achievable in immunoglobulins derives in part from the numerous combinatorial possibilities of joining V and J segments (and, in the case of heavy chain loci, D segments) during rearrangement in B cell development. Additional sequence diversity in the heavy chain variable regions arises from non-uniform rearrangements of the D segments during V-D-J joining and from N region addition. Further, antigen-selection of specific B cell clones selects for higher affinity variants having nongermline mutations in one or both of the heavy and light chain variable regions; a phenomenon referred to as “affinity maturation” or “affinity sharpening”. Typically, these “affinity sharpening” mutations cluster in specific areas of the variable region, most commonly in the complementarity-determining regions (CDRs).
  • CDRs complementarity-determining regions
  • Combinatorial libraries of antibodies have been generated in bacteriophage lambda expression systems which may be screened as bacteriophage plaques or as colonies of lysogens (Huse et al. (1989) Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci . ( U.S.A .) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci . ( U.S.A .) 88: 2432).
  • a bacteriophage antibody display library is screened with a receptor (e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid) that is immobilized (e.g., by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography) and/or labeled (e.g., to screen plaque or colony lifts).
  • a receptor e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid
  • immobilized e.g., by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography
  • labeled e.g., to screen plaque or colony lifts
  • the first step generally involves obtaining the genes encoding V H and V L domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis.
  • the single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly-Gly-Gly-Gly-Ser) 3 or equivalent linker peptide(s).
  • the linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either V H -linker-V L or V L -linker-V H .
  • the scFv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site.
  • scFv fragments are comprised of V H and V L domains linked into a single polypeptide chain by a flexible linker peptide.
  • the scFv genes are assembled, they are cloned into a phagemid and expressed at the tip of the M13 phage (or similar filamentous bacteriophage) as fusion proteins with the bacteriophage pIII (gene 3) coat protein. Enriching for phage expressing an antibody of interest is accomplished by panning the recombinant phage displaying a population scFv for binding to a predetermined epitope (e.g., target antigen, receptor).
  • a predetermined epitope e.g., target antigen, receptor
  • the linked polynucleotide of a library member provides the basis for replication of the library member after a screening or selection procedure, and also provides the basis for the determination, by nucleotide sequencing, of the identity of the displayed peptide sequence or V H and V L amino acid sequence.
  • the displayed peptide(s) or single-chain antibody (e.g., scFv) and/or its V H and V L domains or their CDRs can be cloned and expressed in a suitable expression system.
  • polynucleotides encoding the isolated V H and V L domains will be ligated to polynucleotides encoding constant regions (C H and C L ) to form polynucleotides encoding complete antibodies (e.g., chimeric or fully-human), antibody fragments, and the like.
  • polynucleotides encoding the isolated CDRs will be grafted into polynucleotides encoding a suitable variable region framework (and optionally constant regions) to form polynucleotides encoding complete antibodies (e.g., humanized or fully-human), antibody fragments, and the like.
  • Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography.
  • neoplasia a disease that causes inflammation
  • neoplasia a malignant neoplasia
  • autoimmune disease a malignant neoplasia
  • AIDS a malignant neoplasia
  • cardiovascular disease a malignant neoplasia .
  • infections a malignant neoplasia .
  • stage disease e.g., neoplasia
  • therapeutic application to treat disease such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like.
  • V H and V L cassettes can themselves be diversified, such as by random, pseudorandom, or directed mutagenesis.
  • V H and V L cassettes are diversified in or near the complementarity-determining regions (CDRs), often the third CDR, CDR3.
  • Enzymatic inverse PCR mutagenesis has been shown to be a simple and reliable method for constructing relatively large libraries of scFv site-directed mutants (Stemmer et al. (1993) Biotechniques 14: 256), as has error-prone PCR and chemical mutagenesis (Deng et al. (1994) J. Biol. Chem. 269: 9533).
  • Riechmann et al. (1993) Biochemistry 32: 8848 showed semirational design of an antibody scFv fragment using site-directed randomization by degenerate oligonucleotide PCR and subsequent phage display of the resultant scFv mutants.
  • Barbas et al. (1992) op.cit. attempted to circumvent the problem of limited repertoire sizes resulting from using biased variable region sequences by randomizing the sequence in a synthetic CDR region of a human tetanus toxoid-binding Fab.
  • CDR randomization has the potential to create approximately 1 ⁇ 10 20 CDRs for the heavy chain CDR3 alone, and a roughly similar number of variants of the heavy chain CDR1 and CDR2, and light chain CDR1-3 variants.
  • the combinatorics of CDR randomization of heavy and/or light chains requires generating a prohibitive number of bacteriophage clones to produce a clone library representing all possible combinations, the vast majority of which will be non-binding. Generation of such large numbers of primary transformants is not feasible with current transformation technology and bacteriophage display systems. For example, Barbas et al. (1992) op.cit. only generated 5 ⁇ 10 7 transformants, which represents only a tiny fraction of the potential diversity of a library of thoroughly randomized CDRs.
  • bacteriophage display of scFv have already yielded a variety of useful antibodies and antibody fusion proteins.
  • a bispecific single chain antibody has been shown to mediate efficient tumor cell lysis (Gruber et al. (1994) J. Immunol. 152: 5368).
  • Intracellular expression of an anti-Rev scFv has been shown to inhibit HIV-1 virus replication in vitro (Duan et al. (1994) Proc. Natl. Acad. Sci . ( USA ) 91: 5075), and intracellular expression of an anti-p21 ras scFv has been shown to inhibit meiotic maturation of Xenopus oocytes (Biocca et al. (1993) Biochem.
  • the in vitro and in vivo shuffling methods of the invention are used to recombine CDRs which have been obtained (typically via PCR amplification or cloning) from nucleic acids obtained from selected displayed antibodies.
  • Such displayed antibodies can be displayed on cells, on bacteriophage particles, on polysomes, or any suitable antibody display system wherein the antibody is associated with its encoding nucleic acid(s).
  • the CDRs are initially obtained from mRNA (or cDNA) from antibody-producing cells (e.g., plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO92/03918, WO93/12227, and WO94/25585), including hybridomas derived therefrom.
  • antibody-producing cells e.g., plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO92/03918, WO93/12227, and WO94/25585
  • Polynucleotide sequences selected in a first selection round are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination, especially shuffling of CDRs (typically shuffling heavy chain CDRs with other heavy chain CDRs and light chain CDRs with other light chain CDRs) to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.
  • the recombined selected polynucleotide sequences are expressed in a selection format as a displayed antibody and subjected to at least one subsequent selection round.
  • the polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection until an antibody of the desired binding affinity is obtained.
  • Selected sequences can also be backcrossed with polynucleotide sequences encoding neutral antibody framework sequences (i.e., having insubstantial functional effect on antigen binding), such as for example by backcrossing with a human variable region framework to produce human-like sequence antibodies. Generally, during backcrossing subsequent selection is applied to retain the property of binding to the predetermined antigen.
  • the valency of the target epitope may be varied to control the average binding affinity of selected scFv library members.
  • the target epitope can be bound to a surface or substrate at varying densities, such as by including a competitor epitope, by dilution, or by other method known to those in the art.
  • a high density (valency) of predetermined epitope can be used to enrich for scFv library members which have relatively low affinity, whereas a low density (valency) can preferentially enrich for higher affinity scFv library members.
  • a collection of synthetic oligonucleotides encoding random, pseudorandom, or a defined sequence kernal set of peptide sequences can be inserted by ligation into a predetermined site (e.g., a CDR).
  • a predetermined site e.g., a CDR
  • the sequence diversity of one or more CDRs of the single-chain antibody cassette(s) can be expanded by mutating the CDR(s) with site-directed mutagenesis, CDR-replacement, and the like.
  • the resultant DNA molecules can be propagated in a host for cloning and amplification prior to shuffling, or can be used directly (i.e., may avoid loss of diversity which may occur upon propagation in a host cell) and the selected library members subsequently shuffled.
  • Displayed peptide/polynucleotide complexes which encode a variable segment peptide sequence of interest or a single-chain antibody of interest are selected from the library by an affinity enrichment technique. This is accomplished by means of a immobilized macromolecule or epitope specific for the peptide sequence of interest, such as a receptor, other macromolecule, or other epitope species. Repeating the affinity selection procedure provides an enrichment of library members encoding the desired sequences, which may then be isolated for pooling and shuffling, for sequencing, and/or for further propagation and affinity enrichment.
  • the library members without the desired specificity are removed by washing.
  • the degree and stringency of washing required will be determined for each peptide sequence or single-chain antibody of interest and the immobilized predetermined macromolecule or epitope.
  • a certain degree of control can be exerted over the binding characteristics of the nascent peptide/DNA complexes recovered by adjusting the conditions of the binding incubation and the subsequent washing.
  • the temperature, pH, ionic strength, divalent cations concentration, and the volume and duration of the washing will select for nascent peptide/DNA complexes within particular ranges of affinity for the immobilized macromolecule. Selection based on slow dissociation rate, which is usually predictive of high affinity, is often the most practical route.
  • nascent peptide/DNA or peptide/RNA complex is prevented, and with increasing time, nascent peptide/DNA or peptide/RNA complexes of higher and higher affinity are recovered.
  • affinities of some peptides are dependent on ionic strength or cation concentration. This is a useful characteristic for peptides that will be used in affinity purification of various proteins when gentle conditions for removing the protein from the peptides are required.
  • One variation involves the use of multiple binding targets (multiple epitope species, multiple receptor species), such that a scFv library can be simultaneously screened for a multiplicity of scFv which have different binding specificities.
  • multiple binding targets multiple epitope species, multiple receptor species
  • a scFv library can be simultaneously screened for a multiplicity of scFv which have different binding specificities.
  • multiple target epitope species each encoded on a separate bead (or subset of beads), can be mixed and incubated with a polysome-display scFv library under suitable binding conditions.
  • the collection of beads, comprising multiple epitope species can then be used to isolate, by affinity selection, scFv library members.
  • subsequent affinity screening rounds can include the same mixture of beads, subsets thereof, or beads containing only one or two individual epitope species. This approach affords efficient screening, and is compatible with laboratory automation, batch processing, and high throughput screening methods.
  • a variety of techniques can be used in the present invention to diversify a peptide library or single-chain antibody library, or to diversify, prior to or concomitant with shuffling, around variable segment peptides or V H , V L , or CDRs found in early rounds of panning to have sufficient binding activity to the predetermined macromolecule or epitope.
  • the positive selected peptide/polynucleotide complexes are sequenced to determine the identity of the active peptides.
  • Oligonucleotides are then synthesized based on these active peptide sequences, employing a low level of all bases incorporated at each step to produce slight variations of the primary oligonucleotide sequences.
  • This mixture of (slightly) degenerate oligonucleotides is then cloned into the variable segment sequences at the appropriate locations.
  • This method produces systematic, controlled variations of the starting peptide sequences, which can then be shuffled. It requires, however, that individual positive nascent peptide/polynucleotide complexes be sequenced before mutagenesis, and thus is useful for expanding the diversity of small numbers of recovered complexes and selecting variants having higher binding affinity and/or higher binding specificity.
  • mutagenic PCR amplification of positive selected peptide/polynucleotide complexes is done prior to sequencing.
  • the same general approach can be employed with single-chain antibodies in order to expand the diversity and enhance the binding affinity/specificity, typically by diversifying CDRs or adjacent framework regions prior to or concomitant with shuffling.
  • shuffling reactions can be spiked with mutagenic oligonucleotides capable of in vitro recombination with the selected library members can be included.
  • mixtures of synthetic oligonucleotides and PCR fragments can be added to the in vitro shuffling mix and be incorporated into resulting shuffled library members (shufflants).
  • the present invention of shuffling enables the generation of a vast library of CDR-variant single-chain antibodies.
  • One way to generate such antibodies is to insert synthetic CDRs into the single-chain antibody and/or CDR randomization prior to or concomitant with shuffling.
  • the sequences of the synthetic CDR cassettes are selected by referring to known sequence data of human CDR and are selected in the discretion of the practitioner according to the following guidelines: synthetic CDRs will have at least 40 percent positional sequence identity to known CDR sequences, and preferably will have at least 50 to 70 percent positional sequence identity to known CDR sequences.
  • a collection of synthetic CDR sequences can be generated by synthesizing a collection of oligonucleotide sequences on the basis of naturally-occurring human CDR sequences listed in Kabat et al. (1991) op.cit.; the pool(s) of synthetic CDR sequences are calculated to encode CDR peptide sequences having at least 40 percent sequence identity to at least one known naturally-occurring human CDR sequence.
  • a collection of naturally-occurring CDR sequences may be compared to generate consensus sequences so that amino acids used at a residue position frequently (i.e., in at least 5 percent of known CDR sequences) are incorporated into the synthetic CDRs at the corresponding position(s).
  • oligonucleotides encoding CDR peptide sequences encompassing all or most permutations of the observed natural sequence variations is synthesized.
  • a collection of human V H CDR sequences have carboxy-terminal amino acids which are either Tyr, Val, Phe, or Asp, then the pool(s) of synthetic CDR oligonucleotide sequences are designed to allow the carboxy-terminal CDR residue to be any of these amino acids.
  • residues other than those which naturally-occur at a residue position in the collection of CDR sequences are incorporated: conservative amino acid substitutions are frequently incorporated and up to 5 residue positions may be varied to incorporate non-conservative amino acid substitutions as compared to known naturally-occurring CDR sequences.
  • Such CDR sequences can be used in primary library members (prior to first round screening) and/or can be used to spike in vitro shuffling reactions of selected library member sequences. Construction of such pools of defined and/or degenerate sequences will be readily accomplished by those of ordinary skill in the art.
  • the collection of synthetic CDR sequences comprises at least one member that is not known to be a naturally-occurring CDR sequence. It is within the discretion of the practitioner to include or not include a portion of random or pseudorandom sequence corresponding to N region addition in the heavy chain CDR; the N region sequence ranges from 1 nucleotide to about 4 nucleotides occurring at V-D and D-J junctions.
  • a collection of synthetic heavy chain CDR sequences comprises at least about 100 unique CDR sequences, typically at least about 1,000 unique CDR sequences, preferably at least about 10,000 unique CDR sequences, frequently more than 50,000 unique CDR sequences; however, usually not more than about 1 ⁇ 10 6 unique CDR sequences are included in the collection, although occasionally 1 ⁇ 10 7 to 1 ⁇ 10 8 unique CDR sequences are present, especially if conservative amino acid substitutions are permitted at positions where the conservative amino acid substituent is not present or is rare (i.e., less than 0.1 percent) in that position in naturally-occurring human CDRs.
  • the number of unique CDR sequences included in a library should not exceed the expected number of primary transformants in the library by more than a factor of 10.
  • Such single-chain antibodies generally bind to a predetermined antigen (e.g., the immunogen) with an affinity of about at least 1 ⁇ 10 7 M ⁇ 1 , preferably with an affinity of about at least 5 ⁇ 10 7 M ⁇ 1 , more preferably with an affinity of at least 1 ⁇ 10 8 M ⁇ 1 to 1 ⁇ 10 9 M ⁇ 1 or more, sometimes up to 1 ⁇ 10 10 M ⁇ 1 or more.
  • a predetermined antigen e.g., the immunogen
  • the predetermined antigen is a human protein, such as for example a human cell surface antigen (e.g., CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other human biological macromolecule (e.g., thrombomodulin, protein C, carbohydrate antigen, silyl Lewis antigen, L-selectin), or nonhuman disease associated macromolecule (e.g., bacterial LPS, virion capsid protein or envelope glycoprotein) and the like.
  • a human cell surface antigen e.g., CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor
  • other human biological macromolecule e.g., thrombomodulin, protein C, carbohydrate antigen, silyl Lewis antigen, L-selectin
  • nonhuman disease associated macromolecule e.g., bacterial LPS, virion capsid protein or envelope glycoprotein
  • High affinity single-chain antibodies of the desired specificity can be engineered and expressed in a variety of systems.
  • scFv have been produced in plants (Firek et al. (1993) Plant Mol. Biol. 23: 861) and can be readily made in prokaryotic systems (Owens R J and Young R J (1994) J. Immunol. Meth. 168: 149; Johnson S and Bird R E (1991) Methods Enzymol. 203: 88).
  • the single-chain antibodies can be used as a basis for constructing whole antibodies or various fragments thereof (Kettleborough et al. (1994) Eur. J. Immunol. 24: 952).
  • variable region encoding sequence may be isolated (e.g., by PCR amplification or subcloning) and spliced to a sequence encoding a desired human constant region to encode a human sequence antibody more suitable for human therapeutic uses where immunogenicity is preferably minimized.
  • the polynucleotide(s) having the resultant fully human encoding sequence(s) can be expressed in a host cell (e.g., from an expression vector in a mammalian cell) and purified for pharmaceutical formulation.
  • the DNA expression constructs will typically include an expression control DNA sequence operably linked to the coding sequences, including naturally-associated or heterologous promoter regions.
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the mutant “engineered” antibodies.
  • the DNA sequences will be expressed in hosts after the sequences have been operably linked to an expression control sequence (i.e., positioned to ensure the transcription and translation of the structural gene).
  • expression control sequence i.e., positioned to ensure the transcription and translation of the structural gene.
  • These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein by reference).
  • mammalian tissue cell culture may also be used to produce the polypeptides of the present invention (see, Winnacker, “From Genes to Clones,” VCH Publishers, N.Y., N.Y. (1987), which is incorporated herein by reference).
  • Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, etc, but preferably transformed B-cells or hybridomas.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al. (1986) Immunol. Rev. 89: 49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, Adenovirus, Bovine Papilloma Virus, and the like.
  • Enhancers are cis-acting sequences of between 10 to 300 bp that increase transcription by a promoter. Enhancers can effectively increase transcription when either 5′ or 3′ to the transcription unit. They are also effective if located within an intron or within the coding sequence itself.
  • viral enhancers including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers. Enhancer sequences from mammalian systems are also commonly used, such as the mouse immunoglobulin heavy chain enhancer.
  • Mammalian expression vector systems will also typically include a selectable marker gene.
  • suitable markers include, the dihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), or prokaryotic genes conferring drug resistance.
  • the first two marker genes prefer the use of mutant cell lines that lack the ability to grow without the addition of thymidine to the growth medium. Transformed cells can then be identified by their ability to grow on non-supplemented media.
  • prokaryotic drug resistance genes useful as markers include genes conferring resistance to G418, mycophenolic acid and hygromycin.
  • the vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment. lipofection, or electroporation may be used for other cellular hosts. Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see, generally, Sambrook et al., supra).
  • the antibodies, individual mutated immunoglobulin chains, mutated antibody fragments, and other immunoglobulin polypeptides of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fraction column chromatography, gel electrophoresis and the like (see, generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982)). Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (see, generally, Immunological Methods , Vols. I and II, Eds. Lefrkovits and Pernis, Academic Press, New York, N.Y. (1979 and 1981)).
  • the antibodies generated by the method of the present invention can be used for diagnosis and therapy.
  • they can be used to treat cancer, autoimmune diseases, or viral infections.
  • the antibodies will typically bind to an antigen expressed preferentially on cancer cells, such as erbB-2, CEA, CD33, and many other antigens and binding members well known to those skilled in the art.
  • Shuffling can also be used to recombinatorially diversify a pool of selected library members obtained by screening a two-hybrid screening system to identify library members which bind a predetermined polypeptide sequence.
  • the selected library members are pooled and shuffled by in vitro and/or in vivo recombination.
  • the shuffled pool can then be screened in a yeast two hybrid system to select library members which bind said predetermined polypeptide sequence (e.g., and SH2 domain) or which bind an alternate predetermined polypeptide sequence (e.g., an SH2 domain from another protein species).
  • Polynucleotides encoding two hybrid proteins, one consisting of the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a known protein and the other consisting of the Gal4 activation domain fused to a polypeptide sequence of a second protein, are constructed and introduced into a yeast host cell. Intermolecular binding between the two fusion proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene (e.g., lacZ, HIS3) which is operably linked to a Gal4 binding site.
  • a reporter gene e.g., lacZ, HIS3
  • the two-hybrid method is used to identify novel polypeptide sequences which interact with a known protein (Silver S C and Hunt S W (1993) Mol.
  • X-gal 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside
  • DNAseI deoxyribonuclease
  • PBS phosphate buffered saline
  • the substrate for the reassembly reaction was the dsDNA polymerase chain reaction (“PCR”) product of the wild-type LacZ alpha gene from pUC18.
  • PCR dsDNA polymerase chain reaction
  • the primer sequences were 5′AAAGCGTCGATTTTTGTGAT3′ (SEQ ID NO:1) and 5′ATGGGGTTCCGCGCACATTT3′ (SEQ ID NO:2).
  • the free primers were removed from the PCR product by Wizard PCR prep (Promega, Madison Wis.) according to the manufacturer's directions. The removal of the free primers was found to be important.
  • DNA substrate was digested with 0.15 units of DNAseI (Sigma, St. Louis Mo.) in 100 ⁇ l of [50 mM Tris-HCl pH 7.4, 1 mM MgCl 2 ], for 10-20 minutes at room temperature.
  • the digested DNA was run on a 2% low melting point agarose gel. Fragments of 10-70 basepairs (bp) were purified from the 2% low melting point agarose gels by electrophoresis onto DESI ion exchange paper (Whatman, Hillsborough Oreg.). The DNA fragments were eluted from the paper with 1 M NaCl and ethanol precipitated.
  • the purified fragments were resuspended at a concentration of 10-30 ng/ ⁇ l in PCR Mix (0.2 mM each dNTP, 2.2 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 0.3 ⁇ l Taq DNA polymerase, 50 ⁇ l total volume). No primers were added at this point.
  • a reassembly program of 94° C. for 60 seconds, 30-45 cycles of [94° C. for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds] and 5 minutes at 72° C. was used in an MJ Research (Watertown Mass.) PTC-150 thermocycler.
  • the PCR reassembly of small fragments into larger sequences was followed by taking samples of the reaction after 25, 30, 35, 40 and 45 cycles of reassembly ( FIG. 2 ).
  • the reassembly of 100-200 bp fragments can yield a single PCR product of the correct size
  • 10-50 base fragments typically yield some product of the correct size, as well as products of heterogeneous molecular weights. Most of this size heterogeneity appears to be due to single-stranded sequences at the ends of the products, since after restriction enzyme digestion a single band of the correct size is obtained.
  • the PCR product from step 4 above was digested with the terminal restriction enzymes BamHI and EcoO109 and gel purified as described above in step 2.
  • the reassembled fragments were ligated into pUC18 digested with BamHI and EcoO109.
  • E. coli were transformed with the ligation mixture under standard conditions as recommended by the manufacturer (Stratagene, San Diego Calif.) and plated on agar plates having 100 ⁇ g/ml ampicillin, 0.004% X-gal and 2 mM IPTG.
  • the resulting colonies having the HinDIII-NheI fragment which is diagnostic for the ++recombinant were identified because they appeared blue.
  • the DNA encoding the LacZ gene from the resulting LacZ colonies was sequenced with a sequencing kit (United States Biochemical Co., Cleveland Ohio) according to the manufacturer's instructions and the genes were found to have point mutations due to the reassembly process (Table 1). 11/12 types of substitutions were found, and no frameshifts.
  • DNA sequences can be reassembled from a random mixture of small fragments by a reaction that is surprisingly efficient and simple.
  • One application of this technique is the recombination or shuffling of related sequences based on homology.
  • Oligonucleotides that are mixed into the shuffling mixture can be incorporated into the final product based on the homology of the flanking sequences of the oligonucleotide to the template DNA ( FIG. 4 ).
  • the LacZ ⁇ stop codon mutant (pUC18 ⁇ +) described above was used as the DNAseI digested template.
  • a 66 mer oligonucleotide, including 18 bases of homology to the wild-type LacZ gene at both ends was added into the reaction at a 4-fold molar excess to correct stop codon mutations present in the original gene.
  • the shuffling reaction was conducted under conditions similar to those in step 2 above.
  • the resulting product was digested, ligated and inserted into E. coli as described above.
  • ssDNA appeared to be more efficient than dsDNA, presumably due to competitive hybridization.
  • the degree of incorporation can be varied over a wide range by adjusting the molar excess, annealing temperature, or the length of homology.
  • Plasmid pUC18 was digested with restriction enzymes EcoRI, EcoO109, XmnI and AlwNI, yielding fragments of approximately 370, 460, 770 and 1080 bp. These fragments were electrophoresed and separately purified from a 2% low melting point agarose gel (the 370 and 460 basepair bands could not be separated), yielding a large fragment, a medium fragment and a mixture of two small fragments in 3 separate tubes.
  • Each fragment was digested with DNAseI as described in Example 1, and fragments of 50-130 bp were purified from a 2% low melting point agarose gel for each of the original fragments.
  • PCR mix (as described in Example 1 above) was added to the purified digested fragments to a final concentration of 10 ng/ ⁇ l of fragments. No primers were added.
  • a reassembly reaction was performed for 75 cycles [94° C. for 30 seconds, 60° C. for 30 seconds] separately on each of the three digested DNA fragment mixtures, and the products were analyzed by agarose gel electrophoresis.
  • This example illustrates that crossovers based on homologies of less than 15 bases may be obtained.
  • a human and a murine IL-1 ⁇ gene were shuffled.
  • a murine IL1- ⁇ gene (BBG49) and a human IL1- ⁇ gene with E. coli codon usage (BBG2; R&D Systems, Inc., Minneapolis Minn.) were used as templates in the shuffling reaction.
  • the areas of complete homology between the human and the murine IL-1 ⁇ sequences are on average only 4.1 bases long ( FIG. 5 , regions of heterology are boxed).
  • the first 15 cycles of the shuffling reaction were performed with the Klenow fragment of DNA polymerase I, adding 1 unit of fresh enzyme at each cycle.
  • the DNA was added to the PCR mix of Example 1 which mix lacked the polymerase.
  • the manual program was 94° C. for 1 minute, and then 15 cycles of: [95° C. for 1 minute, 10 seconds on dry ice/ethanol (until frozen), incubate about 20 seconds at 25° C., add 1 U of Klenow fragment and incubate at 25° C. for 2 minutes].
  • the tube was rapidly cooled in dry ice/ethanol and reheated to the annealing temperature. Then the heat-labile polymerase was added.
  • the enzyme needs to be added at every cycle. Using this approach, a high level of crossovers was obtained, based on only a few bases of uninterrupted homology ( FIG. 5 , positions of cross-overs indicated by “
  • Taq polymerase was added and an additional 22 cycles of the shuffling reaction [94° C. for 30 seconds, 35° C. for 30 seconds] without primers were performed.
  • the reaction was then diluted 20-fold.
  • the following primers were added to a final concentration of 0.8 ⁇ M: 5′AACGCCGCATGCAAGCTTGGATCCTTATT3′ (SEQ ID NO:5) and 5′AAAGCCCTCTAGATGATTACGAATTCATAT3′ (SEQ ID NO:6) and a PCR reaction was performed as described above in Example 1.
  • the second primer pair differed from the first pair only because a change in restriction sites was deemed necessary.
  • crossovers were found by DNA sequencing of nine colonies. Some of the crossovers were based on only 1-2 bases of uninterrupted homology.
  • TEM-1 betalactamase is a very efficient enzyme, limited in its reaction rate primarily by diffusion. This example determines whether it is possible to change its reaction specificity and obtain resistance to the drug cefotaxime that it normally does not hydrolyze.
  • the minimum inhibitory concentration (MIC) of cefotaxime on bacterial cells lacking a plasmid was determined by plating 10 ⁇ l of a 10 ⁇ 2 dilution of an overnight bacterial culture (about 1000 cfu) of E. coli XL1-blue cells (Stratagene, San Diego Calif.) on plates with varying levels of cefotaxime (Sigma, St. Louis Mo.), followed by incubation for 24 hours at 37° C.
  • cefotaxime is sensitive to the density of cells, and therefore similar numbers of cells needed to be plated on each plate (obtained by plating on plain LB plates). Platings of 1000 cells were consistently performed.
  • a pUC18 derivative carrying the bacterial TEM-1 betalactamase gene was used (28).
  • the TEM-1 betalactamase gene confers resistance to bacteria against approximately 0.02 ⁇ g/ml of cefotaxime.
  • Sfi1 restriction sites were added 5′ of the promoter and 3′ of the end of the gene by PCR of the vector sequence with two primers:
  • Primer A (SEQ ID NO: 7): 5′TTCTATTGAC GGCC TGTCA GGCC TCATATATACTTTAGATTGATTT3′ and Primer B (SEQ ID NO: 8): 5′TTGACGCACT GGCC ATGGT GGCC AAAAATAAACAAATAGGGGTTCCGC GCACATTT3′ and by PCR of the betalactamase gene sequence with two other primers:
  • Primer C (SEQ ID NO: 9): 5′AACTGACCAC GGCC TGACAGGCCGGTCTGACAGTTACCAATGCTT, and Primer D (SEQ ID NO: 10): 5′AACCTGTCCT GGCC ACCATGGCCTAAATACATTCAAATATGTAT.
  • the two reaction products were digested with SfiI, mixed, ligated and used to transform bacteria.
  • the resulting plasmid was pUC182Sfi.
  • This plasmid contains an Sfi1 fragment carrying the TEM-1 gene and the P-3 promoter.
  • the minimum inhibitory concentration of cefotaxime for E. coli XL1-blue (Stratagene, San Diego Calif.) carrying this plasmid was 0.02 ⁇ g/ml after 24 hours at 37° C.
  • the ability to improve the resistance of the betalactamase gene to cefotaxime without shuffling was determined by stepwise replating of a diluted pool of cells (approximately 10 7 cfu) on 2-fold increasing drug levels. Resistance up to 1.28 ⁇ g/ml could be obtained without shuffling. This represented a 64 fold increase in resistance.
  • the substrate for the first shuffling reaction was dsDNA of 0.9 kb obtained by PCR of pUC182Sfi with primers C and D, both of which contain a SfiI site.
  • the free primers from the PCR product were removed by Wizard PCR prep (Promega, Madison Wis.) at every cycle.
  • DNA substrate(s) was digested with 0.15 units of DNAseI (Sigma, St. Louis Mo.) in 100 ⁇ l of 50 mM Tris-HCl pH 7.4, 1 mM MgCl 2 , for 10 min at room temperature. Fragments of 100-300 bp were purified from 2% low melting point agarose gels by electrophoresis onto DE81 ion exchange paper (Whatman, Hillsborough Oreg.), elution with 1 M NaCl and ethanol precipitation by the method described in Example 1.
  • the purified fragments were resuspended in PCR mix (0.2 mM each dNTP, 2.2 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100), at a concentration of 10-30 ng/ ⁇ l. No primers were added at this point.
  • a reassembly program of 94° C. for 60 seconds, then 40 cycles of [94° C. for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds] and then 72° C. for 5 minutes was used in an MJ Research (Watertown Mass.) PTC-150 thermocycler.
  • the 900 bp product was ligated into the vector pUC182Sfi at the unique SfiI site with T4 DNA ligase (BRL, Gaithersburg Md.).
  • T4 DNA ligase (BRL, Gaithersburg Md.).
  • the mixture was electroporated into E. coli XL1-blue cells and plated on LB plates with 0.32-0.64 ⁇ g/ml of cefotaxime (Sigma, St. Louis Mo.). The cells were grown for up to 24 hours at 37° C. and the resulting colonies were scraped off the plate as a pool and used as the PCR template for the next round of shuffling.
  • the transformants obtained after each of three rounds of shuffling were plated on increasing levels of cefotaxime.
  • the colonies (>100, to maintain diversity) from the plate with the highest level of cefotaxime were pooled and used as the template for the PCR reaction for the next round.
  • a mixture of the cefotaxime r colonies obtained at 0.32-0.64 ⁇ g/ml in Step (5) above were used as the template for the next round of shuffling.
  • 10 ul of cells in LB broth were used as the template in a reassembly program of 10 minutes at 99° C., then 35 cycles of [94° C. for 30 seconds, 52° C. for 30 seconds, 72° C. for 30 seconds] and then 5 minutes at 72° C. as described above.
  • the reassembly products were digested and ligated into pUC182Sfi as described in step (5) above.
  • the mixture was electroporated into E. coli XL1-blue cells and plated on LB plates having 5-10 ⁇ g/ml of cefotaxime.
  • cefotaxime Growth on cefotaxime is dependent on the cell density, requiring that all the MICs be standardized (in our case to about 1,000 cells per plate). At higher cell densities, growth at up to 1280 ⁇ g/ml was obtained. The 5 largest colonies grown at 1,280 ⁇ g/ml were plated for single colonies twice, and the Sfi1 inserts were analyzed by restriction mapping of the colony PCR products.
  • the plasmid of selected clones was transferred back into wild-type E. coli XL1-blue cells (Stratagene, San Diego Calif.) to ensure that none of the measured drug resistance was due to chromosomal mutations.
  • G4205A is located between the ⁇ 35 and ⁇ 10 sites of the betalactamase P3 promoter (31).
  • the promoter up-mutant observed by Chen and Clowes (31) is located outside of the Sfi1 fragment used here, and thus could not have been detected.
  • Four mutations were silent (A3689G, G3713A, G3934A and T3959A), and four resulted in an amino acid change (C3448T resulting in Gly238Ser, A3615G resulting in Met182Thr, C3850T resulting in Glu104Lys, and G4107A resulting in Ala18Val).
  • the resulting transformants were plated on 160 ⁇ g/ml of cefotaxime, and a pool of colonies was replated on increasing levels of cefotaxime up to 1,280 ⁇ g/ml. The largest colony obtained at 1,280 ⁇ g/ml was replated for single colonies.
  • the DNA sequence of the SfiI insert of the backcrossed mutant was determined using a dideoxy DNA sequencing kit (United States Biochemical Co., Cleveland Ohio) according to the manufacturer's instructions (Table 3).
  • the mutant had 9 single base pair mutations. As expected, all four of the previously identified silent mutations were lost, reverting to the sequence of the wild-type gene.
  • the promoter mutation (G4205A) as well as three of the four amino acid mutations (Glu104Lys, Met182Thr, and Gly238Ser) remained in the backcrossed clone, suggesting that they are essential for high level cefotaxime resistance.
  • Both the backcrossed and the non-backcrossed mutants have a promoter mutation (which by itself or in combination results in a 2-3 fold increase in expression level) as well as three common amino acid changes (Glu104Lys, Met182Thr and Gly238Ser).
  • Glu104Lys and Gly238Ser are mutations that are present in several cefotaxime resistant or other TEM-1 derivatives (Table 4).
  • the expression level of the betalactamase gene in the wild-type plasmid, the non-backcrossed mutant and in the backcrossed mutant was compared by SDS-polyacrylamide gel electrophoresis (4-20%; Novex, San Diego Calif.) of periplasmic extracts prepared by osmotic shock according to the method of Witholt, B. (32).
  • TEM-1 betalactamase (Sigma, St. Louis Mo.) was used as a molecular weight standard, and E. coli XL1-blue cells lacking a plasmid were used as a negative control.
  • the mutant and the backcrossed mutant appeared to produce a 2-3 fold higher level of the betalactamase protein compared to the wild-type gene.
  • the promoter mutation appeared to result in a 2-3 times increase in betalactamase.
  • PCR fragments were gel purified away from the synthetic oligonucleotides. 10 ng of each fragment were combined and a reassembly reaction was performed at 94° C. for 1 minute and then 25 cycles; [94° C. for 30 sec, 50° C. for 30 seconds and 72° C. for 45 seconds]. PCR was performed on the reassembly product for 25 cycles in the presence of the SfiI-containing outside primers (primers C and D from Example 5). The DNA was digested with Sfi1 and inserted into the wild-type pUC182Sfi vector. The following mutant combinations were obtained (Table 4).
  • Genotype MIC of MIC TEM-1 Wild-type 0.02 Glu104Lys 0.08 10 Gly238Ser 016 10 TEM-15 Glu104Lys/Gly238Ser* 10 TEM-3 Glu104Lys/Gly238Ser/Gln39Lys 10 37, 15 2-32 ST-4 Glu104Lys/Gly238Ser/Met182 10 Thr* ST-1 Glu104Lys/Gly238Ser/Met182 320 Thr/Ala18Val/T3959A/G3713A/ G3934A/A3689G* ST-2 Glu104Lys/Gly238Ser/Met182Thr/ 640 Ala42Gly/Gly92Ser/Arg241His/ T3842C/A3767G* ST-3 Glu104Lys/Gly238Ser/Met182Thr/ 640 Ala42Gly/Gly92Ser/Arg241His* *All of these mutant
  • Glu104Lys alone was shown to result only in a doubling of the MIC to 0.08 ⁇ g/ml, and Gly238Ser (in several contexts with one additional amino acid change) resulted only in a MIC of 0.16 ⁇ g/ml (26).
  • the double mutant Glu104Lys/Gly238Ser has a MIC of 10 ⁇ g/ml. This mutant corresponds to TEM-15.
  • Glu104Lys and Gly238Ser mutations in combination with Gln39Lys (TEM-3) or Thr263Met (TEM-4) result in a high level of resistance (2-32 ⁇ g/ml for TEM-3 and 8-32 ⁇ g/ml for TEM-4 (34, 35).
  • a mutant containing the three amino acid changes that were conserved after the backcross also had a MIC of 10 ⁇ g/ml. This meant that the mutations that each of the new selected mutants had in addition to the three known mutations were responsible for a further 32 to 64-fold increase in the resistance of the gene to cefotaxime.
  • TEM-1-19 The naturally occurring, clinical TEM-1-derived enzymes (TEM-1-19) each contain a different combination of only 5-7 identical mutations (reviews). Since these mutations are in well separated locations in the gene, a mutant with high cefotaxime resistance cannot be obtained by cassette mutagenesis of a single area. This may explain why the maximum MIC that was obtained by the standard cassette mutagenesis approach is only 0.64 ⁇ g/ml (26). For example, both the Glu104Lys as well as the Gly238Ser mutations were found separately in this study to have MICs below 0.16 ⁇ g/ml. Use of DNA shuffling allowed combinatoriality and thus the Glu104Lys/Gly238Ser combination was found, with a MIC of 10 ⁇ g/ml.
  • An important limitation of this example is the use of a single gene as a starting point. It is contemplated that better combinations can be found if a large number of related, naturally occurring genes are shuffled. The diversity that is present in such a mixture is more meaningful than the random mutations that are generated by mutagenic shuffling. For example, it is contemplated that one could use a repertoire of related genes from a single species, such as the pre-existing diversity of the immune system, or related genes obtained from many different species.
  • the A10B scFv antibody a mouse anti-rabbit IgG
  • Pharmacia Pharmacia (Milwaukee Wis.).
  • the commercially available Pharmacia phage display system was used, which uses the pCANTAB5 phage display vector.
  • the original A10B antibody reproducibly had only a low avidity, since clones that only bound weakly to immobilized antigen (rabbit IgG), (as measured by phage ELISA (Pharmacia assay kit) or by phage titer) were obtained.
  • the concentration of rabbit IgG which yielded 50% inhibition of the A10B antibody binding in a competition assay was 13 picomolar.
  • the observed low avidity may also be due to instability of the A10B clone.
  • the A10B scFv DNA was sequenced (United States Biochemical Co., Cleveland Ohio) according to the manufacturer's instructions. The sequence was similar to existing antibodies, based on comparison to Kabat (33).
  • Phage DNA having the A10B wild-type antibody gene (10 ul) was incubated at 99° C. for 10 min, then at 72° C. for 2 min.
  • PCR mix 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 ⁇ M each dNTP, 1.9 mM MgCl
  • 0.6 ⁇ m of each primer and 0.5 ⁇ l Taq DNA Polymerase was added to the phage DNA.
  • a PCR program was run for 35 cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.].
  • the primers used were: 5′ ATGATTACGCCAAGCTTT 3′ (SEQ ID NO:26) and 5′ TTGTCGTCTTTCCAGACGTT 3′ (SEQ ID NO:27).
  • the 850 bp PCR product was then electrophoresed and purified from a 2% low melting point agarose gel.
  • 300 ng of the gel purified 850 bp band was digested with 0.18 units of DNAse I (Sigma, St. Louis Mo.) in 50 mM Tris-HCl pH 7.5, 10 mM MgCl for 20 minutes at room temperature.
  • the digested DNA was separated on a 2% low melting point agarose gel and bands between 50 and 200 bp were purified from the gel.
  • CDR H means a CDR in the heavy chain
  • CDR L means a CDR in the light chain of the antibody.
  • CDR Oligos with restriction sites CDR H1 (SEQ ID NO: 34) 5′ TTCTGGCTACATCTTCACAGAATTCATCTAGATTTGGGTGAGGCAGA CGCCTGAA3′ CDR H2 (SEQ ID NO: 35) 5′ ACAGGGACTTGAGTGGATTGGAATCACAGTCAAGCTTATCCTTTATC TCAGGTCTCGAGTTCCAAGTACTTAAAGGGCCACACTGAGTGTA 3′ CDR H3 (SEQ ID NO: 36) 5′ TGTCTATTTCTGTGCTAGATCTTGACTGCAGTCTTATACGAGGATCC ATTGGGGCCAAGGGACCAGGTCA 3′ CDR L1 (SEQ ID NO: 37) 5′ AGAGGGTCACCATGACCTGCGGACGTCTTTAAGCGATCGGGCTGATG GCCTGGTACCAACAGAAGCCTGGAT 3′ CDR L2 (SEQ ID NO: 38) 5′ TCCCCCAGACTCCTGATTTATTAAGGGAGATCTAAACAGCTGTTGGT
  • the CDR oligos were added to the purified A10B antibody DNA fragments of between 50 to 200 bp from step (2) above at a 10 fold molar excess.
  • the PCR mix 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton x-100, 1.9 mM MgCl, 200 ⁇ m each dNTP, 0.3 ⁇ l Taq DNA polymerase (Promega, Madison Wis.), 50 ⁇ l total volume
  • the shuffling program run for 1 min at 94° C., 1 min at 72° C., and then 35 cycles: 30 seconds at 94° C., 30 seconds at 55° C., 30 seconds at 72° C.
  • 1 ⁇ l of the shuffled mixture was added to 100 ⁇ l of a PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 ⁇ m each dNTP, 1.9 mM MgCl, 0.6 ⁇ M each of the two outside primers (SEQ ID NO:26 and 27, see below), 0.5 ⁇ l Taq DNA polymerase) and the PCR program was run for 30 cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.].
  • the resulting mixture of DNA fragments of 850 basepair size was phenol/chloroform extracted and ethanol precipitated.
  • the outside primers were:
  • Primer 1 SEQ ID NO: 27 5′ TTGTCGTCTTTCCAGACGTT 3′
  • Primer 2 SEQ ID NO: 26 5′ ATGATTACGCCAAGCTTT 3′
  • the 850 bp PCR product was digested with the restriction enzymes SfiI and NotI, purified from a low melting point agarose gel, and ligated into the pCANTAB5 expression vector obtained from Pharmacia, Milwaukee Wis.
  • the ligated vector was electroporated according to the method set forth by Invitrogen (San Diego Calif.) into TG1 cells (Pharmacia, Milwaukee Wis.) and plated for single colonies.
  • the DNA from the resulting colonies was added to 100 ⁇ l of a PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 ⁇ m each dNTP, 1.9 mM MgCl, 0.6 ⁇ M of Outside primer 1 (SEQ ID No. 27; see below) six inside primers (SEQ ID NOS:40-45; see below), and 0.5 ⁇ l Taq DNA polymerase) and a PCR program was run for 35 cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.]. The sizes of the PCR products were determined by agarose gel electrophoresis, and were used to determine which CDRs with restriction sites were inserted.
  • H 1 (SEQ ID NO: 40) 5′ AGAATTCATCTAGATTTG 3′
  • H 2 (SEQ ID NO: 41) 5′ GCTTATCCTTTATCTCAGGTC 3′
  • H 3 (SEQ ID NO: 42) 5′ ACTGCAGTCTTATACGAGGAT 3′
  • L 1 (SEQ ID NO: 43) 5′ GACGTCTTTAAGCGATCG 3′
  • L 2 (SEQ ID NO: 44) 5′ TAAGGGAGATCTAAACAG 3′
  • L 3 (SEQ ID NO: 45) 5′ TCTGCGCGCTTAAAGGAT 3′
  • the six synthetic CDRs were inserted at the expected locations in the wild-type A10B antibody DNA ( FIG. 7 ). These studies showed that, while each of the six CDRs in a specific clone has a small chance of being a CDR with a restriction site, most of the clones carried at least one CDR with a restriction site, and that any possible combination of CDRs with restriction sites was generated.
  • CDRs Mutant Complementarity Determining Regions
  • the CDRs were synthetically mutagenized at a ratio of 70 (existing base):10:10:10, and were flanked on the 5′ and 3′ sides by about 20 bases of flanking sequence, which provide the homology for the incorporation of the CDRs when mixed into a mixture of unmutagenized antibody gene fragments in a molar excess.
  • the resulting mutant sequences are given below.
  • CDR H1 (SEQ ID NO: 28) 5′ TTCTGGCTACATCTTCACAA CTTATGATATAGACT GGGTGAGGCAGA CGCCTGAA 3′ CDR H2 (SEQ ID NO: 29) 5′ ACAGGGACTTGAGTGGATTGGA TGGATTTTTCCTGGAGAGGGTGGTA CTGAATACAATGAGAAGTTCAAGGGC AGGGCCACACTGAGTGTA 3′ CDR H3 (SEQ ID NO: 30) 5′ TGTCTATTTCTGTGCTAGA GGGGACTACTATAGGCGCTACTTTGACT TG TGGGGCCAAGGGACCACGGTCA 3′ CDR L1 (SEQ ID NO: 31) 5′ AGAGGGTCACCATGACCTGCA GTGCCAGCTCAGGTATACGTTACATA TATT GGTACCAACAGAAGCCTGGAT 3′ CDR L2 (SEQ ID NO: 32) 5′ TCCCCCAGACTCCTGATTTAT GACACATCCAACGTGGCTCCTGGA GT CCCTTTTC
  • a 10 fold molar excess of the CDR mutant oligos were added to the purified A10B antibody DNA fragments between 50 to 200 bp in length from step (2) above.
  • the PCR mix 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton x-100, 1.9 mM MgCl, 200 ⁇ m each dNTP, 0.3 ⁇ l Taq DNA polymerase (Promega, Madison Wis.), 50 ⁇ l total volume
  • 1 ⁇ l of the shuffled mixture was added to 100 ⁇ l of a PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 ⁇ m each dNTP, 1.9 mM MgCl, 0.6 mM each of the two outside primers (SEQ ID NO:26 and 27, see below), 0.5 ⁇ l Taq DNA polymerase) and the PCR program was run for 30 cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.].
  • the resulting mixture of DNA fragments of 850 basepair size was phenol/chloroform extracted and ethanol precipitated.
  • the outside primers were:
  • the 850 bp PCR product was digested with the restriction enzymes SfiI and NotI, purified from a low melting point agarose gel, and ligated into the pCANTAB5 expression vector obtained from Pharmacia, Milwaukee Wis.
  • the ligated vector was electroporated according to the method set forth by Invitrogen (San Diego Calif.) into TG1 cells (Pharmacia, Milwaukee Wis.) and the phage library was grown up using helper phage following the guidelines recommended by the manufacturer.
  • the library that was generated in this fashion was screened for the presence of improved antibodies, using six cycles of selection.
  • 100 ⁇ l of the phage library (1 ⁇ 10 10 cfu) was blocked with 100 ⁇ l of 2% milk for 30 minutes at room temperature, and then added to each of the 15 wells and incubated for 1 hour at 37° C.
  • the best clone has an approximately 100-fold improved expression level compared with the wild-type A10B when tested by the Western assay.
  • the concentration of the rabbit IgG which yielded 50% inhibition in a competition assay with the best clone was 1 picomolar.
  • the best clone was reproducibly specific for rabbit antigen. The number of copies of the antibody displayed by the phage appears to be increased.
  • a plasmid was constructed with two partial, inactive copies of the same gene (beta-lactamase) to demonstrate that recombination between the common areas of these two direct repeats leads to full-length, active recombinant genes.
  • TEM-1 betalactamase gene (“Bla”) confers resistance to bacteria against approximately 0.02 ⁇ g/ml of cefotaxime. Sfi1 restriction sites were added 5′ of the promoter and 3′ of the end of the betalactamase gene by PCR of the vector sequence with two primers:
  • Primer A (SEQ ID NO: 46) 5′ TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT 3′ PRIMER B (SEQ ID NO: 47) 5′ TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCG CGCACATTT 3′ and by PCR of the beta-lactamase gene sequence with two other primers:
  • Primer C (SEQ ID NO: 48) 5′ AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT 3′
  • Primer D (SEQ ID NO: 49) 5′ AACCTGTCCTGCCCACCATGGCCTAAATACATTCAAATATGTAT 3′
  • the two reaction products were digested with Sfi1, mixed, ligated and used to transform competent E. coli bacteria by the procedure described below.
  • the resulting plasmid was pUC182Sfi-Bla-Sfi. This plasmid contains an Sfi1 fragment carrying the Bla gene and the P-3 promoter.
  • cefotaxime for E. coli XL1-blue (Stratagene, San Diego Calif.) carrying pUC182Sfi-Bla-Sfi was 0.02 ⁇ g/ml after 24 hours at 37° C.
  • the tetracycline gene of pBR322 was cloned into pUC18Sfi-Bla-Sfi using the homologous areas, resulting in pBR322TetSfi-Bla-Sfi.
  • the TEM-1 gene was then deleted by restriction digestion of the pBR322TetSfi-Bla-Sfi with SspI and FspI and blunt-end ligation, resulting in pUC322TetSfi-Sfi.
  • Overlapping regions of the TEM-1 gene were amplified using standard PCR techniques and the following primers:
  • Primer 2650 (SEQ ID NO: 50) 5′ TTCTTAGACGTCAGGTGGCACTT 3′ Primer 2493 (SEQ ID NO: 51) 5′ TTT TAA ATC AAT CTA AAG TAT 3′ Primer 2651 (SEQ ID NO: 52) 5′ TGCTCATCCACGAGTGTGGAGAAGTGGTCCTGCAACTTTAT 3′, and Primer 2652 (SEQ ID NO: 53) ACCACTTCTCCACACTCGTGGATGAGCACTTTTAAAGTT
  • the two resulting DNA fragments were digested with Sfi1 and BstX1 and ligated into the Sfi site of pBR322TetSfi-Sfi.
  • the resulting plasmid was called pBR322Sfi-BL-LA-Sfi.
  • a map of the plasmid as well as a schematic of intraplasmidic recombination and reconstitution of functional beta-lactamase is shown in FIG. 9 .
  • E. coli JC8679 is RecBC sbcA (Oliner et al., 1993, NAR 21:5192).
  • the cells were plated on solid agar plates containing tetracycline. Those colonies which grew, were then plated on solid agar plates containing 100 ⁇ g/ml ampicillin and the number of viable colonies counted.
  • beta-lactamase gene inserts in those transformants which exhibited ampicillin resistance were amplified by standard PCR techniques using Primer 2650 (SEQ ID NO: 50) 5′ TTCTTAGACGTCAGGTGGCACTT 3′ and Primer 2493 (SEQ ID NO: 51) 5′ TTTTAAATCAATCTAAAGTAT 3′ and the length of the insert measured.
  • the presence of a 1 kb insert indicates that the gene was successfully recombined, as shown in FIG. 9 and Table 5.
  • a plasmid with two full-length copies of different alleles of the beta-lactamase gene was constructed. Homologous recombination of the two genes resulted in a single recombinant full-length copy of that gene.
  • the two alleles of the beta-lactamase gene were constructed as follows. Two PCR reactions were conducted with pUC18Sfi-Bla-Sfi as the template. One reaction was conducted with the following primers.
  • primer 2650 (SEQ ID NO: 50) 5′ TTCTTAGACGTCAGGTGGCACTT 3′ primer 2649 (SEQ ID NO: 51) 5′ ATGGTAGTCCACGAGTGTGGTAGTGACAGGCCGGTCTGACAGTTA CCAATGCTT 3′
  • primer 2648 (SEQ ID NO: 54) 5′ TGTCACTACCACACTCGTGGACTACCATGGCCTAAATACATTCAAA TATGTAT 3′
  • Primer 2493 (SEQ ID NO: 51) 5′ TTT TAA ATC AAT CTA AAG TAT 3′
  • the plasmid was electroporated into E. coli cells.
  • the cells were plated on solid agar plates containing 15 ⁇ g/ml tetracycline. Those colonies which grew, were then plated on solid agar plates containing 100 ⁇ g/ml ampicillin and the number of viable colonies counted.
  • the Bla inserts in those transformants which exhibited ampicillin resistance were amplified by standard PCR techniques using the method and primers described in Example 8. The presence of a 1 kb insert indicated that the duplicate genes had recombined, as indicated in Table 6.
  • the minus recombination control consisted of a single copy of the betalactamase gene, whereas the plus recombination experiment consisted of inserting two copies of betalactamase as a direct repeat.
  • the tetracycline marker was used to equalize the number of colonies that were selected for cefotaxime resistance in each round, to compensate for ligation efficiencies.
  • pBR322TetSfi-Bla-Sfi was digested with EcrI and subject to PCR with a 1:1 mix (1 ml) of normal and Cadwell PCR mix (Cadwell and Joyce (1992) PCR Methods and Applications 2: 28-33) for error prone PCR.
  • the PCR program was 70° C. for 2 minutes initially and then 30 cycles of 94° C. for 30 seconds, 52° C. for 30 second and 72° C. for 3 minutes and 6 seconds per cycle, followed by 72° C. for 10 minutes.
  • the primers used in the PCR reaction to create the one Bla gene control plasmid were Primer 2650 (SEQ ID NO: 50) and Primer 2719 (SEQ ID NO: 55) 5′ TTAAGGGATTTTGGTCATGAGATT 3′. This resulted in a mixed population of amplified DNA fragments, designated collectively as Fragment #59. These fragments had a number of different mutations.
  • the primers used in two different PCR reactions to create the two Bla gene plasmid were Primer 2650 (SEQ ID NO: 50) and Primer 2649 (SEQ ID NO: 51) for the first gene and Primers 2648 (SEQ ID NO: 54) and Primer 2719 (SEQ ID NO: 55) for the second gene.
  • Fragment #89 amplified with primers 2648 and 2719
  • Fragment #90 amplified with primers 2650 and 2649.
  • a number of different mutations had been introduced the mixed population of each of the fragments.
  • the population of amplified DNA fragment #59 was digested with Sfi1, and then cloned into pBR322TetSfi-Sfi to create a mixed population of the plasmid pBR322Sfi-Bla-Sfi 1 .
  • the population of amplified DNA fragments #90 and #89 was digested with SfiI and BstXI at 50° C., and ligated into pBR322TetSfi-Sfi to create a mixed population of the plasmid pBR322TetSfi-2Bla-Sfi 1 ( FIG. 10 ).
  • the plasmids pBR322Sfi-Bla-Sfi 1 and pBR322Sfi-2Bla-Sfi 1 were electroporated into E. coli JC8679 and placed on agar plates having differing concentrations of cefotaxime to select for resistant strains and on tetracycline plates to titre.
  • Competent E. coli cells containing pUC18Sfi-Bla-Sfi were prepared as described. Plasmid pUC18Sfi-Bla-Sfi contains the standard TEM-1 beta-lactamase gene as described, supra.
  • the complete plasmid pUC18Sfi-cef-Sfi DNA was electroporated into E. coli cells having the plasmid pUC18Sfi-Bla-Sfi.
  • DNA fragment containing the cefotaxime gene from pUC18Sfi-cef-Sfi was amplified by PCR using the primers 2650 (SEQ ID NO: 50) and 2719 (SEQ ID NO: 55).
  • the resulting 1 kb PCR product was digested into DNA fragments of ⁇ 100 bp by DNase and these fragments were electroporated into the competent E. coli cells which already contained pUC18Sfi-Bla-Sfi.
  • competent E. coli cells having the plasmid pUC18Sfi-Kan-Sfi were also used. DNA fragments from the digestion of the PCR product of pUC18Sfi-cef-Sfi were electroporated into these cells. There is no homology between the kanamycin gene and the beta-lactamase gene and thus recombination should not occur.
  • This experiment was designed to determine which format of recombination generated the most recombinants per cycle.
  • the vector PUC18Sfi-Bla-Sfi was amplified with PCR primers to generate a large and small fragment.
  • the large fragment had the plasmid and ends having portions of the Bla gene, and the small fragment coded for the middle of the Bla gene.
  • a third fragment having the complete Bla gene was created using PCR by the method in Example 8.
  • the larger plasmid fragment and the fragment containing the complete Bla gene were electroporated into E. coli JC8679 cells at the same time by the method described above and the transformants plated on differing concentrations of cefotaxime.
  • the vector PUC18Sfi-Bla-Sfi was amplified to produce the large plasmid fragment isolated as in approach 1 above.
  • the two fragments each comprising a portion of the complete Bla gene, such that the two fragments together spanned the complete Bla gene were also obtained by PCR.
  • the large plasmid fragment and the two Bla gene fragments were all electroporated into competent E. coli JC8679 cells and the transformants plated on varying concentrations of cefotaxime.
  • both the vector and the plasmid were electroporated into E. coli JC8679 cells and the transformants were plated on varying concentrations of cefotaxime.
  • the complete Bla gene was electroporated into E. coli JC8679 cells already containing the vector pUCSfi-Sfi and the transformants were plated on varying concentrations of cefotaxime. As controls, the E. coli JC8679 cells were electroporated with either the complete Bla gene or the vector alone.
  • a kit comprising a variety of cassettes which can be shuffled, and optimized shufflants can be selected.
  • FIG. 12 shows schematically one embodiment, with each loci having a plurality of cassettes.
  • FIG. 13 shows example cassettes that are used at the respective loci.
  • Each cassette of a given locus are flanked by substantially identical sequences capable of overlapping the flanking sequence(s) of cassettes of an adjacent locus and preferably also capable of participating in homologous recombination or non-homologous recombination (e.g., lox/cre or flp/frt systems), so as to afford shuffling of cassettes within a locus but substantially not between loci.
  • substantially identical sequences capable of overlapping the flanking sequence(s) of cassettes of an adjacent locus and preferably also capable of participating in homologous recombination or non-homologous recombination (e.g., lox/cre or flp/frt systems), so as to afford shuffling of cassettes within a locus but substantially not between loci.
  • Cassettes are supplied in the kit as PCR fragments, which each cassette type or individual cassette species packaged in a separate tube.
  • Vector libraries are created by combining the contents of tubes to assemble whole plasmids or substantial portions thereof by hybridization of the overlapping flanking sequences of cassettes at each locus with cassettes at the adjacent loci.
  • the assembled vector is ligated to a predetermined gene of interest to form a vector library wherein each library member comprises the predetermined gene of interest and a combination of cassettes determined by the association of cassettes.
  • the vectors are transferred into a suitable host cell and the cells are cultured under conditions suitable for expression, and the desired phenotype is selected.

Abstract

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Description

    BACKGROUND OF THE INVENTION
  • 1. Field of the Invention
  • The present invention relates to a method for the production of polynucleotides conferring a desired phenotype and/or encoding a protein having an advantageous predetermined property which is selectable. In an aspect, the method is used for generating and selecting nucleic acid fragments encoding mutant proteins.
  • 2. Description of the Related Art
  • The complexity of an active sequence of a biological macromolecule, e.g. proteins, DNA etc., has been called its information content (“IC”; 5-9). The information content of a protein has been defined as the resistance of the active protein to amino acid sequence variation, calculated from the minimum number of invariable amino acids (bits) required to describe a family of related sequences with the same function (9, 10). Proteins that are sensitive to random mutagenesis have a high information content. In 1974, when this definition was coined, protein diversity existed only as taxonomic diversity.
  • Molecular biology developments such as molecular libraries have allowed the identification of a much larger number of variable bases, and even to select functional sequences from random libraries. Most residues can be varied, although typically not all at the same time, depending on compensating changes in the context. Thus a 100 amino acid protein can contain only 2,000 different mutations, but 20100 possible combinations of mutations.
  • Information density is the Information Content/unit length of a sequence. Active sites of enzymes tend to have a high information density. By contrast, flexible linkers in enzymes have a low information density (8).
  • Current methods in widespread use for creating mutant proteins in a library format are error-prone polymerase chain reaction (11, 12, 19) and cassette mutagenesis (8, 20, 21, 22, 40, 41, 42), in which the specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide. In both cases, a ‘mutant cloud’ (4) is generated around certain sites in the original sequence.
  • Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. Error prone PCR can be used to mutagenize a mixture of fragments of unknown sequence. However, computer simulations have suggested that point mutagenesis alone may often be too gradual to allow the block changes that are required for continued sequence evolution. The published error-prone PCR protocols do not allow amplification of DNA fragments greater than 0.5 to 1.0 kb, limiting their practical application. Further, repeated cycles of error-prone PCR lead to an accumulation of neutral mutations, which, for example, may make a protein immunogenic.
  • In oligonucleotide-directed mutagenesis, a short sequence is replaced with a synthetically mutagenized oligonucleotide. This approach does not generate combinations of distant mutations and is thus not combinatorial. The limited library size relative to the vast sequence length means that many rounds of selection are unavoidable for protein optimization. Mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round followed by grouping into families, arbitrarily choosing a single family, and reducing it to a consensus motif, which is resynthesized and reinserted into a single gene followed by additional selection. This process constitutes a statistical bottleneck, it is labor intensive and not practical for many rounds of mutagenesis.
  • Error-prone PCR and oligonucleotide-directed mutagenesis are thus useful for single cycles of sequence fine tuning but rapidly become limiting when applied for multiple cycles.
  • Error-prone PCR can be used to mutagenize a mixture of fragments of unknown sequence (11, 12). However, the published error-prone PCR protocols (11, 12) suffer from a low processivity of the polymerase. Therefore, the protocol is unable to result in the random mutagenesis of an average-sized gene. This inability limits the practical application of error-prone PCR.
  • Another serious limitation of error-prone PCR is that the rate of down-mutations grows with the information content of the sequence. At a certain information content, library size, and mutagenesis rate, the balance of down-mutations to up-mutations will statistically prevent the selection of further improvements (statistical ceiling).
  • Finally, repeated cycles of error-prone PCR will also lead to the accumulation of neutral mutations, which can affect, for example, immunogenicity but not binding affinity.
  • Thus error-prone PCR was found to be too gradual to allow the block changes that are required for continued sequence evolution (1, 2).
  • In cassette mutagenesis, a sequence block of a single template is typically replaced by a (partially) randomized sequence. Therefore, the maximum information content that can be obtained is statistically limited by the number of random sequences (i.e., library size). This constitutes a statistical bottleneck, eliminating other sequence families which are not currently best, but which may have greater long term potential.
  • Further, mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round (20). Therefore, this approach is tedious and is not practical for many rounds of mutagenesis.
  • Error-prone PCR and cassette mutagenesis are thus best suited and have been widely used for fine-tuning areas of comparatively low information content. One apparent exception is the selection of an RNA ligase ribozyme from a random library using many rounds of amplification by error-prone PCR and selection (13).
  • It is becoming increasingly clear that the tools for the design of recombinant linear biological sequences such as protein, RNA and DNA are not as powerful as the tools nature has developed. Finding better and better mutants depends on searching more and more sequences within larger and larger libraries, and increasing numbers of cycles of mutagenic amplification and selection are necessary. However as discussed above, the existing mutagenesis methods that are in widespread use have distinct limitations when used for repeated cycles.
  • Evolution of most organisms occurs by natural selection and sexual reproduction. Sexual reproduction ensures mixing and combining of the genes of the offspring of the selected individuals. During meiosis, homologous chromosomes from the parents line up with one another and cross-over part way along their length, thus swapping genetic material. Such swapping or shuffling of the DNA allows organisms to evolve more rapidly (1, 2). In sexual recombination, because the inserted sequences were of proven utility in a homologous environment, the inserted sequences are likely to still have substantial information content once they are inserted into the new sequence.
  • Marton et al., (27) describes the use of PCR in vitro to monitor recombination in a plasmid having directly repeated sequences. Marton et al. discloses that recombination will occur during PCR as a result of breaking or nicking of the DNA. This will give rise to recombinant molecules. Meyerhans et al. (23) also disclose the existence of DNA recombination during in vitro PCR.
  • The term Applied Molecular Evolution (“AME”) means the application of an evolutionary design algorithm to a specific, useful goal. While many different library formats for AME have been reported for polynucleotides (3, 11-14), peptides and proteins (phage (15-17), lacI (18) and polysomes, in none of these formats has recombination by random cross-overs been used to deliberately create a combinatorial library.
  • Theoretically there are 2,000 different single mutants of a 100 amino acid protein. A protein of 100 amino acids has 20100 possible combinations of mutations, a number which is too large to exhaustively explore by conventional methods. It would be advantageous to develop a system which would allow the generation and screening of all of these possible combination mutations.
  • Winter and coworkers (43, 44) have utilized an in vivo site specific recombination system to combine light chain antibody genes with heavy chain antibody genes for expression in a phage system. However, their system relies on specific sites of recombination and thus is limited. Hayashi et al. (48) report simultaneous mutagenesis of antibody CDR regions in single chain antibodies (scFv) by overlap extension and PCR.
  • Caren et al. (45) describe a method for generating a large population of multiple mutants using random in vivo recombination. However, their method requires the recombination of two different libraries of plasmids, each library having a different selectable marker. Thus the method is limited to a finite number of recombinations equal to the number of selectable markers existing, and produces a concomitant linear increase in the number of marker genes linked to the selected sequence(s).
  • Calogero et al. (46) and Galizzi et al. (47) report that in vivo recombination between two homologous but truncated insect-toxin genes on a plasmid can produce a hybrid gene. Radman et al. (49) report in vivo recombination of substantially mismatched DNA sequences in a host cell having defective mismatch repair enzymes, resulting in hybrid molecule formation.
  • It would be advantageous to develop a method for the production of mutant proteins which method allowed for the development of large libraries of mutant nucleic acid sequences which were easily searched. The invention described herein is directed to the use of repeated cycles of point mutagenesis, nucleic acid shuffling and selection which allow for the directed molecular evolution in vitro of highly complex linear sequences, such as proteins through random recombination.
  • Accordingly, it would be advantageous to develop a method which allows for the production of large libraries of mutant DNA, RNA or proteins and the selection of particular mutants for a desired goal. The invention described herein is directed to the use of repeated cycles of mutagenesis, in vivo recombination and selection which allow for the directed molecular evolution in vivo of highly complex linear sequences, such as DNA, RNA or proteins through recombination.
  • Further advantages of the present invention will become apparent from the following description of the invention with reference to the attached drawings.
  • SUMMARY OF THE INVENTION
  • The present invention is directed to a method for generating a selected polynucleotide sequence or population of selected polynucleotide sequences, typically in the form of amplified and/or cloned polynucleotides, whereby the selected polynucleotide sequence(s) possess a desired phenotypic characteristic (e.g., encode a polypeptide, promote transcription of linked polynucleotides, bind a protein, and the like) which can be selected for. One method of identifying polypeptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule (e.g., a receptor), involves the screening of a large library of polypeptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the polypeptide.
  • The present invention provides a method for generating libraries of displayed polypeptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening. The method comprises (1) obtaining a first plurality of selected library members comprising a displayed polypeptide or displayed antibody and an associated polynucleotide encoding said displayed polypeptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequence, optionally introducing mutations into said polynucleotides or copies, and (2) pooling and fragmenting, typically randomly, said associated polynucleotides or copies to form fragments thereof under conditions suitable for PCR amplification, performing PCR amplification and optionally mutagenesis, and thereby homologously recombining said fragments to form a shuffled pool of recombined polynucleotides, whereby a substantial fraction (e.g., greater than 10 percent) of the recombined polynucleotides of said shuffled pool are not present in the first plurality of selected library members, said shuffled pool composing a library of displayed polypeptides or displayed antibodies suitable for affinity interaction screening. Optionally, the method comprises the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind or otherwise interact (e.g., such as catalytic antibodies) with a predetermined macromolecule, such as for example a proteinaceous receptor, peptide, oligosaccharide, virion, or other predetermined compound or structure. The displayed polypeptides, antibodies, peptidomimetic antibodies, and variable region sequences that are identified from such libraries can be used for therapeutic, diagnostic, research, and related purposes (e.g., catalysts, solutes for increasing osmolarity of an aqueous solution, and the like), and/or can be subjected to one or more additional cycles of shuffling and/or affinity selection. The method can be modified such that the step of selecting is for a phenotypic characteristic other than binding affinity for a predetermined molecule (e.g., for catalytic activity, stability, oxidation resistance, drug resistance, or detectable phenotype conferred on a host cell).
  • In one embodiment, the first plurality of selected library members is fragmented and homologously recombined by PCR in vitro.
  • In one embodiment, the first plurality of selected library members is fragmented in vitro, the resultant fragments transferred into a host cell or organism and homologously recombined to form shuffled library members in vivo.
  • In one embodiment, the first plurality of selected library members is cloned or amplified on episomally replicable vectors, a multiplicity of said vectors is transferred into a cell and homologously recombined to form shuffled library members in vivo.
  • In one embodiment, the first plurality of selected library members is not fragmented, but is cloned or amplified on an episomally replicable vector as a direct repeat, which each repeat comprising a distinct species of selected library member sequence, said vector is transferred into a cell and homologously recombined by intra-vector recombination to form shuffled library members in vivo.
  • In an embodiment, combinations of in vitro and in vivo shuffling are provided to enhance combinatorial diversity.
  • The present invention provides a method for generating libraries of displayed antibodies suitable for affinity interaction screening. The method comprises (1) obtaining a first plurality of selected library members comprising a displayed antibody and an associated polynucleotide encoding said displayed antibody, and obtaining said associated polynucleotides or copies thereof, wherein said associated polynucleotides comprise a region of substantially identical variable region framework sequence, and (2) pooling and fragmenting said associated polynucleotides or copies to form fragments thereof under conditions suitable for PCR amplification and thereby homologously recombining said fragments to form a shuffled pool of recombined polynucleotides comprising novel combinations of CDRs, whereby a substantial fraction (e.g., greater than 10 percent) of the recombined polynucleotides of said shuffled pool comprise CDR combinations are not present in the first plurality of selected library members, said shuffled pool composing a library of displayed antibodies comprising CDR permutations and suitable for affinity interaction screening. Optionally, the shuffled pool is subjected to affinity screening to select shuffled library members which bind to a predetermined epitope (antigen) and thereby selecting a plurality of selected shuffled library members. Optionally, the plurality of selected shuffled library members can be shuffled and screened iteratively, from 1 to about 1000 cycles or as desired until library members having a desired binding affinity are obtained.
  • Accordingly, one aspect of the present invention provides a method for introducing one or more mutations into a template double-stranded polynucleotide, wherein the template double-stranded polynucleotide has been cleaved into random fragments of a desired size, by adding to the resultant population of double-stranded fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the template polynucleotide; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at regions of identity between the single-stranded fragments and formation of a mutagenized double-stranded polynucleotide; and repeating the above steps as desired.
  • In another aspect the present invention is directed to a method of producing recombinant proteins having biological activity by treating a sample comprising double-stranded template polynucleotides encoding a wild-type protein under conditions which provide for the cleavage of said template polynucleotides into random double-stranded fragments having a desired size; adding to the resultant population of random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise areas of identity and areas of heterology to the template polynucleotide; denaturing the resultant mixture of double-stranded fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at the areas of identity and formation of a mutagenized double-stranded polynucleotide; repeating the above steps as desired; and then expressing the recombinant protein from the mutagenized double-stranded polynucleotide.
  • A third aspect of the present invention is directed to a method for obtaining a chimeric polynucleotide by treating a sample comprising different double-stranded template polynucleotides wherein said different template polynucleotides contain areas of identity and areas of heterology under conditions which provide for the cleavage of said template polynucleotides into random double-stranded fragments of a desired size; denaturing the resultant random double-stranded fragments contained in the treated sample into single-stranded fragments; incubating the resultant single-stranded fragments with polymerase under conditions which provide for the annealing of the single-stranded fragments at the areas of identity and the formation of a chimeric double-stranded polynucleotide sequence comprising template polynucleotide sequences; and repeating the above steps as desired.
  • A fourth aspect of the present invention is directed to a method of replicating a template polynucleotide by combining in vitro single-stranded template polynucleotides with small random single-stranded fragments resulting from the cleavage and denaturation of the template polynucleotide, and incubating said mixture of nucleic acid fragments in the presence of a nucleic acid polymerase under conditions wherein a population of double-stranded template polynucleotides is formed.
  • The invention also provides the use of polynucleotide shuffling, in vitro and/or in vivo to shuffle polynucleotides encoding polypeptides and/or polynucleotides comprising transcriptional regulatory sequences.
  • The invention also provides the use of polynucleotide shuffling to shuffle a population of viral genes (e.g., capsid proteins, spike glycoproteins, polymerases, proteases, etc.) or viral genomes (e.g., paramyxoviridae, orthomyxoviridae, herpesviruses, retroviruses, reoviruses, rhinoviruses, etc.). In an embodiment, the invention provides a method for shuffling sequences encoding all or portions of immunogenic viral proteins to generate novel combinations of epitopes as well as novel epitopes created by recombination; such shuffled viral proteins may comprise epitopes or combinations of epitopes which are likely to arise in the natural environment as a consequence of viral evolution (e.g., such as recombination of influenza virus strains).
  • The invention also provides a method suitable for shuffling polynucleotide sequences for generating gene therapy vectors and replication-defective gene therapy constructs, such as may be used for human gene therapy, including but not limited to vaccination vectors for DNA-based vaccination, as well as anti-neoplastic gene therapy and other gene therapy formats.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a schematic diagram comparing mutagenic shuffling over error-prone PCR; (a) the initial library; (b) pool of selected sequences in first round of affinity selection; (d) in vitro recombination of the selected sequences (‘shuffling’); (f) pool of selected sequences in second round of affinity selection after shuffling; (c) error-prone PCR; (e) pool of selected sequences in second round of affinity selection after error-prone PCR.
  • FIG. 2 illustrates the reassembly of a 1.0 kb LacZ alpha gene fragment from 10-50 bp random fragments. (a) Photograph of a gel of PCR amplified DNA fragment having the LacZ alpha gene. (b) Photograph of a gel of DNA fragments after digestion with DNAseI. (c) Photograph of a gel of DNA fragments of 10-50 bp purified from the digested LacZ alpha gene DNA fragment; (d) Photograph of a gel of the 10-50 bp DNA fragments after the indicated number of cycles of DNA reassembly; (e) Photograph of a gel of the recombination mixture after amplification by PCR with primers.
  • FIG. 3 is a schematic illustration of the LacZ alpha gene stop codon mutants and their DNA sequences. The boxed regions are heterologous areas, serving as markers. The stop codons are located in smaller boxes or underlined. “+” indicates a wild-type gene and “−” indicates a mutated area in the gene.
  • FIG. 4 is a schematic illustration of the introduction or spiking of a synthetic oligonucleotide into the reassembly process of the LacZ alpha gene.
  • FIG. 5 illustrates the regions of homology between a murine IL1-B gene (M) and a human IL1-B gene (H) with E. coli codon usage. Regions of heterology are boxed. The “ | ” indicate crossovers obtained upon the shuffling of the two genes.
  • FIG. 6 is a schematic diagram of the antibody CDR shut fling model system using the scFv of anti-rabbit IgG antibody (A10B).
  • FIG. 7 illustrates the observed frequency of occurrence of certain combinations of CDRs in the shuffled DNA of the scFv of anti-rabbit IgG antibody (A10B).
  • FIG. 8 illustrates the improved avidity of the scFv anti-rabbit antibody after DNA shuffling and each cycle of selection.
  • FIG. 9 schematically portrays pBR322-Sfi-BL-LA-Sfi and in vivo intraplasmidic recombination via direct repeats, as well as the rate of generation of ampicillin-resistant colonies by intraplasmidic recombination reconstituting a functional beta-lactamase gene.
  • FIG. 10 schematically portrays pBR322-Sfi-2Bla-Sfi and in vivo intraplasmidic recombination via direct repeats, as well as the rate of generation of ampicillin-resistant colonies by intraplasmidic recombination reconstituting a functional beta-lactamase gene.
  • FIG. 11 illustrates the method for testing the efficiency of multiple rounds of homologous recombination after the introduction of polynucleotide fragments into cells for the generation of recombinant proteins.
  • FIG. 12 schematically portrays generation of a library of vectors by shuffling cassettes at the following loci: promoter, leader peptide, terminator, selectable drug resistance gene, and origin of replication. The multiple parallel lines at each locus represents the multiplicity of cassettes for that cassette.
  • FIG. 13 schematically shows some examples of cassettes suitable at various loci for constructing prokaryotic vector libraries by shuffling.
  • DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • The present invention relates to a method for nucleic acid molecule reassembly after random fragmentation and its application to mutagenesis of DNA sequences. Also described is a method for the production of nucleic acid fragments encoding mutant proteins having enhanced biological activity. In particular, the present invention also relates to a method of repeated cycles of mutagenesis, nucleic acid shuffling and selection which allow for the creation of mutant proteins having enhanced biological activity.
  • The present invention is directed to a method for generating a very large library of DNA, RNA or protein mutants. This method has particular advantages in the generation of related DNA fragments from which the desired nucleic acid fragment(s) may be selected. In particular the present invention also relates to a method of repeated cycles of mutagenesis, homologous recombination and selection which allow for the creation of mutant proteins having enhanced biological activity.
  • However, prior to discussing this invention in further detail, the following terms will first be defined.
  • DEFINITIONS
  • As used herein, the following terms have the following meanings:
  • The term “DNA reassembly” is used when recombination occurs between identical sequences.
  • By contrast, the term “DNA shuffling” is used herein to indicate recombination between substantially homologous but non-identical sequences, in some embodiments DNA shuffling may involve crossover via nonhomologous recombination, such as via cre/lox and/or flp/frt systems and the like.
  • The term “amplification” means that the number of copies of a nucleic acid fragment is increased.
  • The term “identical” or “identity” means that two nucleic acid sequences have the same sequence or a complementary sequence. Thus, “areas of identity” means that regions or areas of a nucleic acid fragment or polynucleotide are identical or complementary to another polynucleotide or nucleic acid fragment.
  • The term “corresponds to” is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA”.
  • The following terms are used to describe the sequence relationships between two or more polynucleotides: “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity”, and “substantial identity”. A “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, such as a polynucleotide sequence of FIG. 1 or FIG. 2( b), or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
  • A “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
  • The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • The term “homologous” or “homologous” means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later. Preferably the region of identity is greater than about 5 bp, more preferably the region of identity is greater than 10 bp.
  • The term “heterologous” means that one single-stranded nucleic acid sequence is unable to hybridize to another single-stranded nucleic acid sequence or its complement. Thus areas of heterology means that nucleic acid fragments or polynucleotides have areas or regions in the sequence which are unable to hybridize to another nucleic acid or polynucleotide. Such regions or areas are, for example, areas of mutations.
  • The term “cognate” as used herein refers to a gene sequence that is evolutionarily and functionally related between species. For example but not limitation, in the human genome, the human CD4 gene is the cognate gene to the mouse CD4 gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode a protein which functions in signaling T cell activation through MHC class II-restricted antigen recognition.
  • The term “wild-type” means that the nucleic acid fragment does not comprise any mutations. A “wild-type” protein means that the protein will be active at a level of activity found in nature and will comprise the amino acid sequence found in nature.
  • The term “related polynucleotides” means that regions or areas of the polynucleotides are identical and regions or areas of the polynucleotides are heterologous.
  • The term “chimeric polynucleotide” means that the polynucleotide comprises regions which are wild-type and regions which are mutated. It may also mean that the polynucleotide comprises wild-type regions from one polynucleotide and wild-type regions from another related polynucleotide.
  • The term “cleaving” means digesting the polynucleotide with enzymes or breaking the polynucleotide.
  • The term “population” as used herein means a collection of components such as polynucleotides, nucleic acid fragments or proteins. A “mixed population” means a collection of components which belong to the same family of nucleic acids or proteins (i.e. are related) but which differ in their sequence (i.e. are not identical) and hence in their biological activity.
  • The term “specific nucleic acid fragment” means a nucleic acid fragment having certain end points and having a certain nucleic acid sequence. Two nucleic acid fragments wherein one nucleic acid fragment has the identical sequence as a portion of the second nucleic acid fragment but different ends comprise two different specific nucleic acid fragments.
  • The term “mutations” means changes in the sequence of a wild-type nucleic acid sequence or changes in the sequence of a peptide. Such mutations may be point mutations such as transitions or transversions. The mutations may be deletions, insertions or duplications.
  • In the polypeptide notation used herein, the lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention. Similarly, unless specified otherwise, the lefthand end of single-stranded polynucleotide sequences is the 5′ end; the lefthand direction of double-stranded polynucleotide sequences is referred to as the 5′ direction. The direction of 5′ to 3′ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5′ to the 5′ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA and which are 3′ to the 3′ end of the coding RNA transcript are referred to as “downstream sequences”.
  • The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. Generally, the term naturally-occurring refers to an object as present in a non-pathological (undiseased) individual, such as would be typical for the species.
  • The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, an array of spatially localized compounds (e.g., a VLSIPS peptide array, polynucleotide array, and/or combinatorial small molecule array), a biological macromolecule, a bacteriophage peptide display library, a bacteriophage antibody (e.g., scFv) display library, a polysome peptide display library, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents are evaluated for potential activity as antineoplastics, anti-inflammatories, or apoptosis modulators by inclusion in screening assays described hereinbelow. Agents are evaluated for potential activity as specific protein interaction inhibitors (i.e., an agent which selectively inhibits a binding interaction between two predetermined polypeptides but which does not substantially interfere with cell viability) by inclusion in screening assays described hereinbelow.
  • As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual macromolecular species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species.
  • As used herein the term “physiological conditions” refers to temperature, pH, ionic strength, viscosity, and like biochemical parameters which are compatible with a viable organism, and/or which typically exist intracellularly in a viable cultured yeast cell or mammalian cell. For example, the intracellular conditions in a yeast cell grown under typical laboratory culture conditions are physiological conditions. Suitable in vitro reaction conditions for in vitro transcription cocktails are generally physiological conditions. In general, in vitro physiological conditions comprise 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45° C. and 0.001-10 mM divalent cation (e.g., Mg++, Ca++); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0 percent nonspecific protein (e.g., BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) can often be present, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v). Particular aqueous conditions may be selected by the practitioner according to conventional methods. For general guidance, the following buffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition of divalent cation(s) and/or metal chelators and/or nonionic detergents and/or membrane fractions and/or antifoam agents and/or scintillants.
  • Specific hybridization is defined herein as the formation of hybrids between a first polynucleotide and a second polynucleotide (e.g., a polynucleotide having a distinct but substantially identical sequence to the first polynucleotide), wherein the first polynucleotide preferentially hybridizes to the second polynucleotide under stringent hybridization conditions wherein substantially unrelated polynucleotide sequences do not form hybrids in the mixture.
  • As used herein, the term “single-chain antibody” refers to a polypeptide comprising a VH domain and a VL domain in polypeptide linkage, generally linked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser]x), and which may comprise additional amino acid sequences at the amino- and/or carboxy-termini. For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide. As an example, a scFv is a single-chain antibody. Single-chain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 contiguous amino acids substantially encoded by genes of the immunoglobulin superfamily (e.g., see The Immunoglobulin Gene Superfamily, A. F. Williams and A. N. Barclay, in Immunoglobulin Genes, T. Honjo, F. W. Alt, and T. H. Rabbitts, eds., (1989) Academic Press: San Diego, Calif., pp. 361-387, which is incorporated herein by reference), most frequently encoded by a rodent, non-human primate, avian, porcine, bovine, ovine, goat, or human heavy chain or light chain gene sequence. A functional single-chain antibody generally contains a sufficient portion of an immunoglobulin superfamily gene product so as to retain the property of binding to a specific target molecule, typically a receptor or antigen (epitope).
  • As used herein, the term “complementarity-determining region” and “CDR” refer to the art-recognized term as exemplified by the Kabat and Chothia CDR definitions also generally known as hypervariable regions or hypervariable loops (Chothia and Lesk (1987) J. Mol. Biol. 196: 901; Chothia et al. (1989) Nature 342: 877; E. A. Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) (1987); and Tramontano et al. (1990) J. Mol. Biol. 215: 175). Variable region domains typically comprise the amino-terminal approximately 105-115 amino acids of a naturally-occurring immunoglobulin chain (e.g., amino acids 1-110), although variable domains somewhat shorter or longer are also suitable for forming single-chain antibodies.
  • An immunoglobulin light or heavy chain variable region consists of a “framework” region interrupted by three hypervariable regions, also called CDR's. The extent of the framework region and CDR's have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., 4th Ed., U.S. Department of Health and Human Services, Bethesda, Md. (1987)). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. As used herein, a “human framework region” is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's. The CDR's are primarily responsible for binding to an epitope of an antigen.
  • As used herein, the term “variable segment” refers to a portion of a nascent peptide which comprises a random, pseudorandom, or defined kernal sequence. A variable segment can comprise both variant and invariant residue positions, and the degree of residue variation at a variant residue position may be limited; both options are selected at the discretion of the practitioner. Typically, variable segments are about 5 to 20 amino acid residues in length (e.g., 8 to 10), although variable segments may be longer and may comprise antibody portions or receptor proteins, such as an antibody fragment, a nucleic acid binding protein, a receptor protein, and the like.
  • As used herein, “random peptide sequence” refers to an amino acid sequence composed of two or more amino acid monomers and constructed by a stochastic or random process. A random peptide can include framework or scaffolding motifs, which may comprise invariant sequences.
  • As used herein “random peptide library” refers to a set of polynucleotide sequences that encodes a set of random peptides, and to the set of random peptides encoded by those polynucleotide sequences, as well as the fusion proteins containing those random peptides.
  • As used herein, the term “pseudorandom” refers to a set of sequences that have limited variability, so that for example the degree of residue variability at one position is different than the degree of residue variability at another position, but any pseudorandom position is allowed some degree of residue variation, however circumscribed.
  • As used herein, the term “defined sequence framework” refers to a set of defined sequences that are selected on a nonrandom basis, generally on the basis of experimental data or structural data; for example, a defined sequence framework may comprise a set of amino acid sequences that are predicted to form a β-sheet structure or may comprise a leucine zipper heptad repeat motif, a zinc-finger domain, among other variations. A “defined sequence kernal” is a set of sequences which encompass a limited scope of variability. Whereas (1) a completely random 10-mer sequence of the 20 conventional amino acids can be any of (20)10 sequences, and (2) a pseudorandom 10-mer sequence of the conventional amino acids can be any of (20)10 sequences but will exhibit a bias for certain residues at certain positions and/or overall, (3) a defined sequence kernal is a subset of sequences which is less that the maximum number of potential sequences if each residue position was allowed to be any of the allowable 20 conventional amino acids (and/or allowable unconventional amino/imino acids). A defined sequence kernal generally comprises variant and invariant residue positions and/or comprises variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), and the like, either segmentally or over the entire length of the individual selected library member sequence. Defined sequence kernals can refer to either amino acid sequences or polynucleotide sequences. For illustration and not limitation, the sequences (NNK)10 and (NNM)10, where N represents A, T, G, or C; K represents G or T; and M represents A or C, are defined sequence kernals.
  • As used herein “epitope” refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of an antibody. Typically, such binding interaction is manifested as an intermolecular contact with one or more amino acid residues of a CDR.
  • As used herein, “receptor” refers to a molecule that has an affinity for a given ligand. Receptors can be naturally occurring or synthetic molecules. Receptors can be employed in an unaltered state or as aggregates with other species. Receptors can be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells, or other materials), cell membrane receptors, complex carbohydrates and glycoproteins, enzymes, and hormone receptors.
  • As used herein “ligand” refers to a molecule, such as a random peptide or variable segment sequence, that is recognized by a particular receptor. As one of skill in the art will recognize, a molecule (or macromolecular complex) can be both a receptor and a ligand. In general, the binding partner having a smaller molecular weight is referred to as the ligand and the binding partner having a greater molecular weight is referred to as a receptor.
  • As used herein, “linker” or “spacer” refers to a molecule or group of molecules that connects two molecules, such as a DNA binding protein and a random peptide, and serves to place the two molecules in a preferred configuration, e.g., so that the random peptide can bind to a receptor with minimal steric hindrance from the DNA binding protein.
  • As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • Methodology
  • Nucleic acid shuffling is a method for in vitro or in vivo homologous recombination of pools of nucleic acid fragments or polynucleotides. Mixtures of related nucleic acid sequences or polynucleotides are randomly fragmented, and reassembled to yield a library or mixed population of recombinant nucleic acid molecules or polynucleotides.
  • In contrast to cassette mutagenesis, only shuffling and error-prone PCR allow one to mutate a pool of sequences blindly (without sequence information other than primers).
  • The advantage of the mutagenic shuffling of this invention over error-prone PCR alone for repeated selection can best be explained with an example from antibody engineering. In FIG. 1 is shown a schematic diagram of DNA shuffling as described herein. The initial library can consist of related sequences of diverse origin (i.e. antibodies from naive mRNA) or can be derived by any type of mutagenesis (including shuffling) of a single antibody gene. A collection of selected complementarity determining regions (“CDRs”) is obtained after the first round of affinity selection (FIG. 1). In the diagram the thick CDRs confer onto the antibody molecule increased affinity for the antigen. Shuffling allows the free combinatorial association of all of the CDR1s with all of the CDR2s with all of the CDR3s, etc. (FIG. 1).
  • This method differs from PCR, in that it is an inverse chain reaction. In PCR, the number of polymerase start sites and the number of molecules grows exponentially. However, the sequence of the polymerase start sites and the sequence of the molecules remains essentially the same. In contrast, in nucleic acid reassembly or shuffling of random fragments the number of start sites and the number (but not size) of the random fragments decreases over time. For fragments derived from whole plasmids the theoretical endpoint is a single, large concatemeric molecule.
  • Since cross-overs occur at regions of homology, recombination will primarily occur between members of the same sequence family. This discourages combinations of CDRs that are grossly incompatible (eg. directed against different epitopes of the same antigen). It is contemplated that multiple families of sequences can be shuffled in the same reaction. Further, shuffling conserves the relative order, such that, for example, CDR1 will not be found in the position of CDR2.
  • Rare shufflants will contain a large number of the best (eg. highest affinity) CDRs and these rare shufflants may be selected based on their superior affinity (FIG. 1).
  • CDRs from a pool of 100 different selected antibody sequences can be permutated in up to 1006 different ways. This large number of permutations cannot be represented in a single library of DNA sequences. Accordingly, it is contemplated that multiple cycles of DNA shuffling and selection may be required depending on the length of the sequence and the sequence diversity desired.
  • Error-prone PCR, in contrast, keeps all the selected CDRs in the same relative sequence (FIG. 1), generating a much smaller mutant cloud.
  • The template polynucleotide which may be used in the methods of this invention may be DNA or RNA. It may be of various lengths depending on the size of the gene or DNA fragment to be recombined or reassembled. Preferably the template polynucleotide is from 50 bp to 50 kb. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest can be used in the methods of this invention, and in fact have been successfully used.
  • The template polynucleotide may be obtained by amplification using the PCR reaction (U.S. Pat. Nos. 4,683,202 and 4,683,195) or other amplification or cloning methods. However, the removal of free primers from the PCR product before fragmentation provides a more efficient result. Failure to adequately remove the primers can lead to a low frequency of crossover clones.
  • The template polynucleotide often should be double-stranded. A double-stranded nucleic acid molecule is required to ensure that regions of the resulting single-stranded nucleic acid fragments are complementary to each other and thus can hybridize to form a double-stranded molecule.
  • It is contemplated that single-stranded or double-stranded nucleic acid fragments having regions of identity to the template polynucleotide and regions of heterology to the template polynucleotide may be added to the template polynucleotide at this step. It is also contemplated that two different but related polynucleotide templates can be mixed at this step.
  • The double-stranded polynucleotide template and any added double- or single-stranded fragments are randomly digested into fragments of from about 5 bp to 5 kb or more. Preferably the size of the random fragments is from about 10 bp to 1000 bp, more preferably the size of the DNA fragments is from about 20 bp to 500 bp.
  • Alternatively, it is also contemplated that double-stranded nucleic acid having multiple nicks may be used in the methods of this invention. A nick is a break in one strand of the double-stranded nucleic acid. The distance between such nicks is preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp.
  • The nucleic acid fragment may be digested by a number of different methods. The nucleic acid fragment may be digested with a nuclease, such as DNAseI or RNAse. The nucleic acid may be randomly sheared by the method of sonication or by passage through a tube having a small orifice.
  • It is also contemplated that the nucleic acid may also be partially digested with one or more restriction enzymes, such that certain points of cross-over may be retained statistically.
  • The concentration of any one specific nucleic acid fragment will not be greater than 1% by weight of the total nucleic acid, more preferably the concentration of any one specific nucleic acid sequence will not be greater than 0.1% by weight of the total nucleic acid.
  • The number of different specific nucleic acid fragments in the mixture will be at least about 100, preferably at least about 500, and more preferably at least about 1000.
  • At this step single-stranded or double-stranded nucleic acid fragments, either synthetic or natural, may be added to the random double-stranded nucleic acid fragments in order to increase the heterogeneity of the mixture of nucleic acid fragments.
  • It is also contemplated that populations of double-stranded randomly broken nucleic acid fragments may be mixed or combined at this step.
  • Where insertion of mutations into the template polynucleotide is desired, single-stranded or double-stranded nucleic acid fragments having a region of identity to the template polynucleotide and a region of heterology to the template polynucleotide may be added in a 20 fold excess by weight as compared to the total nucleic acid, more preferably the single-stranded nucleic acid fragments may be added in a 10 fold excess by weight as compared to the total nucleic acid.
  • Where a mixture of different but related template polynucleotides is desired, populations of nucleic acid fragments from each of the templates may be combined at a ratio of less than about 1:100, more preferably the ratio is less than about 1:40. For example, a backcross of the wild-type polynucleotide with a population of mutated polynucleotide may be desired to eliminate neutral mutations (e.g., mutations yielding an insubstantial alteration in the phenotypic property being selected for). In such an example, the ratio of randomly digested wild-type polynucleotide fragments which may be added to the randomly digested mutant polynucleotide fragments is approximately 1:1 to about 100:1, and more preferably from 1:1 to 40:1.
  • The mixed population of random nucleic acid fragments are denatured to form single-stranded nucleic acid fragments and then reannealed. Only those single-stranded nucleic acid fragments having regions of homology with other single-stranded nucleic acid fragments will reanneal.
  • The random nucleic acid fragments may be denatured by heating. One skilled in the art could determine the conditions necessary to completely denature the double stranded nucleic acid. Preferably the temperature is from 80° C. to 100° C., more preferably the temperature is from 90° C. to 96° C. Other methods which may be used to denature the nucleic acid fragments include pressure (36) and pH.
  • The nucleic acid fragments may be reannealed by cooling. Preferably the temperature is from 20° C. to 75° C., more preferably the temperature is from 40° C. to 65° C. If a high frequency of crossovers is needed based on an average of only 4 consecutive bases of homology, recombination can be forced by using a low annealing temperature, although the process becomes more difficult. The degree of renaturation which occurs will depend on the degree of homology between the population of single-stranded nucleic acid fragments.
  • Renaturation can be accelerated by the addition of polyethylene glycol (“PEG”) or salt. The salt concentration is preferably from 0 mM to 200 mM, more preferably the salt concentration is from 10 mM to 100 mM. The salt may be KCl or NaCl. The concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%.
  • The annealed nucleic acid fragments are next incubated in the presence of a nucleic acid polymerase and dNTP's (i.e. dATP, dCTP, dGTP and dTTP). The nucleic acid polymerase may be the Klenow fragment, the Taq polymerase or any other DNA polymerase known in the art.
  • The approach to be used for the assembly depends on the minimum degree of homology that should still yield crossovers. If the areas of identity are large, Taq polymerase can be used with an annealing temperature of between 45-65° C. If the areas of identity are small, Klenow polymerase can be used with an annealing temperature of between 20-30° C. One skilled in the art could vary the temperature of annealing to increase the number of cross-overs achieved.
  • The polymerase may be added to the random nucleic acid fragments prior to annealing, simultaneously with annealing or after annealing.
  • The cycle of denaturation, renaturation and incubation in the presence of polymerase is referred to herein as shuffling or reassembly of the nucleic acid. This cycle is repeated for a desired number of times. Preferably the cycle is repeated from 2 to 50 times, more preferably the sequence is repeated from 10 to 40 times.
  • The resulting nucleic acid is a larger double-stranded polynucleotide of from about 50 bp to about 100 kb, preferably the larger polynucleotide is from 500 bp to 50 kb.
  • This larger polynucleotide fragment may contain a number of copies of a nucleic acid fragment having the same size as the template polynucleotide in tandem. This concatemeric fragment is then digested into single copies of the template polynucleotide. The result will be a population of nucleic acid fragments of approximately the same size as the template polynucleotide. The population will be a mixed population where single or double-stranded nucleic acid fragments having an area of identity and an area of heterology have been added to the template polynucleotide prior to shuffling.
  • These fragment are then cloned into the appropriate vector and the ligation mixture used to transform bacteria.
  • It is contemplated that the single nucleic acid fragments may be obtained from the larger concatemeric nucleic acid fragment by amplification of the single nucleic acid fragments prior to cloning by a variety of methods including PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202) rather than by digestion of the concatemer.
  • The vector used for cloning is not critical provided that it will accept a DNA fragment of the desired size. If expression of the DNA fragment is desired, the cloning vehicle should further comprise transcription and translation signals next to the site of insertion of the DNA fragment to allow expression of the DNA fragment in the host cell. Preferred vectors include the pUC series and the pBR series of plasmids.
  • The resulting bacterial population will include a number of recombinant DNA fragments having random mutations. This mixed population may be tested to identify the desired recombinant nucleic acid fragment. The method of selection will depend on the DNA fragment desired.
  • For example, if a DNA fragment which encodes for a protein with increased binding efficiency to a ligand is desired, the proteins expressed by each of the DNA fragments in the population or library may be tested for their ability to bind to the ligand by methods known in the art (i.e. panning, affinity chromatography). If a DNA fragment which encodes for a protein with increased drug resistance is desired, the proteins expressed by each of the DNA fragments in the population or library may be tested for their ability to confer drug resistance to the host organism. One skilled in the art, given knowledge of the desired protein, could readily test the population to identify DNA fragments which confer the desired properties onto the protein.
  • It is contemplated that one skilled in the art could use a phage display system in which fragments of the protein are expressed as fusion proteins on the phage surface (Pharmacia, Milwaukee Wis.). The recombinant DNA molecules are cloned into the phage DNA at a site which results in the transcription of a fusion protein a portion of which is encoded by the recombinant DNA molecule. The phage containing the recombinant nucleic acid molecule undergoes replication and transcription in the cell. The leader sequence of the fusion protein directs the transport of the fusion protein to the tip of the phage particle. Thus the fusion protein which is partially encoded by the recombinant DNA molecule is displayed on the phage particle for detection and selection by the methods described above.
  • It is further contemplated that a number of cycles of nucleic acid shuffling may be conducted with nucleic acid fragments from a subpopulation of the first population, which subpopulation contains DNA encoding the desired recombinant protein. In this manner, proteins with even higher binding affinities or enzymatic activity could be achieved.
  • It is also contemplated that a number of cycles of nucleic acid shuffling may be conducted with a mixture of wild-type nucleic acid fragments and a subpopulation of nucleic acid from the first or subsequent rounds of nucleic acid shuffling in order to remove any silent mutations from the subpopulation.
  • Any source of nucleic acid, in purified form can be utilized as the starting nucleic acid. Thus the process may employ DNA or RNA including messenger RNA, which DNA or RNA may be single or double stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. The nucleic acid sequence may be of various lengths depending on the size of the nucleic acid sequence to be mutated. Preferably the specific nucleic acid sequence is from 50 to 50000 base pairs. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest may be used in the methods of this invention.
  • The nucleic acid may be obtained from any source, for example, from plasmids such a pBR322, from cloned DNA or RNA or from natural DNA or RNA from any source including bacteria, yeast, viruses and higher organisms such as plants or animals. DNA or RNA may be extracted from blood or tissue material. The template polynucleotide may be obtained by amplification using the polynucleotide chain reaction (PCR) (U.S. Pat. Nos. 4,683,202 and 4,683,195). Alternatively, the polynucleotide may be present in a vector present in a cell and sufficient nucleic acid may be obtained by culturing the cell and extracting the nucleic acid from the cell by methods known in the art.
  • Any specific nucleic acid sequence can be used to produce the population of mutants by the present process. It is only necessary that a small population of mutant sequences of the specific nucleic acid sequence exist or be created prior to the present process.
  • The initial small population of the specific nucleic acid sequences having mutations may be created by a number of different methods. Mutations may be created by error-prone PCR. Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. Alternatively, mutations can be introduced into the template polynucleotide by oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, a short sequence of the polynucleotide is removed from the polynucleotide using restriction enzyme digestion and is replaced with a synthetic polynucleotide in which various bases have been altered from the original sequence. The polynucleotide sequence can also be altered by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Other agents which are analogues of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, these agents are added to the PCR reaction in place of the nucleotide precursor thereby mutating the sequence. Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used. Random mutagenesis of the polynucleotide sequence can also be achieved by irradiation with X-rays or ultraviolet light. Generally, plasmid DNA or DNA fragments so mutagenized are introduced into E. coli and propagated as a pool or library of mutant plasmids.
  • Alternatively the small mixed population of specific nucleic acids may be found in nature in that they may consist of different alleles of the same gene or the same gene from different related species (i.e., cognate genes). Alternatively, they may be related DNA sequences found within one species, for example, the immunoglobulin genes.
  • Once the mixed population of the specific nucleic acid sequences is generated, the polynucleotides can be used directly or inserted into an appropriate cloning vector, using techniques well-known in the art.
  • The choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of this invention. The templates of this invention may be plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof (e.g., coat protein, spike glycoprotein, capsid protein). For example, cosmids and phagemids are preferred where the specific nucleic acid sequence to be mutated is larger because these vectors are able to stably propagate large nucleic acid fragments.
  • If the mixed population of the specific nucleic acid sequence is cloned into a vector it can be clonally amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector. This is referred to as clonal amplification because while the absolute number of nucleic acid sequences increases, the number of mutants does not increase.
  • Utility
  • The DNA shuffling method of this invention can be performed blindly on a pool of unknown sequences. By adding to the reassembly mixture oligonucleotides (with ends that are homologous to the sequences being reassembled) any sequence mixture can be incorporated at any specific position into another sequence mixture. Thus, it is contemplated that mixtures of synthetic oligonucleotides, PCR fragments or even whole genes can be mixed into another sequence library at defined positions. The insertion of one sequence (mixture) is independent from the insertion of a sequence in another part of the template. Thus, the degree of recombination, the homology required, and the diversity of the library can be independently and simultaneously varied along the length of the reassembled DNA.
  • This approach of mixing two genes may be useful for the humanization of antibodies from murine hybridomas. The approach of mixing two genes or inserting mutant sequences into genes may be useful for any therapeutically used protein, for example, interleukin I, antibodies, tPA, growth hormone, etc. The approach may also be useful in any nucleic acid for example, promoters or introns or 3′ untranslated region or 5′ untranslated regions of genes to increase expression or alter specificity of expression of proteins. The approach may also be used to mutate ribozymes or aptamers.
  • Shuffling requires the presence of homologous regions separating regions of diversity. Scaffold-like protein structures may be particularly suitable for shuffling. The conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding. Examples of such scaffolds are the immunoglobulin beta-barrel, and the four-helix bundle (24). This shuffling can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.
  • In Vitro Shuffling
  • The equivalents of some standard genetic matings may also be performed by shuffling in vitro. For example, a ‘molecular backcross’ can be performed by repeated mixing of the mutant's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest. As in traditional breeding, this approach can be used to combine phenotypes from different sources into a background of choice. It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (i.e. immunogenicity). Thus it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not, an advantage which cannot be achieved by error-prone mutagenesis or cassette mutagenesis methods.
  • Large, functional genes can be assembled correctly from a mixture of small random fragments. This reaction may be of use for the reassembly of genes from the highly fragmented DNA of fossils (25). In addition random nucleic acid fragments from fossils may be combined with nucleic acid fragments from similar genes from related species.
  • It is also contemplated that the method of this invention can be used for the in vitro amplification of a whole genome from a single cell as is needed for a variety of research and diagnostic applications. DNA amplification by PCR is in practice limited to a length of about 40 kb. Amplification of a whole genome such as that of E. coli (5,000 kb) by PCR would require about 250 primers yielding 125 forty kb fragments. This approach is not practical due to the unavailability of sufficient sequence data. On the other hand, random digestion of the genome with DNAseI, followed by gel purification of small fragments will provide a multitude of possible primers. Use of this mix of random small fragments as primers in a PCR reaction alone or with the whole genome as the template should result in an inverse chain reaction with the theoretical endpoint of a single concatemer containing many copies of the genome.
  • 100 fold amplification in the copy number and an average fragment size of greater than 50 kb may be obtained when only random fragments are used (see Example 2). It is thought that the larger concatemer is generated by overlap of many smaller fragments. The quality of specific PCR products obtained using synthetic primers will be indistinguishable from the product obtained from unamplified DNA. It is expected that this approach will be useful for the mapping of genomes.
  • The polynucleotide to be shuffled can be fragmented randomly or non-randomly, at the discretion of the practitioner.
  • In Vivo Shuffling
  • In an embodiment of in vivo shuffling, the mixed population of the specific nucleic acid sequence is introduced into bacterial or eukaryotic cells under conditions such that at least two different nucleic acid sequences are present in each host cell. The fragments can be introduced into the host cells by a variety of different methods. The host cells can be transformed with the fragments using methods known in the art, for example treatment with calcium chloride. If the fragments are inserted into a phage genome, the host cell can be transfected with the recombinant phage genome having the specific nucleic acid sequences. Alternatively, the nucleic acid sequences can be introduced into the host cell using electroporation, transfection, lipofection, biolistics, conjugation, and the like.
  • In general, in this embodiment, the specific nucleic acids sequences will be present in vectors which are capable of stably replicating the sequence in the host cell. In addition, it is contemplated that the vectors will encode a marker gene such that host cells having the vector can be selected. This ensures that the mutated specific nucleic acid sequence can be recovered after introduction into the host cell. However, it is contemplated that the entire mixed population of the specific nucleic acid sequences need not be present on a vector sequence. Rather only a sufficient number of sequences need be cloned into vectors to ensure that after introduction of the fragments into the host cells each host cell contains one vector having at least one specific nucleic acid sequence present therein. It is also contemplated that rather than having a subset of the population of the specific nucleic acids sequences cloned into vectors, this subset may be already stably integrated into the host cell.
  • It has been found that when two fragments which have regions of identity are inserted into the host cells homologous recombination occurs between the two fragments. Such recombination between the two mutated specific nucleic acid sequences will result in the production of double or triple mutants in some situations.
  • It has also been found that the frequency of recombination is increased if some of the mutated specific nucleic acid sequences are present on linear nucleic acid molecules. Therefore, in a preferred embodiment, some of the specific nucleic acid sequences are present on linear nucleic acid fragments.
  • After transformation, the host cell transformants are placed under selection to identify those host cell transformants which contain mutated specific nucleic acid sequences having the qualities desired. For example, if increased resistance to a particular drug is desired then the transformed host cells may be subjected to increased concentrations of the particular drug and those transformants producing mutated proteins able to confer increased drug resistance will be selected. If the enhanced ability of a particular protein to bind to a receptor is desired, then expression of the protein can be induced from the transformants and the resulting protein assayed in a ligand binding assay by methods known in the art to identify that subset of the mutated population which shows enhanced binding to the ligand. Alternatively, the protein can be expressed in another system to ensure proper processing.
  • Once a subset of the first recombined specific nucleic acid sequences (daughter sequences) having the desired characteristics are identified, they are then subject to a second round of recombination.
  • In the second cycle of recombination, the recombined specific nucleic acid sequences may be mixed with the original mutated specific nucleic acid sequences (parent sequences) and the cycle repeated as described above. In this way a set of second recombined specific nucleic acids sequences can be identified which have enhanced characteristics or encode for proteins having enhanced properties. This cycle can be repeated a number of times as desired.
  • It is also contemplated that in the second or subsequent recombination cycle, a backcross can be performed. A molecular backcross can be performed by mixing the desired specific nucleic acid sequences with a large number of the wild-type sequence, such that at least one wild-type nucleic acid sequence and a mutated nucleic acid sequence are present in the same host cell after transformation. Recombination with the wild-type specific nucleic acid sequence will eliminate those neutral mutations that may affect unselected characteristics such as immunogenicity but not the selected characteristics.
  • In another embodiment of this invention, it is contemplated that during the first round a subset of the specific nucleic acid sequences can be fragmented prior to introduction into the host cell. The size of the fragments must be large enough to contain some regions of identity with the other sequences so as to homologously recombine with the other sequences. The size of the fragments will range from 0.03 kb to 100 kb more preferably from 0.2 kb to 10 kb. It is also contemplated that in subsequent rounds, all of the specific nucleic acid sequences other than the sequences selected from the previous round may be cleaved into fragments prior to introduction into the host cells.
  • Fragmentation of the sequences can be accomplished by a variety of method known in the art. The sequences can be randomly fragmented or fragmented at specific sites in the nucleic acid sequence. Random fragments can be obtained by breaking the nucleic acid or exposing it to harsh physical treatment (e.g., shearing or irradiation) or harsh chemical agents (e.g., by free radicals; metal ions; acid treatment to depurinate and cleave). Random fragments can also be obtained, in the case of DNA by the use of DNase or like nuclease. The sequences can be cleaved at specific sites by the use of restriction enzymes. The fragmented sequences can be single-stranded or double-stranded. If the sequences were originally single-stranded they can be denatured with heat, chemicals or enzymes prior to insertion into the host cell. The reaction conditions suitable for separating the strands of nucleic acid are well known in the art.
  • The steps of this process can be repeated indefinitely, being limited only by the number of possible mutants which can be achieved. After a certain number of cycles, all possible mutants will have been achieved and further cycles are redundant.
  • In an embodiment the same mutated template nucleic acid is repeatedly recombined and the resulting recombinants selected for the desired characteristic.
  • Therefore, the initial pool or population of mutated template nucleic acid is cloned into a vector capable of replicating in a bacteria such as E. coli. The particular vector is not essential, so long as it is capable of autonomous replication in E. coli. In a preferred embodiment, the vector is designed to allow the expression and production of any protein encoded by the mutated specific nucleic acid linked to the vector. It is also preferred that the vector contain a gene encoding for a selectable marker.
  • The population of vectors containing the pool of mutated nucleic acid sequences is introduced into the E. coli host cells. The vector nucleic acid sequences may be introduced by transformation, transfection or infection in the case of phage. The concentration of vectors used to transform the bacteria is such that a number of vectors is introduced into each cell. Once present in the cell, the efficiency of homologous recombination is such that homologous recombination occurs between the various vectors. This results in the generation of mutants (daughters) having a combination of mutations which differ from the original parent mutated sequences.
  • The host cells are then clonally replicated and selected for the marker gene present on the vector. Only those cells having a plasmid will grow under the selection.
  • The host cells which contain a vector are then tested for the presence of favorable mutations. Such testing may consist of placing the cells under selective pressure, for example, if the gene to be selected is an improved drug resistance gene. If the vector allows expression of the protein encoded by the mutated nucleic acid sequence, then such selection may include allowing expression of the protein so encoded, isolation of the protein and testing of the protein to determine whether, for example, it binds with increased efficiency to the ligand of interest.
  • Once a particular daughter mutated nucleic acid sequence has been identified which confers the desired characteristics, the nucleic acid is isolated either already linked to the vector or separated from the vector. This nucleic acid is then mixed with the first or parent population of nucleic acids and the cycle is repeated.
  • It has been shown that by this method nucleic acid sequences having enhanced desired properties can be selected.
  • In an alternate embodiment, the first generation of mutants are retained in the cells and the parental mutated sequences are added again to the cells. Accordingly, the first cycle of Embodiment I is conducted as described above. However, after the daughter nucleic acid sequences are identified, the host cells containing these sequences are retained.
  • The parent mutated specific nucleic acid population, either as fragments or cloned into the same vector is introduced into the host cells already containing the daughter nucleic acids. Recombination is allowed to occur in the cells and the next generation of recombinants, or granddaughters are selected by the methods described above.
  • This cycle can be repeated a number of times until the nucleic acid or peptide having the desired characteristics is obtained. It is contemplated that in subsequent cycles, the population of mutated sequences which are added to the preferred mutants may come from the parental mutants or any subsequent generation.
  • In an alternative embodiment, the invention provides a method of conducting a “molecular” backcross of the obtained recombinant specific nucleic acid in order to eliminate any neutral mutations. Neutral mutations are those mutations which do not confer onto the nucleic acid or peptide the desired properties. Such mutations may however confer on the nucleic acid or peptide undesirable characteristics. Accordingly, it is desirable to eliminate such neutral mutations. The method of this invention provide a means of doing so.
  • In this embodiment, after the mutant nucleic acid, having the desired characteristics, is obtained by the methods of the embodiments, the nucleic acid, the vector having the nucleic acid or the host cell containing the vector and nucleic acid is isolated.
  • The nucleic acid or vector is then introduced into the host cell with a large excess of the wild-type nucleic acid. The nucleic acid of the mutant and the nucleic acid of the wild-type sequence are allowed to recombine. The resulting recombinants are placed under the same selection as the mutant nucleic acid. Only those recombinants which retained the desired characteristics will be selected. Any silent mutations which do not provide the desired characteristics will be lost through recombination with the wild-type DNA. This cycle can be repeated a number of times until all of the silent mutations are eliminated.
  • Thus the methods of this invention can be used in a molecular backcross to eliminate unnecessary or silent mutations.
  • Utility
  • The in vivo recombination method of this invention can be performed blindly on a pool of unknown mutants or alleles of a specific nucleic acid fragment or sequence. However, it is not necessary to know the actual DNA or RNA sequence of the specific nucleic acid fragment.
  • The approach of using recombination within a mixed population of genes can be useful for the generation of any useful proteins, for example, interleukin I, antibodies, tPA, growth hormone, etc. This approach may be used to generate proteins having altered specificity or activity. The approach may also be useful for the generation of mutant nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 3′ untranslated regions or 5′ untranslated regions of genes. Thus this approach may be used to generate genes having increased rates of expression. This approach may also be useful in the study of repetitive DNA sequences. Finally, this approach may be useful to mutate ribozymes or aptamers.
  • Scaffold-like regions separating regions of diversity in proteins may be particularly suitable for the methods of this invention. The conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding. Examples of such scaffolds are the immunoglobulin beta barrel, and the four-helix bundle. The methods of this invention can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.
  • The equivalents of some standard genetic matings may also be performed by the methods of this invention. For example, a “molecular” backcross can be performed by repeated mixing of the mutant's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest. As in traditional breeding, this approach can be used to combine phenotypes from different sources into a background of choice. It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (i.e. immunogenicity). Thus it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not.
  • Peptide Display Methods
  • The present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by peptide display methods, wherein an associated polynucleotide encodes a displayed peptide which is screened for a phenotype (e.g., for affinity for a predetermined receptor (ligand).
  • An increasingly important aspect of biopharmaceutical drug development and molecular biology is the identification of peptide structures, including the primary amino acid sequences, of peptides or peptidomimetics that interact with biological macromolecules. One method of identifying peptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule (e.g., a receptor), involves the screening of a large library or peptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the peptide.
  • In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods also have been reported. One type involves the display of a peptide sequence, antibody, or other protein on the surface of a bacteriophage particle or cell. Generally, in these methods each bacteriophage particle or cell serves as an individual library member displaying a single species of displayed peptide in addition to the natural bacteriophage or cell protein sequences. Each bacteriophage or cell contains the nucleotide sequence information encoding the particular displayed peptide sequence; thus, the displayed peptide sequence can be ascertained by nucleotide sequence determination of an isolated library member.
  • A well-known peptide display method involves the presentation of a peptide sequence on the surface of a filamentous bacteriophage, typically as a fusion with a bacteriophage coat protein. The bacteriophage library can be incubated with an immobilized, predetermined macromolecule or small molecule (e.g., a receptor) so that bacteriophage particles which present a peptide sequence that binds to the immobilized macromolecule can be differentially partitioned from those that do not present peptide sequences that bind to the predetermined macromolecule. The bacteriophage particles (i.e., library members) which are bound to the immobilized macromolecule are then recovered and replicated to amplify the selected bacteriophage subpopulation for a subsequent round of affinity enrichment and phage replication. After several rounds of affinity enrichment and phage replication, the bacteriophage library members that are thus selected are isolated and the nucleotide sequence encoding the displayed peptide sequence is determined, thereby identifying the sequence(s) of peptides that bind to the predetermined macromolecule (e.g., receptor). Such methods are further described in PCT patent publication Nos. 91/17271, 91/18980, and 91/19818 and 93/08278.
  • The latter PCT publication describes a recombinant DNA method for the display of peptide ligands that involves the production of a library of fusion proteins with each fusion protein composed of a first polypeptide portion, typically comprising a variable sequence, that is available for potential binding to a predetermined macromolecule, and a second polypeptide portion that binds to DNA, such as the DNA vector encoding the individual fusion protein. When transformed host cells are cultured under conditions that allow for expression of the fusion protein, the fusion protein binds to the DNA vector encoding it. Upon lysis of the host cell, the fusion protein/vector DNA complexes can be screened against a predetermined macromolecule in much the same way as bacteriophage particles are screened in the phage-based display system, with the replication and sequencing of the DNA vectors in the selected fusion protein/vector DNA complexes serving as the basis for identification of the selected library peptide sequence(s).
  • Other systems for generating libraries of peptides and like polymers have aspects of both the recombinant and in vitro chemical synthesis methods. In these hybrid methods, cell-free enzymatic machinery is employed to accomplish the in vitro synthesis of the library members (i.e., peptides or polynucleotides). In one type of method, RNA molecules with the ability to bind a predetermined protein or a predetermined dye molecule were selected by alternate rounds of selection and PCR amplification (Tuerk and Gold (1990) Science 249: 505; Ellington and Szostak (1990) Nature 346: 818). A similar technique was used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res. 18: 3203; Beaudry and Joyce (1992) Science 257; 635; PCT patent publication Nos. 92/05258 and 92/14843). In a similar fashion, the technique of in vitro translation has been used to synthesize proteins of interest and has been proposed as a method for generating large libraries of peptides. These methods which rely upon in vitro translation, generally comprising stabilized polysome complexes, are described further in PCT patent publication Nos. 88/08453, 90/05785, 90/07003, 91/02076, 91/05058, and 92/02536. Applicants have described methods in which library members comprise a fusion protein having a first polypeptide portion with DNA binding activity and a second polypeptide portion having the library member unique peptide sequence; such methods are suitable for use in cell-free in vitro selection formats, among others.
  • The displayed peptide sequences can be of varying lengths, typically from 3-5000 amino acids long or longer, frequently from 5-100 amino acids long, and often from about 8-15 amino acids long. A library can comprise library members having varying lengths of displayed peptide sequence, or may comprise library members having a fixed length of displayed peptide sequence. Portions or all of the displayed peptide sequence(s) can be random, pseudorandom, defined set kernal, fixed, or the like. The present display methods include methods for in vitro and in vivo display of single-chain antibodies, such as nascent scFv on polysomes or scFv displayed on phage, which enable large-scale screening of scFv libraries having broad diversity of variable region sequences and binding specificities.
  • The present invention also provides random, pseudorandom, and defined sequence framework peptide libraries and methods for generating and screening those libraries to identify useful compounds (e.g., peptides, including single-chain antibodies) that bind to receptor molecules or epitopes of interest or gene products that modify peptides or RNA in a desired fashion. The random, pseudorandom, and defined sequence framework peptides are produced from libraries of peptide library members that comprise displayed peptides or displayed single-chain antibodies attached to a polynucleotide template from which the displayed peptide was synthesized. The mode of attachment may vary according to the specific embodiment of the invention selected, and can include encapsidation in a phage particle or incorporation in a cell.
  • A method of affinity enrichment allows a very large library of peptides and single-chain antibodies to be screened and the polynucleotide sequence encoding the desired peptide(s) or single-chain antibodies to be selected. The polynucleotide can then be isolated and shuffled to recombine combinatorially the amino acid sequence of the selected peptide(s) (or predetermined portions thereof) or single-chain antibodies (or just VH, VL, or CDR portions thereof). Using these methods, one can identify a peptide or single-chain antibody as having a desired binding affinity for a molecule and can exploit the process of shuffling to converge rapidly to a desired high-affinity peptide or scFv. The peptide or antibody can then be synthesized in bulk by conventional means for any suitable use (e.g., as a therapeutic or diagnostic agent).
  • A significant advantage of the present invention is that no prior information regarding an expected ligand structure is required to isolate peptide ligands or antibodies of interest. The peptide identified can have biological activity, which is meant to include at least specific binding affinity for a selected receptor molecule and, in some instances, will further include the ability to block the binding of other compounds, to stimulate or inhibit metabolic pathways, to act as a signal or messenger, to stimulate or inhibit cellular activity, and the like.
  • The present invention also provides a method for shuffling a pool of polynucleotide sequences selected by affinity screening a library of polysomes displaying nascent peptides (including single-chain antibodies) for library members which bind to a predetermined receptor (e.g., a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as heterodimers and the like) or epitope (e.g., an immobilized protein, glycoprotein, oligosaccharide, and the like).
  • Polynucleotide sequences selected in a first selection round (typically by affinity selection for binding to a receptor (e.g., a ligand) by any of these methods are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences. The recombined selected polynucleotide sequences are subjected to at least one subsequent selection round. The polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection. Selected sequences can also be backcrossed with polynucleotide sequences encoding neutral sequences (i.e., having insubstantial functional effect on binding), such as for example by backcrossing with a wild-type or naturally-occurring sequence substantially identical to a selected sequence to produce native-like functional peptides, which may be less immunogenic. Generally, during backcrossing subsequent selection is applied to retain the property of binding to the predetermined receptor (ligand).
  • Prior to or concomitant with the shuffling of selected sequences, the sequences can be mutagenized. In one embodiment, selected library members are cloned in a prokaryotic vector (e.g., plasmid, phagemid, or bacteriophage) wherein a collection of individual colonies (or plaques) representing discrete library members are produced. Individual selected library members can then be manipulated (e.g., by site-directed mutagenesis, cassette mutagenesis, chemical mutagenesis, PCR mutagenesis, and the like) to generate a collection of library members representing a kernal of sequence diversity based on the sequence of the selected library member. The sequence of an individual selected library member or pool can be manipulated to incorporate random mutation, pseudorandom mutation, defined kernal mutation (i.e., comprising variant and invariant residue positions and/or comprising variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), codon-based mutation, and the like, either segmentally or over the entire length of the individual selected library member sequence. The mutagenized selected library members are then shuffled by in vitro and/or in vivo recombinatorial shuffling as disclosed herein.
  • The invention also provides peptide libraries comprising a plurality of individual library members of the invention, wherein (1) each individual library member of said plurality comprises a sequence produced by shuffling of a pool of selected sequences, and (2) each individual library member comprises a variable peptide segment sequence or single-chain antibody segment sequence which is distinct from the variable peptide segment sequences or single-chain antibody sequences of other individual library members in said plurality (although some library members may be present in more than one copy per library due to uneven amplification, stochastic probability, or the like).
  • The invention also provides a product-by-process, wherein selected polynucleotide sequences having (or encoding a peptide having) a predetermined binding specificity are formed by the process of: (1) screening a displayed peptide or displayed single-chain antibody library against a predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching library members which bind to the predetermined receptor or epitope to produce a pool of selected library members, (2) shuffling by recombination the selected library members (or amplified or cloned copies thereof) which binds the predetermined epitope and has been thereby isolated and/or enriched from the library to generate a shuffled library, and (3) screening the shuffled library against the predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching shuffled library members which bind to the predetermined receptor or epitope to produce a pool of selected shuffled library members.
  • Antibody Display and Screening Methods
  • The present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by antibody display methods, wherein an associated polynucleotide encodes a displayed antibody which is screened for a phenotype (e.g., for affinity for binding a predetermined antigen (ligand).
  • Various molecular genetic approaches have been devised to capture the vast immunological repertoire represented by the extremely large number of distinct variable regions which can be present in immunoglobulin chains. The naturally-occurring germline immunoglobulin heavy chain locus is composed of separate tandem arrays of variable (V) segment genes located upstream of a tandem array of diversity (D) segment genes, which are themselves located upstream of a tandem array of joining (J) region genes, which are located upstream of the constant (CH) region genes. During B lymphocyte development, V-D-J rearrangement occurs wherein a heavy chain variable region gene (VH) is formed by rearrangement to form a fused D-J segment followed by rearrangement with a V segment to form a V-D-J joined product gene which, if productively rearranged, encodes a functional variable region (VH) of a heavy chain. Similarly, light chain loci rearrange one of several V segments with one of several J segments to form a gene encoding the variable region (VL) of a light chain.
  • The vast repertoire of variable regions possible in immunoglobulins derives in part from the numerous combinatorial possibilities of joining V and J segments (and, in the case of heavy chain loci, D segments) during rearrangement in B cell development. Additional sequence diversity in the heavy chain variable regions arises from non-uniform rearrangements of the D segments during V-D-J joining and from N region addition. Further, antigen-selection of specific B cell clones selects for higher affinity variants having nongermline mutations in one or both of the heavy and light chain variable regions; a phenomenon referred to as “affinity maturation” or “affinity sharpening”. Typically, these “affinity sharpening” mutations cluster in specific areas of the variable region, most commonly in the complementarity-determining regions (CDRs).
  • In order to overcome many of the limitations in producing and identifying high-affinity immunoglobulins through antigen-stimulated B cell development (i.e., immunization), various prokaryotic expression systems have been developed that can be manipulated to produce combinatorial antibody libraries which may be screened for high-affinity antibodies to specific antigens. Recent advances in the expression of antibodies in Escherichia coli and bacteriophage systems (see, “Alternative Peptide Display Methods”, infra) have raised the possibility that virtually any specificity can be obtained by either cloning antibody genes from characterized hybridomas or by de novo selection using antibody gene libraries (e.g., from Ig cDNA).
  • Combinatorial libraries of antibodies have been generated in bacteriophage lambda expression systems which may be screened as bacteriophage plaques or as colonies of lysogens (Huse et al. (1989) Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 2432). Various embodiments of bacteriophage antibody display libraries and lambda phage expression libraries have been described (Kang et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363; Clackson et al. (1991) Nature 352: 624; McCafferty et al. (1990) Nature 348: 552; Burton et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 10134; Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133; Chang et al. (1991) J. Immunol. 147: 3610; Breitling et al. (1991) Gene 104: 147; Marks et al. (1991) J. Mol. Biol. 222: 581; Barbas et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89: 4457; Hawkins and Winter (1992) J. Immunol. 22: 867; Marks et al. (1992) Biotechnology 10: 779; Marks et al. (1992) J. Biol. Chem. 267: 16007; Lowman et al (1991) Biochemistry 30: 10832; Lerner et al. (1992) Science 258: 1313, incorporated herein by reference). Typically, a bacteriophage antibody display library is screened with a receptor (e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid) that is immobilized (e.g., by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography) and/or labeled (e.g., to screen plaque or colony lifts).
  • One particularly advantageous approach has been the use of so-called single-chain fragment variable (scFv) libraries (Marks et al. (1992) Biotechnology 10: 779; Winter G and Milstein C (1991) Nature 349: 293; Clackson et al. (1991) op.cit.; Marks et al. (1991) J. Mol. Biol. 222: 581; Chaudhary et al. (1990) Proc. Natl. Acad. Sci. (USA) 87: 1066; Chiswell et al. (1992) TIBTECH 10: 80; McCafferty et al. (1990) op.cit.; and Huston et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 5879). Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described.
  • Beginning in 1988, single-chain analogues of Fv fragments and their fusion proteins have been reliably generated by antibody engineering methods. The first step generally involves obtaining the genes encoding VH and VL domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis. The single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly-Gly-Gly-Gly-Ser)3 or equivalent linker peptide(s). The linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either VH-linker-VL or VL-linker-VH. In principle, the scFv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site.
  • Thus, scFv fragments are comprised of VH and VL domains linked into a single polypeptide chain by a flexible linker peptide. After the scFv genes are assembled, they are cloned into a phagemid and expressed at the tip of the M13 phage (or similar filamentous bacteriophage) as fusion proteins with the bacteriophage pIII (gene 3) coat protein. Enriching for phage expressing an antibody of interest is accomplished by panning the recombinant phage displaying a population scFv for binding to a predetermined epitope (e.g., target antigen, receptor).
  • The linked polynucleotide of a library member provides the basis for replication of the library member after a screening or selection procedure, and also provides the basis for the determination, by nucleotide sequencing, of the identity of the displayed peptide sequence or VH and VL amino acid sequence. The displayed peptide(s) or single-chain antibody (e.g., scFv) and/or its VH and VL domains or their CDRs can be cloned and expressed in a suitable expression system. Often polynucleotides encoding the isolated VH and VL domains will be ligated to polynucleotides encoding constant regions (CH and CL) to form polynucleotides encoding complete antibodies (e.g., chimeric or fully-human), antibody fragments, and the like. Often polynucleotides encoding the isolated CDRs will be grafted into polynucleotides encoding a suitable variable region framework (and optionally constant regions) to form polynucleotides encoding complete antibodies (e.g., humanized or fully-human), antibody fragments, and the like. Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography. Various other uses of such antibodies are to diagnose and/or stage disease (e.g., neoplasia), and for therapeutic application to treat disease, such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like.
  • Various methods have been reported for increasing the combinatorial diversity of a scFv library to broaden the repertoire of binding species (idiotype spectrum). The use of PCR has permitted the variable regions to be rapidly cloned either from a specific hybridoma source or as a gene library from non-immunized cells, affording combinatorial diversity in the assortment of VH and VL cassettes which can be combined. Furthermore, the VH and VL cassettes can themselves be diversified, such as by random, pseudorandom, or directed mutagenesis. Typically, VH and VL cassettes are diversified in or near the complementarity-determining regions (CDRs), often the third CDR, CDR3. Enzymatic inverse PCR mutagenesis has been shown to be a simple and reliable method for constructing relatively large libraries of scFv site-directed mutants (Stemmer et al. (1993) Biotechniques 14: 256), as has error-prone PCR and chemical mutagenesis (Deng et al. (1994) J. Biol. Chem. 269: 9533). Riechmann et al. (1993) Biochemistry 32: 8848 showed semirational design of an antibody scFv fragment using site-directed randomization by degenerate oligonucleotide PCR and subsequent phage display of the resultant scFv mutants. Barbas et al. (1992) op.cit. attempted to circumvent the problem of limited repertoire sizes resulting from using biased variable region sequences by randomizing the sequence in a synthetic CDR region of a human tetanus toxoid-binding Fab.
  • CDR randomization has the potential to create approximately 1×1020 CDRs for the heavy chain CDR3 alone, and a roughly similar number of variants of the heavy chain CDR1 and CDR2, and light chain CDR1-3 variants. Taken individually or together, the combinatorics of CDR randomization of heavy and/or light chains requires generating a prohibitive number of bacteriophage clones to produce a clone library representing all possible combinations, the vast majority of which will be non-binding. Generation of such large numbers of primary transformants is not feasible with current transformation technology and bacteriophage display systems. For example, Barbas et al. (1992) op.cit. only generated 5×107 transformants, which represents only a tiny fraction of the potential diversity of a library of thoroughly randomized CDRs.
  • Despite these substantial limitations, bacteriophage display of scFv have already yielded a variety of useful antibodies and antibody fusion proteins. A bispecific single chain antibody has been shown to mediate efficient tumor cell lysis (Gruber et al. (1994) J. Immunol. 152: 5368). Intracellular expression of an anti-Rev scFv has been shown to inhibit HIV-1 virus replication in vitro (Duan et al. (1994) Proc. Natl. Acad. Sci. (USA) 91: 5075), and intracellular expression of an anti-p21ras scFv has been shown to inhibit meiotic maturation of Xenopus oocytes (Biocca et al. (1993) Biochem. Biophys. Res. Commun. 197: 422. Recombinant scFv which can be used to diagnose HIV infection have also been reported, demonstrating the diagnostic utility of scFv (Lilley et al. (1994) J. Immunol. Meth. 171: 211). Fusion proteins wherein an scFv is linked to a second polypeptide, such as a toxin or fibrinolytic activator protein, have also been reported (Holvost et al. (1992) Eur. J. Biochem. 210: 945; Nicholls et al. (1993) J. Biol. Chem. 268: 5302).
  • If it were possible to generate scFv libraries having broader antibody diversity and overcoming many of the limitations of conventional CDR mutagenesis and randomization methods which can cover only a very tiny fraction of the potential sequence combinations, the number and quality of scFv antibodies suitable for therapeutic and diagnostic use could be vastly improved. To address this, the in vitro and in vivo shuffling methods of the invention are used to recombine CDRs which have been obtained (typically via PCR amplification or cloning) from nucleic acids obtained from selected displayed antibodies. Such displayed antibodies can be displayed on cells, on bacteriophage particles, on polysomes, or any suitable antibody display system wherein the antibody is associated with its encoding nucleic acid(s). In a variation, the CDRs are initially obtained from mRNA (or cDNA) from antibody-producing cells (e.g., plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO92/03918, WO93/12227, and WO94/25585), including hybridomas derived therefrom.
  • Polynucleotide sequences selected in a first selection round (typically by affinity selection for displayed antibody binding to an antigen (e.g., a ligand) by any of these methods are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination, especially shuffling of CDRs (typically shuffling heavy chain CDRs with other heavy chain CDRs and light chain CDRs with other light chain CDRs) to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences. The recombined selected polynucleotide sequences are expressed in a selection format as a displayed antibody and subjected to at least one subsequent selection round. The polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection until an antibody of the desired binding affinity is obtained. Selected sequences can also be backcrossed with polynucleotide sequences encoding neutral antibody framework sequences (i.e., having insubstantial functional effect on antigen binding), such as for example by backcrossing with a human variable region framework to produce human-like sequence antibodies. Generally, during backcrossing subsequent selection is applied to retain the property of binding to the predetermined antigen.
  • Alternatively, or in combination with the noted variations, the valency of the target epitope may be varied to control the average binding affinity of selected scFv library members. The target epitope can be bound to a surface or substrate at varying densities, such as by including a competitor epitope, by dilution, or by other method known to those in the art. A high density (valency) of predetermined epitope can be used to enrich for scFv library members which have relatively low affinity, whereas a low density (valency) can preferentially enrich for higher affinity scFv library members.
  • For generating diverse variable segments, a collection of synthetic oligonucleotides encoding random, pseudorandom, or a defined sequence kernal set of peptide sequences can be inserted by ligation into a predetermined site (e.g., a CDR). Similarly, the sequence diversity of one or more CDRs of the single-chain antibody cassette(s) can be expanded by mutating the CDR(s) with site-directed mutagenesis, CDR-replacement, and the like. The resultant DNA molecules can be propagated in a host for cloning and amplification prior to shuffling, or can be used directly (i.e., may avoid loss of diversity which may occur upon propagation in a host cell) and the selected library members subsequently shuffled.
  • Displayed peptide/polynucleotide complexes (library members) which encode a variable segment peptide sequence of interest or a single-chain antibody of interest are selected from the library by an affinity enrichment technique. This is accomplished by means of a immobilized macromolecule or epitope specific for the peptide sequence of interest, such as a receptor, other macromolecule, or other epitope species. Repeating the affinity selection procedure provides an enrichment of library members encoding the desired sequences, which may then be isolated for pooling and shuffling, for sequencing, and/or for further propagation and affinity enrichment.
  • The library members without the desired specificity are removed by washing. The degree and stringency of washing required will be determined for each peptide sequence or single-chain antibody of interest and the immobilized predetermined macromolecule or epitope. A certain degree of control can be exerted over the binding characteristics of the nascent peptide/DNA complexes recovered by adjusting the conditions of the binding incubation and the subsequent washing. The temperature, pH, ionic strength, divalent cations concentration, and the volume and duration of the washing will select for nascent peptide/DNA complexes within particular ranges of affinity for the immobilized macromolecule. Selection based on slow dissociation rate, which is usually predictive of high affinity, is often the most practical route. This may be done either by continued incubation in the presence of a saturating amount of free predetermined macromolecule, or by increasing the volume, number, and length of the washes. In each case, the rebinding of dissociated nascent peptide/DNA or peptide/RNA complex is prevented, and with increasing time, nascent peptide/DNA or peptide/RNA complexes of higher and higher affinity are recovered.
  • Additional modifications of the binding and washing procedures may be applied to find peptides with special characteristics. The affinities of some peptides are dependent on ionic strength or cation concentration. This is a useful characteristic for peptides that will be used in affinity purification of various proteins when gentle conditions for removing the protein from the peptides are required.
  • One variation involves the use of multiple binding targets (multiple epitope species, multiple receptor species), such that a scFv library can be simultaneously screened for a multiplicity of scFv which have different binding specificities. Given that the size of a scFv library often limits the diversity of potential scFv sequences, it is typically desirable to us scFv libraries of as large a size as possible. The time and economic considerations of generating a number of very large polysome scFv-display libraries can become prohibitive. To avoid this substantial problem, multiple predetermined epitope species (receptor species) can be concomitantly screened in a single library, or sequential screening against a number of epitope species can be used. In one variation, multiple target epitope species, each encoded on a separate bead (or subset of beads), can be mixed and incubated with a polysome-display scFv library under suitable binding conditions. The collection of beads, comprising multiple epitope species, can then be used to isolate, by affinity selection, scFv library members. Generally, subsequent affinity screening rounds can include the same mixture of beads, subsets thereof, or beads containing only one or two individual epitope species. This approach affords efficient screening, and is compatible with laboratory automation, batch processing, and high throughput screening methods.
  • A variety of techniques can be used in the present invention to diversify a peptide library or single-chain antibody library, or to diversify, prior to or concomitant with shuffling, around variable segment peptides or VH, VL, or CDRs found in early rounds of panning to have sufficient binding activity to the predetermined macromolecule or epitope. In one approach, the positive selected peptide/polynucleotide complexes (those identified in an early round of affinity enrichment) are sequenced to determine the identity of the active peptides. Oligonucleotides are then synthesized based on these active peptide sequences, employing a low level of all bases incorporated at each step to produce slight variations of the primary oligonucleotide sequences. This mixture of (slightly) degenerate oligonucleotides is then cloned into the variable segment sequences at the appropriate locations. This method produces systematic, controlled variations of the starting peptide sequences, which can then be shuffled. It requires, however, that individual positive nascent peptide/polynucleotide complexes be sequenced before mutagenesis, and thus is useful for expanding the diversity of small numbers of recovered complexes and selecting variants having higher binding affinity and/or higher binding specificity. In a variation, mutagenic PCR amplification of positive selected peptide/polynucleotide complexes (especially of the variable region sequences, the amplification products of which are shuffled in vitro and/or in vivo and one or more additional rounds of screening is done prior to sequencing. The same general approach can be employed with single-chain antibodies in order to expand the diversity and enhance the binding affinity/specificity, typically by diversifying CDRs or adjacent framework regions prior to or concomitant with shuffling. If desired, shuffling reactions can be spiked with mutagenic oligonucleotides capable of in vitro recombination with the selected library members can be included. Thus, mixtures of synthetic oligonucleotides and PCR fragments (synthesized by error-prone or high-fidelity methods) can be added to the in vitro shuffling mix and be incorporated into resulting shuffled library members (shufflants).
  • The present invention of shuffling enables the generation of a vast library of CDR-variant single-chain antibodies. One way to generate such antibodies is to insert synthetic CDRs into the single-chain antibody and/or CDR randomization prior to or concomitant with shuffling. The sequences of the synthetic CDR cassettes are selected by referring to known sequence data of human CDR and are selected in the discretion of the practitioner according to the following guidelines: synthetic CDRs will have at least 40 percent positional sequence identity to known CDR sequences, and preferably will have at least 50 to 70 percent positional sequence identity to known CDR sequences. For example, a collection of synthetic CDR sequences can be generated by synthesizing a collection of oligonucleotide sequences on the basis of naturally-occurring human CDR sequences listed in Kabat et al. (1991) op.cit.; the pool(s) of synthetic CDR sequences are calculated to encode CDR peptide sequences having at least 40 percent sequence identity to at least one known naturally-occurring human CDR sequence. Alternatively, a collection of naturally-occurring CDR sequences may be compared to generate consensus sequences so that amino acids used at a residue position frequently (i.e., in at least 5 percent of known CDR sequences) are incorporated into the synthetic CDRs at the corresponding position(s). Typically, several (e.g., 3 to about 50) known CDR sequences are compared and observed natural sequence variations between the known CDRs are tabulated, and a collection of oligonucleotides encoding CDR peptide sequences encompassing all or most permutations of the observed natural sequence variations is synthesized. For example but not for limitation, if a collection of human VH CDR sequences have carboxy-terminal amino acids which are either Tyr, Val, Phe, or Asp, then the pool(s) of synthetic CDR oligonucleotide sequences are designed to allow the carboxy-terminal CDR residue to be any of these amino acids. In some embodiments, residues other than those which naturally-occur at a residue position in the collection of CDR sequences are incorporated: conservative amino acid substitutions are frequently incorporated and up to 5 residue positions may be varied to incorporate non-conservative amino acid substitutions as compared to known naturally-occurring CDR sequences. Such CDR sequences can be used in primary library members (prior to first round screening) and/or can be used to spike in vitro shuffling reactions of selected library member sequences. Construction of such pools of defined and/or degenerate sequences will be readily accomplished by those of ordinary skill in the art.
  • The collection of synthetic CDR sequences comprises at least one member that is not known to be a naturally-occurring CDR sequence. It is within the discretion of the practitioner to include or not include a portion of random or pseudorandom sequence corresponding to N region addition in the heavy chain CDR; the N region sequence ranges from 1 nucleotide to about 4 nucleotides occurring at V-D and D-J junctions. A collection of synthetic heavy chain CDR sequences comprises at least about 100 unique CDR sequences, typically at least about 1,000 unique CDR sequences, preferably at least about 10,000 unique CDR sequences, frequently more than 50,000 unique CDR sequences; however, usually not more than about 1×106 unique CDR sequences are included in the collection, although occasionally 1×107 to 1×108 unique CDR sequences are present, especially if conservative amino acid substitutions are permitted at positions where the conservative amino acid substituent is not present or is rare (i.e., less than 0.1 percent) in that position in naturally-occurring human CDRs. In general, the number of unique CDR sequences included in a library should not exceed the expected number of primary transformants in the library by more than a factor of 10. Such single-chain antibodies generally bind to a predetermined antigen (e.g., the immunogen) with an affinity of about at least 1×107 M−1, preferably with an affinity of about at least 5×107 M−1, more preferably with an affinity of at least 1×108 M−1 to 1×109 M−1 or more, sometimes up to 1×1010M−1 or more. Frequently, the predetermined antigen is a human protein, such as for example a human cell surface antigen (e.g., CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other human biological macromolecule (e.g., thrombomodulin, protein C, carbohydrate antigen, silyl Lewis antigen, L-selectin), or nonhuman disease associated macromolecule (e.g., bacterial LPS, virion capsid protein or envelope glycoprotein) and the like.
  • High affinity single-chain antibodies of the desired specificity can be engineered and expressed in a variety of systems. For example, scFv have been produced in plants (Firek et al. (1993) Plant Mol. Biol. 23: 861) and can be readily made in prokaryotic systems (Owens R J and Young R J (1994) J. Immunol. Meth. 168: 149; Johnson S and Bird R E (1991) Methods Enzymol. 203: 88). Furthermore, the single-chain antibodies can be used as a basis for constructing whole antibodies or various fragments thereof (Kettleborough et al. (1994) Eur. J. Immunol. 24: 952). The variable region encoding sequence may be isolated (e.g., by PCR amplification or subcloning) and spliced to a sequence encoding a desired human constant region to encode a human sequence antibody more suitable for human therapeutic uses where immunogenicity is preferably minimized. The polynucleotide(s) having the resultant fully human encoding sequence(s) can be expressed in a host cell (e.g., from an expression vector in a mammalian cell) and purified for pharmaceutical formulation.
  • The DNA expression constructs will typically include an expression control DNA sequence operably linked to the coding sequences, including naturally-associated or heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the mutant “engineered” antibodies.
  • As stated previously, the DNA sequences will be expressed in hosts after the sequences have been operably linked to an expression control sequence (i.e., positioned to ensure the transcription and translation of the structural gene). These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein by reference).
  • In addition to eukaryotic microorganisms such as yeast, mammalian tissue cell culture may also be used to produce the polypeptides of the present invention (see, Winnacker, “From Genes to Clones,” VCH Publishers, N.Y., N.Y. (1987), which is incorporated herein by reference). Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, etc, but preferably transformed B-cells or hybridomas. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al. (1986) Immunol. Rev. 89: 49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, Adenovirus, Bovine Papilloma Virus, and the like.
  • Eukaryotic DNA transcription can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting sequences of between 10 to 300 bp that increase transcription by a promoter. Enhancers can effectively increase transcription when either 5′ or 3′ to the transcription unit. They are also effective if located within an intron or within the coding sequence itself. Typically, viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers. Enhancer sequences from mammalian systems are also commonly used, such as the mouse immunoglobulin heavy chain enhancer.
  • Mammalian expression vector systems will also typically include a selectable marker gene. Examples of suitable markers include, the dihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), or prokaryotic genes conferring drug resistance. The first two marker genes prefer the use of mutant cell lines that lack the ability to grow without the addition of thymidine to the growth medium. Transformed cells can then be identified by their ability to grow on non-supplemented media. Examples of prokaryotic drug resistance genes useful as markers include genes conferring resistance to G418, mycophenolic acid and hygromycin.
  • The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment. lipofection, or electroporation may be used for other cellular hosts. Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see, generally, Sambrook et al., supra).
  • Once expressed, the antibodies, individual mutated immunoglobulin chains, mutated antibody fragments, and other immunoglobulin polypeptides of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fraction column chromatography, gel electrophoresis and the like (see, generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982)). Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (see, generally, Immunological Methods, Vols. I and II, Eds. Lefrkovits and Pernis, Academic Press, New York, N.Y. (1979 and 1981)).
  • The antibodies generated by the method of the present invention can be used for diagnosis and therapy. By way of illustration and not limitation, they can be used to treat cancer, autoimmune diseases, or viral infections. For treatment of cancer, the antibodies will typically bind to an antigen expressed preferentially on cancer cells, such as erbB-2, CEA, CD33, and many other antigens and binding members well known to those skilled in the art.
  • Yeast Two-Hybrid Screening Assays
  • Shuffling can also be used to recombinatorially diversify a pool of selected library members obtained by screening a two-hybrid screening system to identify library members which bind a predetermined polypeptide sequence. The selected library members are pooled and shuffled by in vitro and/or in vivo recombination. The shuffled pool can then be screened in a yeast two hybrid system to select library members which bind said predetermined polypeptide sequence (e.g., and SH2 domain) or which bind an alternate predetermined polypeptide sequence (e.g., an SH2 domain from another protein species).
  • An approach to identifying polypeptide sequences which bind to a predetermined polypeptide sequence has been to use a so-called “two-hybrid” system wherein the predetermined polypeptide sequence is present in a fusion protein (Chien et al. (1991) Proc. Natl. Acad. Sci. (USA) 88: 9578). This approach identifies protein-protein interactions in vivo through reconstitution of a transcriptional activator (Fields S and Song O (1989) Nature 340: 245), the yeast Gal4 transcription protein. Typically, the method is based on the properties of the yeast Gal4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation. Polynucleotides encoding two hybrid proteins, one consisting of the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a known protein and the other consisting of the Gal4 activation domain fused to a polypeptide sequence of a second protein, are constructed and introduced into a yeast host cell. Intermolecular binding between the two fusion proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene (e.g., lacZ, HIS3) which is operably linked to a Gal4 binding site. Typically, the two-hybrid method is used to identify novel polypeptide sequences which interact with a known protein (Silver S C and Hunt S W (1993) Mol. Biol. Rep. 17: 155; Durfee et al. (1993) Genes Devel. 7; 555; Yang et al. (1992) Science 257: 680; Luban et al. (1993) Cell 73: 1067; Hardy et al. (1992) Genes Devel. 6; 801; Bartel et al. (1993) Biotechniques 14: 920; and Vojtek et al. (1993) Cell 74: 205). However, variations of the two-hybrid method have been used to identify mutations of a known protein that affect its binding to a second known protein (Li B and Fields S (1993) FASEB J. 7: 957; Lalo et al. (1993) Proc. Natl. Acad. Sci. (USA) 90: 5524; Jackson et al. (1993) Mol. Cell. Biol. 13; 2899; and Madura et al. (1993) J. Biol. Chem. 268: 12046). Two-hybrid systems have also been used to identify interacting structural domains of two known proteins (Bardwell et al. (1993) med. Microbiol. 8: 1177; Chakraborty et al. (1992) J. Biol. Chem. 267: 17498; Staudinger et al. (1993) J. Biol. Chem. 268: 4608; and Milne G T and Weaver D T (1993) Genes Devel. 7; 1755) or domains responsible for oligomerization of a single protein (Iwabuchi et al. (1993) Oncogene 8; 1693; Bogerd et al. (1993) J. Virol. 67: 5030). Variations of two-hybrid systems have been used to study the in vivo activity of a proteolytic enzyme (Dasmahapatra et al. (1992) Proc. Natl. Acad. Sci. (USA) 89: 4159). Alternatively, an E. coli/BCCP interactive screening system (Germino et al. (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90: 933; Guarente L (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90: 1639) can be used to identify interacting protein sequences (i.e., protein sequences which heterodimerize or form higher order heteromultimers). Sequences selected by a two-hybrid system can be pooled and shuffled and introduced into a two-hybrid system for one or more subsequent rounds of screening to identify polypeptide sequences which bind to the hybrid containing the predetermined binding sequence. The sequences thus identified can be compared to identify consensus sequence(s) and consensus sequence kernals.
  • As can be appreciated from the disclosure above, the present invention has a wide variety of applications. Accordingly, the following examples are offered by way of illustration, not by way of limitation.
  • In the examples below, the following abbreviations have the following meanings. If not defined below, then the abbreviations have their art recognized meanings.
  • ml = milliliter
    μl = microliters
    μM = micromolar
    nM = nanomolar
    PBS = phosphate buffered saline
    ng = nanograms
    μg = micrograms
    IPTG = isopropylthio-β-D-galactoside
    bp = basepairs
    kb = kilobasepairs
    dNTP = deoxynucleoside triphosphates
    PCR = polymerase chain reaction
    X-gal = 5-bromo-4-chloro-3-indolyl-β-D-galactoside
    DNAseI = deoxyribonuclease
    PBS = phosphate buffered saline
    CDR = complementarity determining regions
    MIC = minimum inhibitory concentration
    scFv = single-chain Fv fragment of an antibody
  • In general, standard techniques of recombination DNA technology are described in various publications, e.g. Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; Ausubel et al., 1987, Current Protocols in Molecular Biology, vols. 1 and 2 and supplements, and Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif., each of which is incorporated herein in their entirety by reference. Restriction enzymes and polynucleotide modifying enzymes were used according to the manufacturers recommendations. Oligonucleotides were synthesized on an Applied Biosystems Inc. Model 394 DNA synthesizer using ABI chemicals. If desired, PCR amplimers for amplifying a predetermined DNA sequence may be selected at the discretion of the practitioner.
  • EXAMPLES Example 1 LacZ Alpha Gene Reassembly 1) Substrate Preparation
  • The substrate for the reassembly reaction was the dsDNA polymerase chain reaction (“PCR”) product of the wild-type LacZ alpha gene from pUC18. (FIG. 2) (28; Gene Bank No. X02514) The primer sequences were 5′AAAGCGTCGATTTTTGTGAT3′ (SEQ ID NO:1) and 5′ATGGGGTTCCGCGCACATTT3′ (SEQ ID NO:2). The free primers were removed from the PCR product by Wizard PCR prep (Promega, Madison Wis.) according to the manufacturer's directions. The removal of the free primers was found to be important.
  • 2) DNAseI Digestion
  • About 5 μg of the DNA substrate was digested with 0.15 units of DNAseI (Sigma, St. Louis Mo.) in 100 μl of [50 mM Tris-HCl pH 7.4, 1 mM MgCl2], for 10-20 minutes at room temperature. The digested DNA was run on a 2% low melting point agarose gel. Fragments of 10-70 basepairs (bp) were purified from the 2% low melting point agarose gels by electrophoresis onto DESI ion exchange paper (Whatman, Hillsborough Oreg.). The DNA fragments were eluted from the paper with 1 M NaCl and ethanol precipitated.
  • 3) DNA Reassembly
  • The purified fragments were resuspended at a concentration of 10-30 ng/μl in PCR Mix (0.2 mM each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 0.3 μl Taq DNA polymerase, 50 μl total volume). No primers were added at this point. A reassembly program of 94° C. for 60 seconds, 30-45 cycles of [94° C. for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds] and 5 minutes at 72° C. was used in an MJ Research (Watertown Mass.) PTC-150 thermocycler. The PCR reassembly of small fragments into larger sequences was followed by taking samples of the reaction after 25, 30, 35, 40 and 45 cycles of reassembly (FIG. 2).
  • Whereas the reassembly of 100-200 bp fragments can yield a single PCR product of the correct size, 10-50 base fragments typically yield some product of the correct size, as well as products of heterogeneous molecular weights. Most of this size heterogeneity appears to be due to single-stranded sequences at the ends of the products, since after restriction enzyme digestion a single band of the correct size is obtained.
  • 4) PCR with Primers
  • After dilution of the reassembly product into the PCR Mix with 0.8 μM of each of the above primers (SEQ ID Nos: 1 and 2) and about 15 cycles of PCR, each cycle consisting of [94° C. for 30 seconds, 50° C. for 30 seconds and 72° C. for 30 seconds], a single product of the correct size was obtained (FIG. 2).
  • 5) Cloning and Analysis
  • The PCR product from step 4 above was digested with the terminal restriction enzymes BamHI and EcoO109 and gel purified as described above in step 2. The reassembled fragments were ligated into pUC18 digested with BamHI and EcoO109. E. coli were transformed with the ligation mixture under standard conditions as recommended by the manufacturer (Stratagene, San Diego Calif.) and plated on agar plates having 100 μg/ml ampicillin, 0.004% X-gal and 2 mM IPTG. The resulting colonies having the HinDIII-NheI fragment which is diagnostic for the ++recombinant were identified because they appeared blue.
  • This Example illustrates that a 1.0 kb sequence carrying the LacZ alpha gene can be digested into 10-70 bp fragments, and that these gel purified 10-70 bp fragments can be reassembled to a single product of the correct size, such that 84% (N=377) of the resulting colonies are LacZ+ (versus 94% without shuffling; FIG. 2).
  • The DNA encoding the LacZ gene from the resulting LacZ colonies was sequenced with a sequencing kit (United States Biochemical Co., Cleveland Ohio) according to the manufacturer's instructions and the genes were found to have point mutations due to the reassembly process (Table 1). 11/12 types of substitutions were found, and no frameshifts.
  • TABLE 1
    Mutations introduced by mutagenic shuffling
    Transitions Frequency Transversions Frequency
    G-A 6 A-T 1
    A-G 4 A-C 2
    C-T 7 C-A 1
    T-C 3 C-G 0
    G-C 3
    G-T 2
    T-A 1
    T-G 2
  • A total of 4,437 bases of shuffled lacZ DNA were sequenced.
  • The rate of point mutagenesis during DNA reassembly from 10-70 bp pieces was determined from DNA sequencing to be 0.7% (N=4,473), which is similar to error-prone PCR. Without being limited to any theory it is believed that the rate of point mutagenesis may be lower if larger fragments are used for the reassembly, or if a proofreading polymerase is added.
  • When plasmid DNA from 14 of these point-mutated LacZ-colonies were combined and again reassembled/shuffled by the method described above, 34% (N=291) of the resulting colonies were LacZ+, and these colonies presumably arose by recombination of the DNA from different colonies.
  • The expected rate of reversal of a single point mutation by error-prone PCR, assuming a mutagenesis rate of 0.7% (10), would be expected to be <1%.
  • Thus large DNA sequences can be reassembled from a random mixture of small fragments by a reaction that is surprisingly efficient and simple. One application of this technique is the recombination or shuffling of related sequences based on homology.
  • Example 2 LacZ Gene and Whole Plasmid DNA Shuffling 1) LacZ Gene Shuffling
  • Crossover between two markers separated by 75 bases was measured using two LacZ gene constructs. Stop codons were inserted in two separate areas of the LacZ alpha gene to serve as negative markers. Each marker is a 25 bp non-homologous sequence with four stop codons, of which two are in the LacZ gene reading frame. The 25 bp non-homologous sequence is indicated in FIG. 3 by a large box. The stop codons are either boxed or underlined. A 1:1 mixture of the two 1.0 kb LacZ templates containing the +− and −+ versions of the LacZ alpha gene (FIG. 3) was digested with DNAseI and 100-200 bp fragments were purified as described in Example 1. The shuffling program was conducted under conditions similar to those described for reassembly in Example 1 except 0.5 μl of polymerase was added and the total volume was 100 μl.
  • After cloning, the number of blue colonies obtained was 24%; (N=386) which is close to the theoretical maximum number of blue colonies (i.e. 25%), indicating that recombination between the two markers was complete. All of the 10 blue colonies contained the expected HindIII-NheI restriction fragment.
  • 2) Whole Plasmid DNA Shuffling
  • Whole 2.7 kb plasmids (pUC18−+ and pUC18+−) were also tested. A 1:1 mixture of the two 2.9 kb plasmids containing the +− and −+ versions of the LacZ alpha gene (FIG. 3) was digested with DNAseI and 100-200 bp fragments were purified as described in Example 1. The shuffling program was conducted under conditions similar to those described for reassembly in step (1) above except the program was for 60 cycles [94° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 30 seconds]. Gel analysis showed that after the shuffling program most of the product was greater than 20 kb. Thus, whole 2.7 kb plasmids (pUC18−+ and pUC18+−) were efficiently reassembled from random 100-200 bp fragments without added primers.
  • After digestion with a restriction enzyme having a unique site on the plasmid (EcoO109), most of the product consisted of a single band of the expected size. This band was gel purified, religated and the DNA used to transform E. coli. The transformants were plated on 0.004% X-gal plates as described in Example 1. 11% (N=328) of the resulting plasmids were blue and thus ++ recombinants.
  • 3) Spiked DNA Shuffling
  • Oligonucleotides that are mixed into the shuffling mixture can be incorporated into the final product based on the homology of the flanking sequences of the oligonucleotide to the template DNA (FIG. 4). The LacZ stop codon mutant (pUC18−+) described above was used as the DNAseI digested template. A 66 mer oligonucleotide, including 18 bases of homology to the wild-type LacZ gene at both ends was added into the reaction at a 4-fold molar excess to correct stop codon mutations present in the original gene. The shuffling reaction was conducted under conditions similar to those in step 2 above. The resulting product was digested, ligated and inserted into E. coli as described above.
  • TABLE 2
    % blue colonies
    Control 0.0 (N > 1000)
    Top strand spike 8.0 (N = 855)
    Bottom strand spike 9.3 (N = 620)
    Top and bottom strand spike 2.1 (N = 537)
  • ssDNA appeared to be more efficient than dsDNA, presumably due to competitive hybridization. The degree of incorporation can be varied over a wide range by adjusting the molar excess, annealing temperature, or the length of homology.
  • Example 3 DNA Reassembly in the Complete Absence of Primers
  • Plasmid pUC18 was digested with restriction enzymes EcoRI, EcoO109, XmnI and AlwNI, yielding fragments of approximately 370, 460, 770 and 1080 bp. These fragments were electrophoresed and separately purified from a 2% low melting point agarose gel (the 370 and 460 basepair bands could not be separated), yielding a large fragment, a medium fragment and a mixture of two small fragments in 3 separate tubes.
  • Each fragment was digested with DNAseI as described in Example 1, and fragments of 50-130 bp were purified from a 2% low melting point agarose gel for each of the original fragments.
  • PCR mix (as described in Example 1 above) was added to the purified digested fragments to a final concentration of 10 ng/μl of fragments. No primers were added. A reassembly reaction was performed for 75 cycles [94° C. for 30 seconds, 60° C. for 30 seconds] separately on each of the three digested DNA fragment mixtures, and the products were analyzed by agarose gel electrophoresis.
  • The results clearly showed that the 1080, 770 and the 370 and 460 bp bands reformed efficiently from the purified fragments, demonstrating that shuffling does not require the use of any primers at all.
  • Example 4 IL-1β Gene Shuffling
  • This example illustrates that crossovers based on homologies of less than 15 bases may be obtained. As an example, a human and a murine IL-1β gene were shuffled.
  • A murine IL1-β gene (BBG49) and a human IL1-β gene with E. coli codon usage (BBG2; R&D Systems, Inc., Minneapolis Minn.) were used as templates in the shuffling reaction. The areas of complete homology between the human and the murine IL-1β sequences are on average only 4.1 bases long (FIG. 5, regions of heterology are boxed).
  • Preparation of dsDNA PCR products for each of the genes, removal of primers, DNAseI digestion and purification of 10-50 bp fragments was similar to that described above in Example 1. The sequences of the primers used in the PCR reaction were 5′TTAGGCACCCCAGGCTTT3′ (SEQ ID NO:3) and 5′ATGTGCTGCAAGGCGATT3′ (SEQ ID NO:4).
  • The first 15 cycles of the shuffling reaction were performed with the Klenow fragment of DNA polymerase I, adding 1 unit of fresh enzyme at each cycle. The DNA was added to the PCR mix of Example 1 which mix lacked the polymerase. The manual program was 94° C. for 1 minute, and then 15 cycles of: [95° C. for 1 minute, 10 seconds on dry ice/ethanol (until frozen), incubate about 20 seconds at 25° C., add 1 U of Klenow fragment and incubate at 25° C. for 2 minutes]. In each cycle after the denaturation step, the tube was rapidly cooled in dry ice/ethanol and reheated to the annealing temperature. Then the heat-labile polymerase was added. The enzyme needs to be added at every cycle. Using this approach, a high level of crossovers was obtained, based on only a few bases of uninterrupted homology (FIG. 5, positions of cross-overs indicated by “ | ”).
  • After these 15 manual cycles, Taq polymerase was added and an additional 22 cycles of the shuffling reaction [94° C. for 30 seconds, 35° C. for 30 seconds] without primers were performed.
  • The reaction was then diluted 20-fold. The following primers were added to a final concentration of 0.8 μM: 5′AACGCCGCATGCAAGCTTGGATCCTTATT3′ (SEQ ID NO:5) and 5′AAAGCCCTCTAGATGATTACGAATTCATAT3′ (SEQ ID NO:6) and a PCR reaction was performed as described above in Example 1. The second primer pair differed from the first pair only because a change in restriction sites was deemed necessary.
  • After digestion of the PCR product with XbaI and SphI, the fragments were ligated into XbaI-SphI-digested pUC18. The sequences of the inserts from several colonies were determined by a dideoxy DNA sequencing kit (United States Biochemical Co., Cleveland Ohio) according to the manufacturer's instructions.
  • A total of 17 crossovers were found by DNA sequencing of nine colonies. Some of the crossovers were based on only 1-2 bases of uninterrupted homology.
  • It was found that to force efficient crossovers based on short homologies, a very low effective annealing temperature is required. With any heat-stable polymerase, the cooling time of the PCR machine (94° C. to 25° C. at 1-2 degrees/second) causes the effective annealing temperature to be higher than the set annealing temperature. Thus, none of the protocols based on Taq polymerase yielded crossovers, even when a ten-fold excess of one of the IL1-β genes was used. In contrast, a heat-labile polymerase, such as the Klenow fragment of DNA polymerase I, can be used to accurately obtain a low annealing temperature.
  • Example 5 DNA Shuffling of the TEM-1 betalactamase Gene
  • The utility of mutagenic DNA shuffling for directed molecular evolution was tested in a betalactamase model system. TEM-1 betalactamase is a very efficient enzyme, limited in its reaction rate primarily by diffusion. This example determines whether it is possible to change its reaction specificity and obtain resistance to the drug cefotaxime that it normally does not hydrolyze.
  • The minimum inhibitory concentration (MIC) of cefotaxime on bacterial cells lacking a plasmid was determined by plating 10 μl of a 10−2 dilution of an overnight bacterial culture (about 1000 cfu) of E. coli XL1-blue cells (Stratagene, San Diego Calif.) on plates with varying levels of cefotaxime (Sigma, St. Louis Mo.), followed by incubation for 24 hours at 37° C.
  • Growth on cefotaxime is sensitive to the density of cells, and therefore similar numbers of cells needed to be plated on each plate (obtained by plating on plain LB plates). Platings of 1000 cells were consistently performed.
  • 1) Initial Plasmid Construction
  • A pUC18 derivative carrying the bacterial TEM-1 betalactamase gene was used (28). The TEM-1 betalactamase gene confers resistance to bacteria against approximately 0.02 μg/ml of cefotaxime. Sfi1 restriction sites were added 5′ of the promoter and 3′ of the end of the gene by PCR of the vector sequence with two primers:
  • Primer A (SEQ ID NO: 7):
    5′TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT3′
    and
    Primer B (SEQ ID NO: 8):
    5′TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCGC
    GCACATTT3′

    and by PCR of the betalactamase gene sequence with two other primers:
  • Primer C (SEQ ID NO: 9):
    5′AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT,
    and
    Primer D (SEQ ID NO: 10):
    5′AACCTGTCCTGGCCACCATGGCCTAAATACATTCAAATATGTAT.
  • The two reaction products were digested with SfiI, mixed, ligated and used to transform bacteria.
  • The resulting plasmid was pUC182Sfi. This plasmid contains an Sfi1 fragment carrying the TEM-1 gene and the P-3 promoter.
  • The minimum inhibitory concentration of cefotaxime for E. coli XL1-blue (Stratagene, San Diego Calif.) carrying this plasmid was 0.02 μg/ml after 24 hours at 37° C.
  • The ability to improve the resistance of the betalactamase gene to cefotaxime without shuffling was determined by stepwise replating of a diluted pool of cells (approximately 107 cfu) on 2-fold increasing drug levels. Resistance up to 1.28 μg/ml could be obtained without shuffling. This represented a 64 fold increase in resistance.
  • 2) DNAseI Digestion
  • The substrate for the first shuffling reaction was dsDNA of 0.9 kb obtained by PCR of pUC182Sfi with primers C and D, both of which contain a SfiI site.
  • The free primers from the PCR product were removed by Wizard PCR prep (Promega, Madison Wis.) at every cycle.
  • About 5 μg of the DNA substrate(s) was digested with 0.15 units of DNAseI (Sigma, St. Louis Mo.) in 100 μl of 50 mM Tris-HCl pH 7.4, 1 mM MgCl2, for 10 min at room temperature. Fragments of 100-300 bp were purified from 2% low melting point agarose gels by electrophoresis onto DE81 ion exchange paper (Whatman, Hillsborough Oreg.), elution with 1 M NaCl and ethanol precipitation by the method described in Example 1.
  • 3) Gene Shuffling
  • The purified fragments were resuspended in PCR mix (0.2 mM each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100), at a concentration of 10-30 ng/μl. No primers were added at this point. A reassembly program of 94° C. for 60 seconds, then 40 cycles of [94° C. for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds] and then 72° C. for 5 minutes was used in an MJ Research (Watertown Mass.) PTC-150 thermocycler.
  • 4) Amplification of Reassembly Product with Primers
  • After dilution of the reassembly product into the PCR mix with 0.8 μM of each primer (C and D) and 20 PCR cycles [94° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 30 seconds] a single product 900 bp in size was obtained.
  • 5) Cloning and Analysis
  • After digestion of the 900 bp product with the terminal restriction enzyme SfiI and agarose gel purification, the 900 bp product was ligated into the vector pUC182Sfi at the unique SfiI site with T4 DNA ligase (BRL, Gaithersburg Md.). The mixture was electroporated into E. coli XL1-blue cells and plated on LB plates with 0.32-0.64 μg/ml of cefotaxime (Sigma, St. Louis Mo.). The cells were grown for up to 24 hours at 37° C. and the resulting colonies were scraped off the plate as a pool and used as the PCR template for the next round of shuffling.
  • 6) Subsequent Reassembly Rounds
  • The transformants obtained after each of three rounds of shuffling were plated on increasing levels of cefotaxime. The colonies (>100, to maintain diversity) from the plate with the highest level of cefotaxime were pooled and used as the template for the PCR reaction for the next round.
  • A mixture of the cefotaximer colonies obtained at 0.32-0.64 μg/ml in Step (5) above were used as the template for the next round of shuffling. 10 ul of cells in LB broth were used as the template in a reassembly program of 10 minutes at 99° C., then 35 cycles of [94° C. for 30 seconds, 52° C. for 30 seconds, 72° C. for 30 seconds] and then 5 minutes at 72° C. as described above.
  • The reassembly products were digested and ligated into pUC182Sfi as described in step (5) above. The mixture was electroporated into E. coli XL1-blue cells and plated on LB plates having 5-10 μg/ml of cefotaxime.
  • Colonies obtained at 5-10 μg/ml were used for a third round similar to the first and second rounds except the cells were plated on LB plates having 80-160 μg/ml of cefotaxime. After the third round, colonies were obtained at 80-160 μg/ml, and after replating on increasing concentrations of cefotaxime, colonies could be obtained at up to 320 μg/ml after 24 hours at 37° C. (MIC=320 μg/ml).
  • Growth on cefotaxime is dependent on the cell density, requiring that all the MICs be standardized (in our case to about 1,000 cells per plate). At higher cell densities, growth at up to 1280 μg/ml was obtained. The 5 largest colonies grown at 1,280 μg/ml were plated for single colonies twice, and the Sfi1 inserts were analyzed by restriction mapping of the colony PCR products.
  • One mutant was obtained with a 16,000 fold increased resistance to cefotaxime (MIC=0.02 μg/ml to MIC=320 μg/ml).
  • After selection, the plasmid of selected clones was transferred back into wild-type E. coli XL1-blue cells (Stratagene, San Diego Calif.) to ensure that none of the measured drug resistance was due to chromosomal mutations.
  • Three cycles of shuffling and selection yielded a 1.6×104-fold increase in the minimum inhibitory concentration of the extended broad spectrum antibiotic cefotaxime for the TEM-1 betalactamase. In contrast, repeated plating without shuffling resulted in only a 16-fold increase in resistance (error-prone PCR or cassette mutagenesis).
  • 7) Sequence Analysis
  • All 5 of the largest colonies grown at 1,280 μg/ml had a restriction map identical to the wild-type TEM-1 enzyme. The SfiI insert of the plasmid obtained from one of these colonies was sequenced by dideoxy DNA sequencing (United States Biochemical Co., Cleveland Ohio) according to the manufacturer's instructions. All the base numbers correspond to the revised pBR322 sequence (29), and the amino acid numbers correspond to the ABL standard numbering scheme (30). The amino acids are designated by their three letter codes and the nucleotides by their one letter codes. The term G4205A means that nucleotide 4205 was changed from guanidine to adenine.
  • Nine single base substitutions were found. G4205A is located between the −35 and −10 sites of the betalactamase P3 promoter (31). The promoter up-mutant observed by Chen and Clowes (31) is located outside of the Sfi1 fragment used here, and thus could not have been detected. Four mutations were silent (A3689G, G3713A, G3934A and T3959A), and four resulted in an amino acid change (C3448T resulting in Gly238Ser, A3615G resulting in Met182Thr, C3850T resulting in Glu104Lys, and G4107A resulting in Ala18Val).
  • 8) Molecular Backcross
  • Molecular backcrossing with an excess of the wild-type DNA was then used in order to eliminate non-essential mutations.
  • Molecular backcrossing was conducted on a selected plasmid from the third round of DNA shuffling by the method identical to normal shuffling as described above, except that the DNAseI digestion and shuffling reaction were performed in the presence of a 40-fold excess of wild-type TEM-1 gene fragment. To make the backcross more efficient, very small DNA fragments (30 to 100 bp) were used in the shuffling reaction. The backcrossed mutants were again selected on LB plates with 80-160 μg/ml of cefotaxime (Sigma, St. Louis Mo.).
  • This backcross shuffling was repeated with DNA from colonies from the first backcross round in the presence of a 40-fold excess of wild-type TEM-1 DNA. Small DNA fragments (30-100 bp) were used to increase the efficiency of the backcross. The second round of backcrossed mutants were again selected on LB plates with 80-160 μg/ml of cefotaxime.
  • The resulting transformants were plated on 160 μg/ml of cefotaxime, and a pool of colonies was replated on increasing levels of cefotaxime up to 1,280 μg/ml. The largest colony obtained at 1,280 μg/ml was replated for single colonies.
  • This backcrossed mutant was 32,000 fold more resistant than wild-type. (MIC=640 μg/ml) The mutant strain is 64-fold more resistant to cefotaxime than previously reported clinical or engineered TEM-1-derived strains. Thus, it appears that DNA shuffling is a fast and powerful tool for at least several cycles of directed molecular evolution.
  • The DNA sequence of the SfiI insert of the backcrossed mutant was determined using a dideoxy DNA sequencing kit (United States Biochemical Co., Cleveland Ohio) according to the manufacturer's instructions (Table 3). The mutant had 9 single base pair mutations. As expected, all four of the previously identified silent mutations were lost, reverting to the sequence of the wild-type gene. The promoter mutation (G4205A) as well as three of the four amino acid mutations (Glu104Lys, Met182Thr, and Gly238Ser) remained in the backcrossed clone, suggesting that they are essential for high level cefotaxime resistance. However, two new silent mutations (T3842C and A3767G), as well as three new mutations resulting in amino acid changes were found (C3441T resulting in Arg241His, C3886T resulting in Gly92Ser, and G4035C resulting in Ala42Gly). While these two silent mutations do not affect the protein primary sequence, they may influence protein expression level (for example by mRNA structure) and possibly even protein folding (by changing the codon usage and therefore the pause site, which has been implicated in protein folding).
  • TABLE 3
    Mutations in Betalactamase
    Mutation Type Non-Backcrossed Backcrossed
    amino acid Ala18Lys
    change Glu104Lys Glu104Lys
    Met182Thr Met182Thr
    Gly238Ser Gly238Ser
    Ala42Gly
    Gly92Ser
    silent T3959A
    G3934A
    G3713A
    A3689G
    T3842C
    A3767G
    promoter G4205A G4205A
  • Both the backcrossed and the non-backcrossed mutants have a promoter mutation (which by itself or in combination results in a 2-3 fold increase in expression level) as well as three common amino acid changes (Glu104Lys, Met182Thr and Gly238Ser). Glu104Lys and Gly238Ser are mutations that are present in several cefotaxime resistant or other TEM-1 derivatives (Table 4).
  • 9) Expression Level Comparison
  • The expression level of the betalactamase gene in the wild-type plasmid, the non-backcrossed mutant and in the backcrossed mutant was compared by SDS-polyacrylamide gel electrophoresis (4-20%; Novex, San Diego Calif.) of periplasmic extracts prepared by osmotic shock according to the method of Witholt, B. (32).
  • Purified TEM-1 betalactamase (Sigma, St. Louis Mo.) was used as a molecular weight standard, and E. coli XL1-blue cells lacking a plasmid were used as a negative control.
  • The mutant and the backcrossed mutant appeared to produce a 2-3 fold higher level of the betalactamase protein compared to the wild-type gene. The promoter mutation appeared to result in a 2-3 times increase in betalactamase.
  • Example 6 Construction of Mutant Combinations of the TEM-1 betalactamase Gene
  • To determine the resistance of different combinations of mutations and to compare the new mutants to published mutants, several mutants were constructed into an identical plasmid background. Two of the mutations, Glu104Lys and Gly238Ser, are known as cefotaxime mutants. All mutant combinations constructed had the promoter mutation, to allow comparison to selected mutants. The results are shown in Table 4.
  • Specific combinations of mutations were introduced into the wild-type pUC182Sfi by PCR, using two oligonucleotides per mutation.
  • The oligonucleotides to obtain the following mutations were:
  • Ala42Gly
    (SEQ ID NO: 11)
    AGTTGGGTGGACGAGTGGGTTACATCGAACT
    and
    (SEQ ID NO: 12)
    AACCCACTCGTCCACCCAACTGATCTTCAGCAT;
    Gln39Lys:
    (SEQ ID NO: 13)
    AGTAAAAGATGCTGAAGATAAGTTGGGTGCAC GAGTGGGTT
    and
    (SEQ ID NO: 14)
    ACTTATCTTCAGCATCTTTTACTT;
    Gly92Ser:
    (SEQ ID NO: 15)
    AAGAGCAACTCAGTCGCCGCATACACTATTCT
    and
    (SEQ ID NO: 16)
    ATGGCGGCGACTGAGTTGCTCTTGCCCGGCGTCAAT;
    Glu104Lys:
    (SEQ ID NO: 17)
    TATTCTCAGAATGACTTGGTTAAGTACTCACCAGT CACAGAA
    and
    (SEQ ID NO: 18)
    TTAACCAAGTCATTCTGAGAAT;
    Met182Thr:
    (SEQ ID NO: 19)
    AACGACGAGCGTGACACCACGACGCCTGTAGCAATG
    and
    (SEQ ID NO: 20)
    TCGTGGTGTCACGCTCGTCGTT;
    Gly238Ser alone:
    (SEQ ID NO: 21)
    TTGCTGATAAATCTGGAGCCAGTGAGCGTGGGTCTC GCGGTA
    and
    (SEQ ID NO: 22)
    TGGCTCCAGATTTATCAGCAA;
    Gly238Ser and Arg241HiS (combined):
    (SEQ ID NO: 23)
    ATGCTCACTGGCTCCAGATTTATCAGCAAT
    and
    (SEQ ID NO: 24)
    TCTGGAGCCAGTGAGCATGGGTCTCGCGGTATCATT;
    G4205A:
    (SEQ ID NO: 25)
    AACCTGTCCTGGCCACCATGGCCTAAATACAATCAAA
    TATGTATCCGCTTATGAGACAATAACCCTGATA.
  • These separate PCR fragments were gel purified away from the synthetic oligonucleotides. 10 ng of each fragment were combined and a reassembly reaction was performed at 94° C. for 1 minute and then 25 cycles; [94° C. for 30 sec, 50° C. for 30 seconds and 72° C. for 45 seconds]. PCR was performed on the reassembly product for 25 cycles in the presence of the SfiI-containing outside primers (primers C and D from Example 5). The DNA was digested with Sfi1 and inserted into the wild-type pUC182Sfi vector. The following mutant combinations were obtained (Table 4).
  • TABLE 4
    Source
    Name Genotype MIC of MIC
    TEM-1 Wild-type 0.02
    Glu104Lys 0.08 10
    Gly238Ser 016 10
    TEM-15 Glu104Lys/Gly238Ser* 10
    TEM-3 Glu104Lys/Gly238Ser/Gln39Lys 10 37, 15
    2-32
    ST-4 Glu104Lys/Gly238Ser/Met182 10
    Thr*
    ST-1 Glu104Lys/Gly238Ser/Met182 320
    Thr/Ala18Val/T3959A/G3713A/
    G3934A/A3689G*
    ST-2 Glu104Lys/Gly238Ser/Met182Thr/ 640
    Ala42Gly/Gly92Ser/Arg241His/
    T3842C/A3767G*
    ST-3 Glu104Lys/Gly238Ser/Met182Thr/ 640
    Ala42Gly/Gly92Ser/Arg241His*
    *All of these mutants additionally contain the G4205A promoter mutation.
  • It was concluded that conserved mutations account for 9 of 15 doublings in the MIC.
  • Glu104Lys alone was shown to result only in a doubling of the MIC to 0.08 μg/ml, and Gly238Ser (in several contexts with one additional amino acid change) resulted only in a MIC of 0.16 μg/ml (26). The double mutant Glu104Lys/Gly238Ser has a MIC of 10 μg/ml. This mutant corresponds to TEM-15.
  • These same Glu104Lys and Gly238Ser mutations, in combination with Gln39Lys (TEM-3) or Thr263Met (TEM-4) result in a high level of resistance (2-32 μg/ml for TEM-3 and 8-32 μg/ml for TEM-4 (34, 35).
  • A mutant containing the three amino acid changes that were conserved after the backcross (Glu104Lys/Met182Thr/Gly238Ser) also had a MIC of 10 μg/ml. This meant that the mutations that each of the new selected mutants had in addition to the three known mutations were responsible for a further 32 to 64-fold increase in the resistance of the gene to cefotaxime.
  • The naturally occurring, clinical TEM-1-derived enzymes (TEM-1-19) each contain a different combination of only 5-7 identical mutations (reviews). Since these mutations are in well separated locations in the gene, a mutant with high cefotaxime resistance cannot be obtained by cassette mutagenesis of a single area. This may explain why the maximum MIC that was obtained by the standard cassette mutagenesis approach is only 0.64 μg/ml (26). For example, both the Glu104Lys as well as the Gly238Ser mutations were found separately in this study to have MICs below 0.16 μg/ml. Use of DNA shuffling allowed combinatoriality and thus the Glu104Lys/Gly238Ser combination was found, with a MIC of 10 μg/ml.
  • An important limitation of this example is the use of a single gene as a starting point. It is contemplated that better combinations can be found if a large number of related, naturally occurring genes are shuffled. The diversity that is present in such a mixture is more meaningful than the random mutations that are generated by mutagenic shuffling. For example, it is contemplated that one could use a repertoire of related genes from a single species, such as the pre-existing diversity of the immune system, or related genes obtained from many different species.
  • Example 7 Improvement of Antibody A10B by DNA Shuffling of a Library of all Six Mutant CDRs
  • The A10B scFv antibody, a mouse anti-rabbit IgG, was a gift from Pharmacia (Milwaukee Wis.). The commercially available Pharmacia phage display system was used, which uses the pCANTAB5 phage display vector.
  • The original A10B antibody reproducibly had only a low avidity, since clones that only bound weakly to immobilized antigen (rabbit IgG), (as measured by phage ELISA (Pharmacia assay kit) or by phage titer) were obtained. The concentration of rabbit IgG which yielded 50% inhibition of the A10B antibody binding in a competition assay was 13 picomolar. The observed low avidity may also be due to instability of the A10B clone.
  • The A10B scFv DNA was sequenced (United States Biochemical Co., Cleveland Ohio) according to the manufacturer's instructions. The sequence was similar to existing antibodies, based on comparison to Kabat (33).
  • 1) Preparation of Phage DNA
  • Phage DNA having the A10B wild-type antibody gene (10 ul) was incubated at 99° C. for 10 min, then at 72° C. for 2 min. PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μM each dNTP, 1.9 mM MgCl), 0.6 μm of each primer and 0.5 μl Taq DNA Polymerase (Promega, Madison Wis.) was added to the phage DNA. A PCR program was run for 35 cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.]. The primers used were: 5′ ATGATTACGCCAAGCTTT 3′ (SEQ ID NO:26) and 5′ TTGTCGTCTTTCCAGACGTT 3′ (SEQ ID NO:27).
  • The 850 bp PCR product was then electrophoresed and purified from a 2% low melting point agarose gel.
  • 2) Fragmentation
  • 300 ng of the gel purified 850 bp band was digested with 0.18 units of DNAse I (Sigma, St. Louis Mo.) in 50 mM Tris-HCl pH 7.5, 10 mM MgCl for 20 minutes at room temperature. The digested DNA was separated on a 2% low melting point agarose gel and bands between 50 and 200 bp were purified from the gel.
  • 3) Construction of Test Library
  • The purpose of this experiment was to test whether the insertion of the CDRs would be efficient.
  • The following CDR sequences having internal restriction enzyme sites were synthesized. “CDR H” means a CDR in the heavy chain and “CDR L” means a CDR in the light chain of the antibody.
  • CDR Oligos with restriction sites:
    CDR H1 (SEQ ID NO: 34)
    5′ TTCTGGCTACATCTTCACAGAATTCATCTAGATTTGGGTGAGGCAGA
    CGCCTGAA3′
    CDR H2 (SEQ ID NO: 35)
    5′ ACAGGGACTTGAGTGGATTGGAATCACAGTCAAGCTTATCCTTTATC
    TCAGGTCTCGAGTTCCAAGTACTTAAAGGGCCACACTGAGTGTA
    3′
    CDR H3 (SEQ ID NO: 36)
    5′ TGTCTATTTCTGTGCTAGATCTTGACTGCAGTCTTATACGAGGATCC
    ATTGGGGCCAAGGGACCAGGTCA
    3′
    CDR L1 (SEQ ID NO: 37)
    5′ AGAGGGTCACCATGACCTGCGGACGTCTTTAAGCGATCGGGCTGATG
    GCCTGGTACCAACAGAAGCCTGGAT
    3′
    CDR L2 (SEQ ID NO: 38)
    5′ TCCCCCAGACTCCTGATTTATTAAGGGAGATCTAAACAGCTGTTGGT
    CCCTTTTCGCTTCAGT
    3′
    CDR L3 (SEQ ID NO: 39)
    5′ ATGCTGCCACTTATTACTGCTTCTGCGCGCTTAAAGGATATCTTCAT
    TTCGGAGGGGGGACCAAGCT
    3′
  • The CDR oligos were added to the purified A10B antibody DNA fragments of between 50 to 200 bp from step (2) above at a 10 fold molar excess. The PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton x-100, 1.9 mM MgCl, 200 μm each dNTP, 0.3 μl Taq DNA polymerase (Promega, Madison Wis.), 50 μl total volume) was added and the shuffling program run for 1 min at 94° C., 1 min at 72° C., and then 35 cycles: 30 seconds at 94° C., 30 seconds at 55° C., 30 seconds at 72° C.
  • 1 μl of the shuffled mixture was added to 100 μl of a PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μm each dNTP, 1.9 mM MgCl, 0.6 μM each of the two outside primers (SEQ ID NO:26 and 27, see below), 0.5 μl Taq DNA polymerase) and the PCR program was run for 30 cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.]. The resulting mixture of DNA fragments of 850 basepair size was phenol/chloroform extracted and ethanol precipitated.
  • The outside primers were:
  • outside Primer 1: SEQ ID NO: 27
    5′ TTGTCGTCTTTCCAGACGTT 3′
    Outside Primer 2: SEQ ID NO: 26
    5′ ATGATTACGCCAAGCTTT 3′
  • The 850 bp PCR product was digested with the restriction enzymes SfiI and NotI, purified from a low melting point agarose gel, and ligated into the pCANTAB5 expression vector obtained from Pharmacia, Milwaukee Wis. The ligated vector was electroporated according to the method set forth by Invitrogen (San Diego Calif.) into TG1 cells (Pharmacia, Milwaukee Wis.) and plated for single colonies.
  • The DNA from the resulting colonies was added to 100 μl of a PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μm each dNTP, 1.9 mM MgCl, 0.6 μM of Outside primer 1 (SEQ ID No. 27; see below) six inside primers (SEQ ID NOS:40-45; see below), and 0.5 μl Taq DNA polymerase) and a PCR program was run for 35 cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.]. The sizes of the PCR products were determined by agarose gel electrophoresis, and were used to determine which CDRs with restriction sites were inserted.
  • CDR Inside Primers:
  • H 1 (SEQ ID NO: 40) 5′ AGAATTCATCTAGATTTG 3′,
    H 2 (SEQ ID NO: 41) 5′ GCTTATCCTTTATCTCAGGTC 3′,
    H 3 (SEQ ID NO: 42) 5′ ACTGCAGTCTTATACGAGGAT 3′
    L 1 (SEQ ID NO: 43) 5′ GACGTCTTTAAGCGATCG 3′,
    L 2 (SEQ ID NO: 44) 5′ TAAGGGAGATCTAAACAG 3′,
    L 3 (SEQ ID NO: 45) 5′ TCTGCGCGCTTAAAGGAT 3′
  • The six synthetic CDRs were inserted at the expected locations in the wild-type A10B antibody DNA (FIG. 7). These studies showed that, while each of the six CDRs in a specific clone has a small chance of being a CDR with a restriction site, most of the clones carried at least one CDR with a restriction site, and that any possible combination of CDRs with restriction sites was generated.
  • 4) Construction of Mutant Complementarity Determining Regions (“CDRs”)
  • Based on our sequence data six oligonucleotides corresponding to the six CDRs were made. The CDRs (Kabat definition) were synthetically mutagenized at a ratio of 70 (existing base):10:10:10, and were flanked on the 5′ and 3′ sides by about 20 bases of flanking sequence, which provide the homology for the incorporation of the CDRs when mixed into a mixture of unmutagenized antibody gene fragments in a molar excess. The resulting mutant sequences are given below.
  • Oligos for CDR Library
  • CDR H1 (SEQ ID NO: 28)
    5′ TTCTGGCTACATCTTCACAA CTTATGATATAGACT GGGTGAGGCAGA
    CGCCTGAA
    3′
    CDR H2 (SEQ ID NO: 29)
    5′ ACAGGGACTTGAGTGGATTGGA TGGATTTTTCCTGGAGAGGGTGGTA
    CTGAATACAATGAGAAGTTCAAGGGC AGGGCCACACTGAGTGTA
    3′
    CDR H3 (SEQ ID NO: 30)
    5′ TGTCTATTTCTGTGCTAGA GGGGACTACTATAGGCGCTACTTTGACT
    TG TGGGGCCAAGGGACCACGGTCA
    3′
    CDR L1 (SEQ ID NO: 31)
    5′ AGAGGGTCACCATGACCTGCA GTGCCAGCTCAGGTATACGTTACATA
    TATT GGTACCAACAGAAGCCTGGAT
    3′
    CDR L2 (SEQ ID NO: 32)
    5′ TCCCCCAGACTCCTGATTTAT GACACATCCAACGTGGCTCCTGGA GT
    CCCTTTTCGCTTCAGT
    3′
    CDR L3 (SEQ ID NO: 33)
    5′ ATGCTGCCACTTATTACTTGCC AGGAGTGGAGTGGTTATCCGTACAC
    GT TCGGAGGGGGGACCAAGCT
    3′.

    Bold and underlined sequences were the mutant sequences synthesized using a mixture of nucleosides of 70:10:10:10 where 70% was the wild-type nucleoside.
  • A 10 fold molar excess of the CDR mutant oligos were added to the purified A10B antibody DNA fragments between 50 to 200 bp in length from step (2) above. The PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton x-100, 1.9 mM MgCl, 200 μm each dNTP, 0.3 μl Taq DNA polymerase (Promega, Madison Wis.), 50 μl total volume) was added and the shuffling program run for 1 min at 94° C., 1 min at 72° C., and then 35 cycles: [30 seconds at 94° C., 30 seconds at 55° C., 30 seconds at 72° C.].
  • 1 μl of the shuffled mixture was added to 100 μl of a PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μm each dNTP, 1.9 mM MgCl, 0.6 mM each of the two outside primers (SEQ ID NO:26 and 27, see below), 0.5 μl Taq DNA polymerase) and the PCR program was run for 30 cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.]. The resulting mixture of DNA fragments of 850 basepair size was phenol/chloroform extracted and ethanol precipitated.
  • The outside primers were:
  • outside Primer 1:
    SEQ ID NO: 27 5′ TTGTCGTCTTTCCAGACGTT 3′
    outside Primer 2:
    SEQ ID NO: 26 5′ ATGATTACGCCAAGCTTT 3′

    5) Cloning of the scFv Antibody DNA into pCANTAB5
  • The 850 bp PCR product was digested with the restriction enzymes SfiI and NotI, purified from a low melting point agarose gel, and ligated into the pCANTAB5 expression vector obtained from Pharmacia, Milwaukee Wis. The ligated vector was electroporated according to the method set forth by Invitrogen (San Diego Calif.) into TG1 cells (Pharmacia, Milwaukee Wis.) and the phage library was grown up using helper phage following the guidelines recommended by the manufacturer.
  • The library that was generated in this fashion was screened for the presence of improved antibodies, using six cycles of selection.
  • 6) Selection of High Affinity Clones
  • 15 wells of a 96 well microtiter plate were coated with Rabbit IgG (Jackson Immunoresearch, Bar Harbor Me.) at 10 μg/well for 1 hour at 37° C., and then blocked with 2% non-fat dry milk in PBS for 1 hour at 37° C.
  • 100 μl of the phage library (1×1010 cfu) was blocked with 100 μl of 2% milk for 30 minutes at room temperature, and then added to each of the 15 wells and incubated for 1 hour at 37° C.
  • Then the wells were washed three times with PBS containing 0.5% Tween-20 at 37° C. for 10 minutes per wash. Bound phage was eluted with 100 μl elution buffer (Glycine-HCl, pH 2.2), followed by immediate neutralization with 2M Tris pH 7.4 and transfection for phage production. This selection cycle was repeated six times.
  • After the sixth cycle, individual phage clones were picked and the relative affinities were compared by phage ELISA, and the specificity for the rabbit IgG was assayed with a kit from Pharmacia (Milwaukee Wis.) according to the methods recommended by the manufacturer.
  • The best clone has an approximately 100-fold improved expression level compared with the wild-type A10B when tested by the Western assay. The concentration of the rabbit IgG which yielded 50% inhibition in a competition assay with the best clone was 1 picomolar. The best clone was reproducibly specific for rabbit antigen. The number of copies of the antibody displayed by the phage appears to be increased.
  • Example 8 In Vivo Recombination Via Direct Repeats of Partial Genes
  • A plasmid was constructed with two partial, inactive copies of the same gene (beta-lactamase) to demonstrate that recombination between the common areas of these two direct repeats leads to full-length, active recombinant genes.
  • A pUC18 derivative carrying the bacterial TEM-1 betalactamase gene was used (Yanish-Perron et al., 1985, Gene 33:103-119). The TEM-1 betalactamase gene (“Bla”) confers resistance to bacteria against approximately 0.02 μg/ml of cefotaxime. Sfi1 restriction sites were added 5′ of the promoter and 3′ of the end of the betalactamase gene by PCR of the vector sequence with two primers:
  • Primer A (SEQ ID NO: 46)
    5′ TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT
    3′
    PRIMER B (SEQ ID NO: 47)
    5′ TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCG
    CGCACATTT
    3′

    and by PCR of the beta-lactamase gene sequence with two other primers:
  • Primer C (SEQ ID NO: 48)
    5′ AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT
    3′
    Primer D (SEQ ID NO: 49)
    5′ AACCTGTCCTGCCCACCATGGCCTAAATACATTCAAATATGTAT 3′
  • The two reaction products were digested with Sfi1, mixed, ligated and used to transform competent E. coli bacteria by the procedure described below. The resulting plasmid was pUC182Sfi-Bla-Sfi. This plasmid contains an Sfi1 fragment carrying the Bla gene and the P-3 promoter.
  • The minimum inhibitory concentration of cefotaxime for E. coli XL1-blue (Stratagene, San Diego Calif.) carrying pUC182Sfi-Bla-Sfi was 0.02 μg/ml after 24 hours at 37° C.
  • The tetracycline gene of pBR322 was cloned into pUC18Sfi-Bla-Sfi using the homologous areas, resulting in pBR322TetSfi-Bla-Sfi. The TEM-1 gene was then deleted by restriction digestion of the pBR322TetSfi-Bla-Sfi with SspI and FspI and blunt-end ligation, resulting in pUC322TetSfi-Sfi.
  • Overlapping regions of the TEM-1 gene were amplified using standard PCR techniques and the following primers:
  • Primer 2650 (SEQ ID NO: 50)
    5′ TTCTTAGACGTCAGGTGGCACTT 3′
    Primer 2493 (SEQ ID NO: 51)
    5′ TTT TAA ATC AAT CTA AAG TAT 3′
    Primer 2651 (SEQ ID NO: 52)
    5′ TGCTCATCCACGAGTGTGGAGAAGTGGTCCTGCAACTTTAT 3′,
    and
    Primer 2652 (SEQ ID NO: 53)
    ACCACTTCTCCACACTCGTGGATGAGCACTTTTAAAGTT
  • The two resulting DNA fragments were digested with Sfi1 and BstX1 and ligated into the Sfi site of pBR322TetSfi-Sfi. The resulting plasmid was called pBR322Sfi-BL-LA-Sfi. A map of the plasmid as well as a schematic of intraplasmidic recombination and reconstitution of functional beta-lactamase is shown in FIG. 9.
  • The plasmid was electroporated into either TG-1 or JC8679 E. coli cells. E. coli JC8679 is RecBC sbcA (Oliner et al., 1993, NAR 21:5192). The cells were plated on solid agar plates containing tetracycline. Those colonies which grew, were then plated on solid agar plates containing 100 μg/ml ampicillin and the number of viable colonies counted. The beta-lactamase gene inserts in those transformants which exhibited ampicillin resistance were amplified by standard PCR techniques using Primer 2650 (SEQ ID NO: 50) 5′ TTCTTAGACGTCAGGTGGCACTT 3′ and Primer 2493 (SEQ ID NO: 51) 5′ TTTTAAATCAATCTAAAGTAT 3′ and the length of the insert measured. The presence of a 1 kb insert indicates that the gene was successfully recombined, as shown in FIG. 9 and Table 5.
  • TABLE 5
    Cell Tet Colonies Amp colonies Colony PCR
    TG-1 131 21 3/3 at 1 kb
    JC8679 123 31 4/4 at 1 kb
    vector
    51 0
    control
  • About 17-25% of the tetracycline-resistant colonies were also ampicillin-resistant and all of the Ampicillin resistant colonies had correctly recombined, as determined by colony PCR. Therefore, partial genes located on the same plasmid will successfully recombine to create a functional gene.
  • Example 9 In Vivo Recombination Via Direct Repeats of Full-Length Genes
  • A plasmid with two full-length copies of different alleles of the beta-lactamase gene was constructed. Homologous recombination of the two genes resulted in a single recombinant full-length copy of that gene.
  • The construction of pBR322TetSfi-Sfi and pBR322TetSfi-Bla-Sfi was described above.
  • The two alleles of the beta-lactamase gene were constructed as follows. Two PCR reactions were conducted with pUC18Sfi-Bla-Sfi as the template. One reaction was conducted with the following primers.
  • One reaction was conducted with the following
    primers.
    primer 2650 (SEQ ID NO: 50)
    5′ TTCTTAGACGTCAGGTGGCACTT 3′
    primer 2649 (SEQ ID NO: 51)
    5′ ATGGTAGTCCACGAGTGTGGTAGTGACAGGCCGGTCTGACAGTTA
    CCAATGCTT
    3′
    The second PCR reaction was conducted with the
    following primers:
    primer 2648 (SEQ ID NO: 54)
    5′ TGTCACTACCACACTCGTGGACTACCATGGCCTAAATACATTCAAA
    TATGTAT
    3′
    Primer 2493 (SEQ ID NO: 51)
    5′ TTT TAA ATC AAT CTA AAG TAT 3′
  • This yielded two Bla genes, one with a 5′ Sfi1 site and a 3′ BstX1 site, the other with a 5′ BstX1 site and a 3′ Sfi1 site.
  • After digestion of these two genes with BstX1 and Sfi1, and ligation into the Sfi1-digested plasmid pBR322TetSfi-Sfi, a plasmid (pBR322-Sfi-2BLA-Sfi) with a tandem repeat of the Bla gene was obtained. (See FIG. 10).
  • The plasmid was electroporated into E. coli cells. The cells were plated on solid agar plates containing 15 μg/ml tetracycline. Those colonies which grew, were then plated on solid agar plates containing 100 μg/ml ampicillin and the number of viable colonies counted. The Bla inserts in those transformants which exhibited ampicillin resistance were amplified by standard PCR techniques using the method and primers described in Example 8. The presence of a 1 kb insert indicated that the duplicate genes had recombined, as indicated in Table 6.
  • TABLE 6
    Cell Tet Colonies Amp Colonies Colony PCR
    TG-1 28 54 7/7 at 1 kb
    JC8679
    149 117 3/3 at 1 kb
    vector
    51 0
    control

    Colony PCR confirmed that the tandem repeat was efficiently recombined to form a single recombinant gene.
  • Example 10 Multiple Cycles of Direct Repeat Recombination-Interplasmidic
  • In order to determine whether multiple cycles of recombination could be used to produce resistant cells more quickly, multiple cycles of the method described in Example 9 were performed.
  • The minus recombination control consisted of a single copy of the betalactamase gene, whereas the plus recombination experiment consisted of inserting two copies of betalactamase as a direct repeat. The tetracycline marker was used to equalize the number of colonies that were selected for cefotaxime resistance in each round, to compensate for ligation efficiencies.
  • In the first round, pBR322TetSfi-Bla-Sfi was digested with EcrI and subject to PCR with a 1:1 mix (1 ml) of normal and Cadwell PCR mix (Cadwell and Joyce (1992) PCR Methods and Applications 2: 28-33) for error prone PCR. The PCR program was 70° C. for 2 minutes initially and then 30 cycles of 94° C. for 30 seconds, 52° C. for 30 second and 72° C. for 3 minutes and 6 seconds per cycle, followed by 72° C. for 10 minutes.
  • The primers used in the PCR reaction to create the one Bla gene control plasmid were Primer 2650 (SEQ ID NO: 50) and Primer 2719 (SEQ ID NO: 55) 5′ TTAAGGGATTTTGGTCATGAGATT 3′. This resulted in a mixed population of amplified DNA fragments, designated collectively as Fragment #59. These fragments had a number of different mutations.
  • The primers used in two different PCR reactions to create the two Bla gene plasmid were Primer 2650 (SEQ ID NO: 50) and Primer 2649 (SEQ ID NO: 51) for the first gene and Primers 2648 (SEQ ID NO: 54) and Primer 2719 (SEQ ID NO: 55) for the second gene. This resulted in a mixed population of each of the two amplified DNA fragments: Fragment #89 (amplified with primers 2648 and 2719) and Fragment #90 (amplified with primers 2650 and 2649). In each case a number of different mutations had been introduced the mixed population of each of the fragments.
  • After error prone PCR, the population of amplified DNA fragment #59 was digested with Sfi1, and then cloned into pBR322TetSfi-Sfi to create a mixed population of the plasmid pBR322Sfi-Bla-Sfi1.
  • After error prone PCR, the population of amplified DNA fragments #90 and #89 was digested with SfiI and BstXI at 50° C., and ligated into pBR322TetSfi-Sfi to create a mixed population of the plasmid pBR322TetSfi-2Bla-Sfi1 (FIG. 10).
  • The plasmids pBR322Sfi-Bla-Sfi1 and pBR322Sfi-2Bla-Sfi1 were electroporated into E. coli JC8679 and placed on agar plates having differing concentrations of cefotaxime to select for resistant strains and on tetracycline plates to titre.
  • An equal number of colonies (based on the number of colonies growing on tetracycline) were picked, grown in LB-tet and DNA extracted from the colonies. This was one round of the recombination. This DNA was digested with EcrI and used for a second round of error-prone PCR as described above.
  • After five rounds the MIC (minimum inhibitory concentration) for cefotaxime for the one fragment plasmid was 0.32 whereas the MIC for the two fragment plasmid was 1.28. The results show that after five cycles the resistance obtained with recombination was four-fold higher in the presence of in vivo recombination.
  • Example 11 In Vivo Recombination Via Electroporation of Fragments
  • Competent E. coli cells containing pUC18Sfi-Bla-Sfi were prepared as described. Plasmid pUC18Sfi-Bla-Sfi contains the standard TEM-1 beta-lactamase gene as described, supra.
  • A TEM-1 derived cefotaxime resistance gene from pUC18Sfi-cef-Sfi, (clone ST2) (Stemmer WPC (1994) Nature 370: 389-91, incorporated herein by reference) which confers on E. coli carrying the plasmid an MIC of 640 μg/ml for cefotaxime, was obtained. In one experiment the complete plasmid pUC18Sfi-cef-Sfi DNA was electroporated into E. coli cells having the plasmid pUC18Sfi-Bla-Sfi.
  • In another experiment the DNA fragment containing the cefotaxime gene from pUC18Sfi-cef-Sfi was amplified by PCR using the primers 2650 (SEQ ID NO: 50) and 2719 (SEQ ID NO: 55). The resulting 1 kb PCR product was digested into DNA fragments of <100 bp by DNase and these fragments were electroporated into the competent E. coli cells which already contained pUC18Sfi-Bla-Sfi.
  • The transformed cells from both experiments were then assayed for their resistance to cefotaxime by plating the transformed cells onto agar plates having varying concentrations of cefotaxime. The results are indicated in Table 7.
  • TABLE 7
    Colonies/Cefotaxime Concentration
    0.16 0.32 1.28 5.0 10.0
    no DNA control 14
    ST-2 mutant, whole 4000 2000 800 400
    ST-2 mutant, fragments 1000 120 22 7
    Wildtype, whole 27
    Wildtype, fragments 18
  • From the results it appears that the whole ST-2 Cef gene was inserted into either the bacterial genome or the plasmid after electroporation. Because most insertions are homologous, it is expected that the gene was inserted into the plasmid, replacing the wildtype gene. The fragments of the Cef gene from St-2 also inserted efficiently into the wild-type gene in the plasmid. No sharp increase in cefotaxime resistance was observed with the introduction of the wildtype gene (whole or in fragments) and no DNA. Therefore, the ST-2 fragments were shown to yield much greater cefotaxime resistance than the wild-type fragments.
  • It was contemplated that repeated insertions of fragments, prepared from increasing resistant gene pools would lead to increasing resistance.
  • Accordingly, those colonies that produced increased cefotaxime resistance with the St-2 gene fragments were isolated and the plasmid DNA extracted. This DNA was amplified using PCR by the method described above. The amplified DNA was digested with DNase into fragments (<100 bp) and 2-4 μg of the fragments were electroporated into competent E. coli cells already containing pUC322Sfi-Bla-Sfi as described above. The transformed cells were plated on agar containing varying concentrations of cefotaxime.
  • As a control, competent E. coli cells having the plasmid pUC18Sfi-Kan-Sfi were also used. DNA fragments from the digestion of the PCR product of pUC18Sfi-cef-Sfi were electroporated into these cells. There is no homology between the kanamycin gene and the beta-lactamase gene and thus recombination should not occur.
  • This experiment was repeated for 2 rounds and the results are shown in Table 8.
  • TABLE 8
    Cef resistant
    Round Cef conc. KAN control colonies
    1 0.16-0.64 lawn lawn
    replate 0.32 10 small 1000
    2 10 10 400
    Replate 100 sm @ 2.5 50 @ 10
    3 40 100 sm
    1280 100 sm
  • Example 12 Determination of Recombination Formats
  • This experiment was designed to determine which format of recombination generated the most recombinants per cycle.
  • In the first approach, the vector PUC18Sfi-Bla-Sfi was amplified with PCR primers to generate a large and small fragment. The large fragment had the plasmid and ends having portions of the Bla gene, and the small fragment coded for the middle of the Bla gene. A third fragment having the complete Bla gene was created using PCR by the method in Example 8. The larger plasmid fragment and the fragment containing the complete Bla gene were electroporated into E. coli JC8679 cells at the same time by the method described above and the transformants plated on differing concentrations of cefotaxime.
  • In approach 2, the vector PUC18Sfi-Bla-Sfi was amplified to produce the large plasmid fragment isolated as in approach 1 above. The two fragments each comprising a portion of the complete Bla gene, such that the two fragments together spanned the complete Bla gene were also obtained by PCR. The large plasmid fragment and the two Bla gene fragments were all electroporated into competent E. coli JC8679 cells and the transformants plated on varying concentrations of cefotaxime.
  • In the third approach, both the vector and the plasmid were electroporated into E. coli JC8679 cells and the transformants were plated on varying concentrations of cefotaxime.
  • In the fourth approach, the complete Bla gene was electroporated into E. coli JC8679 cells already containing the vector pUCSfi-Sfi and the transformants were plated on varying concentrations of cefotaxime. As controls, the E. coli JC8679 cells were electroporated with either the complete Bla gene or the vector alone.
  • The results are presented in FIG. 11. The efficiency of the insertion of two fragments into the vector is 100× lower than when one fragment having the complete Bla gene is used. Approach 3 indicated that the efficiency of insertion does depend on the presence of free DNA ends since no recombinants were obtained with this approach. However, the results of approach 3 were also due to the low efficiency of electroporation of the vector. When the expression vector is already in the competent cells, the efficiency of the vector electroporation is not longer a factor and efficient homologous recombination can be achieved even with uncut vector.
  • Example 12 Kit for Cassette Shuffling to Optimize Vector Performance
  • In order to provide a vector capable of conferring an optimized phenotype (e.g., maximal expression of a vector-encoded sequence, such as a cloned gene), a kit is provided comprising a variety of cassettes which can be shuffled, and optimized shufflants can be selected. FIG. 12 shows schematically one embodiment, with each loci having a plurality of cassettes. For example, in a bacterial expression system, FIG. 13 shows example cassettes that are used at the respective loci. Each cassette of a given locus (e.g., all promoters in this example) are flanked by substantially identical sequences capable of overlapping the flanking sequence(s) of cassettes of an adjacent locus and preferably also capable of participating in homologous recombination or non-homologous recombination (e.g., lox/cre or flp/frt systems), so as to afford shuffling of cassettes within a locus but substantially not between loci.
  • Cassettes are supplied in the kit as PCR fragments, which each cassette type or individual cassette species packaged in a separate tube. Vector libraries are created by combining the contents of tubes to assemble whole plasmids or substantial portions thereof by hybridization of the overlapping flanking sequences of cassettes at each locus with cassettes at the adjacent loci. The assembled vector is ligated to a predetermined gene of interest to form a vector library wherein each library member comprises the predetermined gene of interest and a combination of cassettes determined by the association of cassettes. The vectors are transferred into a suitable host cell and the cells are cultured under conditions suitable for expression, and the desired phenotype is selected.
  • While the present invention has been described with reference to what are considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
  • REFERENCES
  • The following references are cited in this application at the relevant portion of the application.
    • 1. Holland, J. H. (1992) Sci. Am. July, 66-72.
    • 2. Holland, J. H. (1992) “Adaptation in natural and artificial systems”. Second edition, MIT Press, Cambridge.
    • 3. Joyce, G. F. (1992) Scientific American, December, 90-97.
    • 4. Kauffman, S. A. (1993) “The origins of order”. Oxford University Press, New York.
    • 5. Stormo, G. D. (1991) Methods Enzymol. 208:458-468.
    • 6. Schneider, T. D. et al., (1986) J. Mol. Biol. 188:415-431.
    • 7. Reidhaar-Olson, J. F and Sauer, R. T. (1988) Science 241:53-57.
    • 8. Stemmer, W. P. C. et al., (1992) Biotechniques 14:256-265.
    • 9. Yockey, H. P. (1977) J. Theor. Biol. 67:345-376.
    • 10. Yockey, H. P. (1974) J. Theor. Biol. 46:369-380.
    • 11. Leung, D. W. et al., (1989) Technique 1:11-15.
    • 12. Caldwell, R. C. and Joyce, G. F. (1992) PCR Methods and Applications 2:28-33.
    • 13. Bartel, D. P., and Szostak, J. W. (1993) Science 261:1411-1418.
    • 14. Bock, L. C. et al., (1992) Nature 355:564-566.
    • 15. Scott, J. K. and Smith, G. P. (1990) Science 249:386-390.
    • 16. Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382.
    • 17. McCafferty, J. et al. (1990) Nature 348:552-554.
    • 18. Cull, M. G. et al., (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869.
    • 19. Gramm, H. et al., (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580.
    • 20. Arkin, A. and Youvan, D. C. (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815.
    • 21. Oliphant, A. R. et al., (1986) Gene 44:177-183.
    • 22. Hermes, J. D. et al., (1990) Proc. Natl. Acad. Sci. USA 87:696-700.
    • 23. Meyerhans, A. et al., (1990) Nucleic Acids Res. 18:1687-1691.
    • 24. Osterhout, J. J. et al., (1992) J. Am. Chem. Soc. 114:331-337.
    • 25. Cano, R. J. et al., (1993) Nature 363:536-538.
    • 26. Palzkill and Botstein, (1992) J. Bacteriol. 174:5237-5243.
    • 27. Marton et al., Nucleic Acids Res. 19:2423.
    • 28. Yanish-Perron et al., [1985] Gene 33:103-119.
    • 29. Watson (1988) Gene 70:399-403.
    • 30. Ambler et al. (1991) Biochem J. 276:269-272.
    • 31. Chen and Clowes, (1984) Nucleic Acid Res. 12:3219-3234.
    • 32. Witholt, B. ([1987] Anal. Biochem. 164(2):320-330
    • 33. Kabat et al., (1991) “Sequences of Proteins of Immunological Interest” U.S. Department of Health and Human Services, NIH Publication 91-3242.
    • 34. Philippon et al., (1989) Antimicrob Agents Chemother 33:1131-1136.
    • 35. Jacoby and Medeiros (1991) Antimicrob. Agents Chemother. 35:167-1704.
    • 36. Coelhosampaio (1993) Biochem. 32:10929-10935
    • 37. Tuerk, C. et al., (1992) Proc. Natl. Acad. Sci. USA 89:6988-6992.
    • 38. U.S. Pat. No. 4,683,195
    • 39. U.S. Pat. No. 4,683,202
    • 40. Delagrave et al. (1993) Protein Engineering 6: 327-331
    • 41. Delgrave et al. (1993) Bio/Technology 11: 1548-1552
    • 42. Goldman, E R and Youvan D C (1992) Bio/Technology 10:1557-1561
    • 43. Nissim et al. (1994) EMBO J. 13: 692-698
    • 44. Winter et al. (1994) Ann. Rev. Immunol. 12: 433-55
    • 45. Caren et al. (1994) Bio/Technology 12: 517-520
    • 46. Calogero et al. (1992) FEMS Microbiology Lett. 97: 41-44
    • 47. Galizzi et al. WO91/01087
    • 48. Hayashi et al. (1994) Biotechniques 17: 310-315
    • 49. Radman et al. WO90/07576
  • All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims (32)

1. A method for introducing one or more mutations into a template double-stranded polynucleotide, wherein the template double-stranded polynucleotide has been cleaved into double-stranded random fragments of a desired size, comprising:
a) adding to the resultant population of double-stranded fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the template polynucleotide;
b) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments;
c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at regions of identity between the single-stranded fragments and formation of a mutagenized double-stranded polynucleotide; and
d) repeating steps (b) and (c).
2. The method of claim 1 wherein the concentration of a specific double-stranded fragment in the mixture of double-stranded fragments is less than 1% by weight of the total DNA.
3. The method of claim 1 wherein the number of different specific double-stranded fragments comprises at least about 100.
4. The method of claim 1 wherein the size of the double-stranded fragments is from about 5 bp to 5 kb.
5. The method of claim 1 wherein the size of the mutagenized double-stranded polynucleotide comprises from 50 bp to 100 kb.
6. A method of producing recombinant proteins having biological activity comprising:
a) treating a sample comprising double-stranded template polynucleotides encoding a wild-type protein under conditions which provide for the cleavage of said template polynucleotides into random double-stranded fragments having a desired size;
b) adding to the resultant population of random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise areas of identity and areas of heterology to the template polynucleotide;
c) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments;
d) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at the areas of identity and formation of a mutagenized double-stranded polynucleotide;
e) repeating steps (c) and (d); and
f) expressing the recombinant protein from the mutagenized double-stranded polynucleotide.
7. The method of claim 6 wherein the concentration of a specific double-stranded fragment in the mixture of double-stranded fragments in step (a) is less than 1% by weight of the total DNA.
8. The method of claim 6 where the number of different specific double-stranded fragments in step (a) comprises at least about 100.
9. The method of claim 6 wherein the size of the double-stranded fragments is from about 5 bp to 5 kb.
10. The method of claim 6 wherein the size of the mutagenized double-stranded polynucleotide comprises from 50 bp to 100 kb.
11. The method of claim 6 further comprising selecting the desired recombinant protein from the population of recombinant proteins.
12. A method for obtaining a chimeric polynucleotide comprising:
a) treating a sample comprising different double-stranded template polynucleotides wherein said different template polynucleotides contain areas of identity and areas of heterology under conditions which provide for the cleavage of said template polynucleotides into random double-stranded fragments of a desired size;
b) denaturing the resultant random double-stranded template fragments contained in the treated sample produced by step (a) into single-stranded fragments;
c) incubating the resultant single-stranded fragments with polymerase under conditions which provide for the annealing of the target single-stranded fragments at the areas of identity and the formation of a chimeric double-stranded polynucleotide sequence comprising template polynucleotide sequences; and
d) repeating steps (b) and (c) as desired.
13. The method of claim 12 wherein the concentration of a specific double-stranded fragment in the mixture of double-stranded fragments in step (a) is less than 1% by weight of the total DNA.
14. The method of claim 12 where the number of different specific double-stranded fragments in step (a) comprises at least about 100.
15. The method of claim 12 wherein the size of the double-stranded fragments is from about 5 bp to 5 kb.
16. The method of claim 12 wherein the size of the mutagenized double-stranded polynucleotide comprises from 50 bp to 100 kb.
17. A method of replicating a template polynucleotide which method comprises combining in vitro single-stranded template polynucleotides with small random single-stranded fragments resulting from the cleavage and denaturation of the template polynucleotide, and incubating said mixture of nucleic acid fragments in the presence of a nucleic acid polymerase under conditions wherein a population of double-stranded template polynucleotides is formed.
18. A method for generating libraries of displayed peptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening, the method comprising:
(1) obtaining a first plurality of selected library members comprising a displayed peptide or displayed antibody and an associated polynucleotide encoding said displayed peptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequence, and
(2) pooling and fragmenting said associated polynucleotides or copies to form fragments thereof under conditions suitable for PCR amplification, performing PCR amplification, and thereby homologously recombining said fragments to form a shuffled pool of recombined polynucleotides, whereby a substantial fraction of the recombined polynucleotides of said shuffled pool are not present in the first plurality of selected library members.
19. The method of claim 18, further comprising introducing mutations into said polynucleotides or copies.
20. The method of claim 19, wherein the mutations are introduced by performing PCR amplification.
21. The method of claim 20, wherein the PCR amplification is error-prone PCR.
22. The method of claim 18, comprising the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind with a predetermined macromolecule.
23. The method of claim 18, wherein the first plurality of selected library members is obtained by selecting for a phenotypic characteristic other than binding affinity for a predetermined molecule.
24. The method of claim 18, wherein the first plurality of selected library members is pooled and fragmented and homologously recombined by PCR in vitro.
25. The method of claim 18, wherein the first plurality of selected library members is pooled and fragmented in vitro, the resultant fragments transferred into a host cell or organism and homologously recombined to form shuffled library members in vivo.
26. The method of claim 18, wherein the first plurality of selected library members is cloned or amplified on episomally replicable vectors, a multiplicity of said vectors is transferred into a cell and homologously recombined to form shuffled library members in vivo.
27. A method for generating libraries of displayed peptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening, the method comprising:
(1) obtaining a first plurality of selected library members comprising a displayed peptide or displayed antibody and an associated polynucleotide encoding said displayed peptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequence, and
(2) cloning or amplifying said associated polynucleotides or copies on episomally replicable vectors and transferring a multiplicity of said vectors into a cell and homologously recombined to form shuffled library members in vivo.
28. The method of claim 27, further comprising introducing mutations into said polynucleotides or copies thereof.
29. The method of claim 27, wherein said episomally replicable vectors comprise a direct repeat of a plurality of associated polynucleotides or copies thereof.
30. A method for generating libraries of displayed antibodies suitable for affinity interaction screening, the method comprising:
(1) obtaining a first plurality of selected library members comprising a displayed antibody and an associated polynucleotide encoding said displayed antibody, and obtaining said associated polynucleotides or copies thereof, wherein said associated polynucleotides comprise a region of substantially identical variable region framework sequence, and
(2) pooling and fragmenting said associated polynucleotides or copies to form fragments thereof under conditions suitable for PCR amplification, performing PCR amplification, and thereby homologously recombining said fragments to form a shuffled pool of recombined polynucleotides comprising novel combinations of CDRs, whereby a substantial fraction of the recombined polynucleotides of said shuffled pool comprise CDR combinations are not present in the first plurality of selected library members.
31. The method of claim 30, comprising the additional step wherein the shuffled pool is subjected to affinity screening to select shuffled library members which bind to a predetermined epitope and thereby selecting a plurality of selected shuffled library members.
32. The method of claim 31, comprising the additional step of shuffling the plurality of selected shuffled library members and screening, from 1 to about 1000 cycles.
US11/843,351 1994-02-17 2007-08-22 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination Abandoned US20080261833A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US11/843,351 US20080261833A1 (en) 1994-02-17 2007-08-22 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US12/557,519 US7981614B2 (en) 1994-02-17 2009-09-11 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US08/198,431 US5605793A (en) 1994-02-17 1994-02-17 Methods for in vitro recombination
US08/537,874 US5830721A (en) 1994-02-17 1995-02-17 DNA mutagenesis by random fragmentation and reassembly
PCT/US1995/002126 WO1995022625A1 (en) 1994-02-17 1995-02-17 Dna mutagenesis by random fragmentation and reassembly
US09/100,856 US6132970A (en) 1994-02-17 1998-06-19 Methods of shuffling polynucleotides
US23116199A 1999-01-15 1999-01-15
US09/713,920 US6602986B1 (en) 1994-02-17 2000-11-15 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US11/843,351 US20080261833A1 (en) 1994-02-17 2007-08-22 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
US09/713,920 Continuation US6602986B1 (en) 1994-02-17 2000-11-15 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US10/623,036 Continuation US7288375B2 (en) 1994-02-17 2003-07-18 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US11/405,038 Continuation US20070092887A1 (en) 1994-02-17 2006-04-14 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/557,519 Continuation US7981614B2 (en) 1994-02-17 2009-09-11 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Publications (1)

Publication Number Publication Date
US20080261833A1 true US20080261833A1 (en) 2008-10-23

Family

ID=22733354

Family Applications (14)

Application Number Title Priority Date Filing Date
US08/198,431 Expired - Lifetime US5605793A (en) 1994-02-17 1994-02-17 Methods for in vitro recombination
US08/537,874 Expired - Lifetime US5830721A (en) 1994-02-17 1995-02-17 DNA mutagenesis by random fragmentation and reassembly
US08/564,955 Expired - Lifetime US5811238A (en) 1994-02-17 1995-11-30 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/100,856 Expired - Lifetime US6132970A (en) 1994-02-17 1998-06-19 Methods of shuffling polynucleotides
US09/133,508 Expired - Lifetime US6287861B1 (en) 1994-02-17 1998-08-12 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/232,863 Expired - Lifetime US6277638B1 (en) 1994-02-17 1999-01-15 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/231,253 Expired - Fee Related US6420175B1 (en) 1994-02-17 1999-01-15 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/501,698 Expired - Lifetime US6297053B1 (en) 1994-02-17 2000-02-10 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/713,920 Expired - Fee Related US6602986B1 (en) 1994-02-17 2000-11-15 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/718,764 Expired - Fee Related US6576467B1 (en) 1994-02-17 2000-11-21 Methods for producing recombined antibodies
US09/724,958 Expired - Fee Related US6444468B1 (en) 1994-02-17 2000-11-28 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US10/623,036 Expired - Fee Related US7288375B2 (en) 1994-02-17 2003-07-18 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US11/843,351 Abandoned US20080261833A1 (en) 1994-02-17 2007-08-22 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US12/557,519 Expired - Fee Related US7981614B2 (en) 1994-02-17 2009-09-11 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Family Applications Before (12)

Application Number Title Priority Date Filing Date
US08/198,431 Expired - Lifetime US5605793A (en) 1994-02-17 1994-02-17 Methods for in vitro recombination
US08/537,874 Expired - Lifetime US5830721A (en) 1994-02-17 1995-02-17 DNA mutagenesis by random fragmentation and reassembly
US08/564,955 Expired - Lifetime US5811238A (en) 1994-02-17 1995-11-30 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/100,856 Expired - Lifetime US6132970A (en) 1994-02-17 1998-06-19 Methods of shuffling polynucleotides
US09/133,508 Expired - Lifetime US6287861B1 (en) 1994-02-17 1998-08-12 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/232,863 Expired - Lifetime US6277638B1 (en) 1994-02-17 1999-01-15 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/231,253 Expired - Fee Related US6420175B1 (en) 1994-02-17 1999-01-15 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/501,698 Expired - Lifetime US6297053B1 (en) 1994-02-17 2000-02-10 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/713,920 Expired - Fee Related US6602986B1 (en) 1994-02-17 2000-11-15 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US09/718,764 Expired - Fee Related US6576467B1 (en) 1994-02-17 2000-11-21 Methods for producing recombined antibodies
US09/724,958 Expired - Fee Related US6444468B1 (en) 1994-02-17 2000-11-28 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US10/623,036 Expired - Fee Related US7288375B2 (en) 1994-02-17 2003-07-18 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Family Applications After (1)

Application Number Title Priority Date Filing Date
US12/557,519 Expired - Fee Related US7981614B2 (en) 1994-02-17 2009-09-11 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Country Status (12)

Country Link
US (14) US5605793A (en)
EP (4) EP1205547A3 (en)
JP (6) JPH10500561A (en)
CN (1) CN1145641A (en)
AT (2) ATE216722T1 (en)
AU (3) AU703264C (en)
CA (1) CA2182393C (en)
DE (5) DE69526497T2 (en)
DK (2) DK0934999T3 (en)
ES (2) ES2165652T3 (en)
RU (1) RU2157851C2 (en)
WO (1) WO1995022625A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8168380B2 (en) 1997-02-12 2012-05-01 Life Technologies Corporation Methods and products for analyzing polymers
US8202831B2 (en) 2008-06-06 2012-06-19 The Procter & Gamble Company Detergent composition comprising a variant of a family 44 xyloglucanase
US8314216B2 (en) 2000-12-01 2012-11-20 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US8603792B2 (en) 2009-03-27 2013-12-10 Life Technologies Corporation Conjugates of biomolecules to nanoparticles

Families Citing this family (2267)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69132905T2 (en) * 1990-12-06 2002-08-01 Affymetrix Inc N D Ges D Staat Detection of nucleic acid sequences
US6150141A (en) * 1993-09-10 2000-11-21 Trustees Of Boston University Intron-mediated recombinant techniques and reagents
US6309883B1 (en) * 1994-02-17 2001-10-30 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
US5837458A (en) * 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US20060257890A1 (en) * 1996-05-20 2006-11-16 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US6117679A (en) * 1994-02-17 2000-09-12 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6335160B1 (en) * 1995-02-17 2002-01-01 Maxygen, Inc. Methods and compositions for polypeptide engineering
US6395547B1 (en) * 1994-02-17 2002-05-28 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6406855B1 (en) * 1994-02-17 2002-06-18 Maxygen, Inc. Methods and compositions for polypeptide engineering
US6165793A (en) 1996-03-25 2000-12-26 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6995017B1 (en) 1994-02-17 2006-02-07 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6808904B2 (en) * 1994-06-16 2004-10-26 Syngenta Participations Ag Herbicide-tolerant protox genes produced by DNA shuffling
US7262055B2 (en) 1998-08-25 2007-08-28 Gendaq Limited Regulated gene expression in plants
GB9824544D0 (en) 1998-11-09 1999-01-06 Medical Res Council Screening system
US7285416B2 (en) * 2000-01-24 2007-10-23 Gendaq Limited Regulated gene expression in plants
USRE39229E1 (en) 1994-08-20 2006-08-08 Gendaq Limited Binding proteins for recognition of DNA
US6063562A (en) * 1994-09-16 2000-05-16 Sepracor, Inc. In vitro method for predicting the evolutionary response of HIV protease to a drug targeted thereagainst
US7820798B2 (en) * 1994-11-07 2010-10-26 Human Genome Sciences, Inc. Tumor necrosis factor-gamma
US7597886B2 (en) * 1994-11-07 2009-10-06 Human Genome Sciences, Inc. Tumor necrosis factor-gamma
WO1996017932A1 (en) * 1994-12-09 1996-06-13 Wakunaga Seiyaku Kabushiki Kaisha Method of inhibiting non specific hybridization in primer extension
US20010053523A1 (en) * 1995-06-02 2001-12-20 M&E Biotech A/S. Method for identification of biologically active peptides and nucleic acids
US6936289B2 (en) 1995-06-07 2005-08-30 Danisco A/S Method of improving the properties of a flour dough, a flour dough improving composition and improved food products
US6964861B1 (en) * 1998-11-13 2005-11-15 Invitrogen Corporation Enhanced in vitro recombinational cloning of using ribosomal proteins
US6143557A (en) * 1995-06-07 2000-11-07 Life Technologies, Inc. Recombination cloning using engineered recombination sites
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6495357B1 (en) * 1995-07-14 2002-12-17 Novozyme A/S Lipolytic enzymes
US6004788A (en) * 1995-07-18 1999-12-21 Diversa Corporation Enzyme kits and libraries
US5958672A (en) 1995-07-18 1999-09-28 Diversa Corporation Protein activity screening of clones having DNA from uncultivated microorganisms
US6455254B1 (en) 1995-07-18 2002-09-24 Diversa Corporation Sequence based screening
US6057103A (en) 1995-07-18 2000-05-02 Diversa Corporation Screening for novel bioactivities
ATE216427T1 (en) * 1995-08-11 2002-05-15 Novozymes As METHOD FOR PRODUCING POLYPEPTIDE DERIVATIVES
DE69621940T2 (en) 1995-08-18 2003-01-16 Morphosys Ag PROTEIN - / (POLY) PEPTIDE LIBRARIES
US6352842B1 (en) 1995-12-07 2002-03-05 Diversa Corporation Exonucease-mediated gene assembly in directed evolution
GB9621295D0 (en) * 1995-12-07 1996-11-27 Cambridge Antibody Tech Specific binding members,materials and methods
US6939689B2 (en) 1995-12-07 2005-09-06 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
US5965408A (en) 1996-07-09 1999-10-12 Diversa Corporation Method of DNA reassembly by interrupting synthesis
US6790605B1 (en) * 1995-12-07 2004-09-14 Diversa Corporation Methods for obtaining a desired bioactivity or biomolecule using DNA libraries from an environmental source
US6764835B2 (en) * 1995-12-07 2004-07-20 Diversa Corporation Saturation mutageneis in directed evolution
US20030219752A1 (en) * 1995-12-07 2003-11-27 Diversa Corporation Novel antigen binding molecules for therapeutic, diagnostic, prophylactic, enzymatic, industrial, and agricultural applications, and methods for generating and screening thereof
US6537776B1 (en) 1999-06-14 2003-03-25 Diversa Corporation Synthetic ligation reassembly in directed evolution
US6713279B1 (en) 1995-12-07 2004-03-30 Diversa Corporation Non-stochastic generation of genetic vaccines and enzymes
US6358709B1 (en) 1995-12-07 2002-03-19 Diversa Corporation End selection in directed evolution
US7018793B1 (en) * 1995-12-07 2006-03-28 Diversa Corporation Combinatorial screening of mixed populations of organisms
US20020164580A1 (en) * 1995-12-07 2002-11-07 Diversa Corporation Combinatorial screening of mixed populations of organisms
US20020028443A1 (en) * 1999-09-27 2002-03-07 Jay M. Short Method of dna shuffling with polynucleotides produced by blocking or interrupting a synthesis or amplification process
US6489145B1 (en) 1996-07-09 2002-12-03 Diversa Corporation Method of DNA shuffling
US5830696A (en) 1996-12-05 1998-11-03 Diversa Corporation Directed evolution of thermophilic enzymes
US20020031771A1 (en) * 1995-12-07 2002-03-14 Short Jay M. Sequence based screening
US6740506B2 (en) 1995-12-07 2004-05-25 Diversa Corporation End selection in directed evolution
US5939250A (en) * 1995-12-07 1999-08-17 Diversa Corporation Production of enzymes having desired activities by mutagenesis
US6238884B1 (en) 1995-12-07 2001-05-29 Diversa Corporation End selection in directed evolution
US6361974B1 (en) * 1995-12-07 2002-03-26 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
US6344328B1 (en) 1995-12-07 2002-02-05 Diversa Corporation Method for screening for enzyme activity
US7888466B2 (en) 1996-01-11 2011-02-15 Human Genome Sciences, Inc. Human G-protein chemokine receptor HSATU68
US5976846A (en) * 1996-01-13 1999-11-02 Passmore; Steven E. Method for multifragment in vivo cloning and mutation mapping
AU1874997A (en) * 1996-02-15 1997-09-02 Pangenetics B.V. Molecules for the induction of immunological tolerance
US6506602B1 (en) 1996-03-25 2003-01-14 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6096548A (en) 1996-03-25 2000-08-01 Maxygen, Inc. Method for directing evolution of a virus
US6372480B1 (en) 1996-04-19 2002-04-16 Mycogen Corporation Pesticidal proteins
US6242211B1 (en) 1996-04-24 2001-06-05 Terragen Discovery, Inc. Methods for generating and screening novel metabolic pathways
US7465571B1 (en) 1996-05-22 2008-12-16 Verenium Corporation Endoglucanases
US20020120118A1 (en) * 1996-05-22 2002-08-29 Short Jay M. Enzymes having endoglucanase activity and methods of use thereof
US6617434B1 (en) * 1996-05-31 2003-09-09 North Shore Long Island Jewish Research Institute Identificiaton of differentially methylated and mutated nucleic acids
US5871917A (en) 1996-05-31 1999-02-16 North Shore University Hospital Research Corp. Identification of differentially methylated and mutated nucleic acids
AU4113797A (en) 1996-07-10 1998-02-02 Lonza A.G. Method of preparing (s) - or (r) -3,3,3-trifluoro-2-hydroxy-2- methylpropionic acid
JPH1066576A (en) * 1996-08-07 1998-03-10 Novo Nordisk As Double-stranded dna having protruding terminal and shuffling method using the same
JPH1066577A (en) * 1996-08-07 1998-03-10 Novo Nordisk As Nucleic acid pool and its production
GB9618960D0 (en) 1996-09-11 1996-10-23 Medical Science Sys Inc Proteases
EP0963434A4 (en) * 1996-09-27 2000-10-25 Maxygen Inc Methods for optimization of gene therapy by recursive sequence shuffling and selection
US5811381A (en) * 1996-10-10 1998-09-22 Mark A. Emalfarb Cellulase compositions and methods of use
US7883872B2 (en) * 1996-10-10 2011-02-08 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US6838238B1 (en) * 1996-10-17 2005-01-04 Invitrogen Corporation Morphatides: novel shape and structure libraries
US6242669B1 (en) 1996-10-30 2001-06-05 Mycogen Corporation Pesticidal toxins and nucleotide sequences which encode these toxins
IL119586A (en) * 1996-11-07 2001-09-13 Univ Ramot Discontinuous library of a single biological unit and a method for its preparation
US20070009930A1 (en) * 1996-12-18 2007-01-11 Maxygen, Inc. Methods and compositions for polypeptide engineering
WO1998031816A1 (en) 1997-01-17 1998-07-23 Regents Of The University Of Minnesota Dna molecules and protein displaying improved triazine compound degrading ability
AU1022402A (en) * 1997-01-17 2002-03-14 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US7148054B2 (en) * 1997-01-17 2006-12-12 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
EP1007732B1 (en) * 1997-01-17 2006-07-26 Maxygen, Inc. EVOLUTION OF procaryotic WHOLE CELLS BY RECURSIVE SEQUENCE RECOMBINATION
US6326204B1 (en) 1997-01-17 2001-12-04 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US8207093B2 (en) * 1997-01-21 2012-06-26 The General Hospital Corporation Selection of proteins using RNA-protein fusions
US6261804B1 (en) * 1997-01-21 2001-07-17 The General Hospital Corporation Selection of proteins using RNA-protein fusions
ES2373110T3 (en) 1997-01-21 2012-01-31 The General Hospital Corporation SELECTION OF PROTEINS USING ARN-PROTEIN FUSIONS.
GB9701425D0 (en) * 1997-01-24 1997-03-12 Bioinvent Int Ab A method for in vitro molecular evolution of protein function
EP1302540A3 (en) * 1997-01-31 2003-11-05 Cosmix Molecular Biologicals GmbH Generation of diversity in combinatorial libraries
US5851808A (en) 1997-02-28 1998-12-22 Baylor College Of Medicine Rapid subcloning using site-specific recombination
AU6443298A (en) 1997-02-28 1998-09-18 Nature Technology Corporation Self-assembling genes, vectors and uses thereof
DE69840382D1 (en) * 1997-03-18 2009-02-05 Novozymes As METHOD FOR THE PRODUCTION OF A LIBRARY THROUGH DNA SHUFFLING
DK1015575T3 (en) * 1997-03-18 2010-08-23 Novozymes As Shuffling of heterologous DNA sequences
US6159687A (en) * 1997-03-18 2000-12-12 Novo Nordisk A/S Methods for generating recombined polynucleotides
US6159688A (en) 1997-03-18 2000-12-12 Novo Nordisk A/S Methods of producing polynucleotide variants
US6291165B1 (en) * 1997-03-18 2001-09-18 Novo Nordisk A/S Shuffling of heterologous DNA sequences
US5948653A (en) * 1997-03-21 1999-09-07 Pati; Sushma Sequence alterations using homologous recombination
US6153410A (en) * 1997-03-25 2000-11-28 California Institute Of Technology Recombination of polynucleotide sequences using random or defined primers
JP2002514919A (en) * 1997-04-04 2002-05-21 バイオサイト ダイアグノスティックス,インコーポレイテッド Multivalent and polyclonal libraries
JP2002510966A (en) 1997-04-11 2002-04-09 カリフォルニア・インスティテュート・オブ・テクノロジー Apparatus and method for automatic protein design
IL124275A (en) * 1997-05-02 2002-03-10 Bio Merieux Vitek Inc Method for generating nucleic acid sequences
US6558901B1 (en) * 1997-05-02 2003-05-06 Biomerieux Vitek Nucleic acid assays
GB9712512D0 (en) 1997-06-16 1997-08-20 Bioinvent Int Ab A method for in vitro molecular evolution of protein function
US7087420B1 (en) 1997-07-17 2006-08-08 Cambia Microbial β-glucuronidase genes, gene products and uses thereof
US6391547B1 (en) * 1997-09-09 2002-05-21 Center For The Application Of Molecular Biology To International Agriculture Microbial β-glucuronidase genes, gene products and uses thereof
DE19731990A1 (en) * 1997-07-25 1999-01-28 Studiengesellschaft Kohle Mbh Process for the production and identification of new hydrolases with improved properties
WO1999011818A1 (en) * 1997-08-28 1999-03-11 Isao Karube Method for detecting highly functional polypeptides or nucleic acids
AU754312C (en) * 1997-09-19 2005-11-10 Promega Corporation Thermostable luciferases and methods of production
US6602677B1 (en) * 1997-09-19 2003-08-05 Promega Corporation Thermostable luciferases and methods of production
ES2536878T3 (en) 1997-10-13 2015-05-29 Novozymes A/S Alpha-amylase mutants
GB9722131D0 (en) 1997-10-20 1997-12-17 Medical Res Council Method
CN101125873A (en) * 1997-10-24 2008-02-20 茵维特罗根公司 Recombinational cloning using nucleic acids having recombination sites
CN100342008C (en) * 1997-10-24 2007-10-10 茵维特罗根公司 Recombinational cloning using nucleic acids having recombinatin sites
US7351578B2 (en) * 1999-12-10 2008-04-01 Invitrogen Corp. Use of multiple recombination sites with unique specificity in recombinational cloning
AU1124499A (en) 1997-10-28 1999-05-17 Maxygen, Inc. Human papillomavirus vectors
US20030129146A1 (en) * 1997-10-31 2003-07-10 Vincent Fischetti The use of bacterial phage associated lysing proteins for treating bacterial dental caries
US20020136712A1 (en) * 1997-10-31 2002-09-26 Fischetti Vincent Bacterial phage associated lysing enzymes for the prophylactic and therapeutic treatment of colonization and infections caused by streptococcus pneumoniae
US7232576B2 (en) 1997-10-31 2007-06-19 New Horizons Diagnostics Corp Throat lozenge for the treatment of Streptococcus Group A
US6399097B1 (en) 1997-10-31 2002-06-04 New Horizons Diagnostics Corporation Composition for treatment of a bacterial infection of the digestive tract
EP1690868A1 (en) 1997-10-31 2006-08-16 Maxygen, Inc. Modification of virus tropism and host range by viral genome shuffling
US6423299B1 (en) 1997-10-31 2002-07-23 Vincent Fischetti Composition for treatment of a bacterial infection of an upper respiratory tract
US6399098B1 (en) 1997-10-31 2002-06-04 New Horizons Diagnostics Corp Composition for treating dental caries caused by streptococcus mutans
US6428784B1 (en) 1997-10-31 2002-08-06 New Horizons Diagnostics Corp Vaginal suppository for treating group B Streptococcus infection
US6277399B1 (en) 1997-10-31 2001-08-21 New Horizon Diagnostics Corporation Composition incorporating bacterial phage associated lysing enzymes for treating dermatological infections
US6752988B1 (en) * 2000-04-28 2004-06-22 New Horizons Diagnostic Corp Method of treating upper resiratory illnesses
US6056954A (en) 1997-10-31 2000-05-02 New Horizons Diagnostics Corp Use of bacterial phage associated lysing enzymers for the prophylactic and therapeutic treatment of various illnesses
US20030129147A1 (en) * 1997-10-31 2003-07-10 Vincent Fischetti Use of bacterial phage associated lysing proteins for treating bacterial dental caries
US6432444B1 (en) 1997-10-31 2002-08-13 New Horizons Diagnostics Corp Use of bacterial phage associated lysing enzymes for treating dermatological infections
US6406692B1 (en) 1997-10-31 2002-06-18 New Horizons Diagnostics Corp Composition for treatment of an ocular bacterial infection
US20030082110A1 (en) * 1997-10-31 2003-05-01 Vincent Fischetti Use of bacterial phage associated lysing proteins for treating bacterial dental caries
US7321023B2 (en) * 1997-11-07 2008-01-22 Incyte Corporation SP16 protein
EP1034260B1 (en) * 1997-12-05 2003-06-04 Europäisches Laboratorium Für Molekularbiologie (Embl) Novel dna cloning method relying on the e. coli rece/rect recombination system
AU746786B2 (en) 1997-12-08 2002-05-02 California Institute Of Technology Method for creating polynucleotide and polypeptide sequences
AR017831A1 (en) * 1997-12-10 2001-10-24 Pioneer Hi Bred Int METHOD FOR ALTERING THE COMPOSITION OF AMINO ACIDS OF A NATIVE PROTEIN OF INTEREST, PREPARED PROTEIN, AND POLINUCLEOTIDE
US6303848B1 (en) 1998-01-16 2001-10-16 Large Scale Biology Corporation Method for conferring herbicide, pest, or disease resistance in plant hosts
US20030166169A1 (en) * 1998-01-16 2003-09-04 Padgett Hal S. Method for constructing viral nucleic acids in a cell-free manner
US20020164585A1 (en) * 1998-01-16 2002-11-07 Sean Chapman Method for enhancing RNA or protein production using non-native 5' untranslated sequences in recombinant viral nucleic acids
US6426185B1 (en) * 1998-01-16 2002-07-30 Large Scale Biology Corporation Method of compiling a functional gene profile in a plant by transfecting a nucleic acid sequence of a donor plant into a different host plant in an anti-sense orientation
US6468745B1 (en) 1998-01-16 2002-10-22 Large Scale Biology Corporation Method for expressing a library of nucleic acid sequence variants and selecting desired traits
US20030027173A1 (en) * 1998-01-16 2003-02-06 Della-Cioppa Guy Method of determining the function of nucleotide sequences and the proteins they encode by transfecting the same into a host
US7390619B1 (en) 1998-02-11 2008-06-24 Maxygen, Inc. Optimization of immunomodulatory properties of genetic vaccines
US6541011B2 (en) 1998-02-11 2003-04-01 Maxygen, Inc. Antigen library immunization
KR20010041943A (en) * 1998-03-17 2001-05-25 한스 루돌프 하우스, 헨리테 브룬너, 베아트리체 귄터 Genes encoding MLO proteins and conferring fungal resistance upon plants
EP1073670A1 (en) 1998-05-01 2001-02-07 Maxygen, Inc. Optimization of pest resistance genes using dna shuffling
US6287765B1 (en) * 1998-05-20 2001-09-11 Molecular Machines, Inc. Methods for detecting and identifying single molecules
US6902918B1 (en) 1998-05-21 2005-06-07 California Institute Of Technology Oxygenase enzymes and screening method
US20030207345A1 (en) * 1998-05-21 2003-11-06 California Institute Of Technology Oxygenase enzymes and screening method
EP2284272B1 (en) 1998-06-10 2014-08-13 Novozymes A/S Mannanases
US7153655B2 (en) * 1998-06-16 2006-12-26 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function involving the use of exonuclease enzyme and two populations of parent polynucleotide sequence
KR20010052894A (en) 1998-06-17 2001-06-25 맥시겐, 인크. Method for producing polynucleotides with desired properties
US6365408B1 (en) 1998-06-19 2002-04-02 Maxygen, Inc. Methods of evolving a polynucleotides by mutagenesis and recombination
US6846655B1 (en) 1998-06-29 2005-01-25 Phylos, Inc. Methods for generating highly diverse libraries
ES2188190T5 (en) 1998-07-21 2007-11-16 Danisco A/S FOOD PRODUCT.
US20030153042A1 (en) * 1998-07-28 2003-08-14 California Institute Of Technology Expression of functional eukaryotic proteins
WO2000009727A2 (en) * 1998-08-12 2000-02-24 Maxygen, Inc. Dna shuffling to produce herbicide selective crops
US6951719B1 (en) * 1999-08-11 2005-10-04 Proteus S.A. Process for obtaining recombined nucleotide sequences in vitro, libraries of sequences and sequences thus obtained
US20030104417A1 (en) * 1998-08-12 2003-06-05 Proteus S.A. Template-mediated, ligation-oriented method of nonrandomly shuffling polynucleotides
US20030092023A1 (en) * 1998-08-12 2003-05-15 Daniel Dupret Method of shuffling polynucleotides using templates
US6991922B2 (en) * 1998-08-12 2006-01-31 Proteus S.A. Process for in vitro creation of recombinant polynucleotide sequences by oriented ligation
US20040191772A1 (en) * 1998-08-12 2004-09-30 Dupret Daniel Marc Method of shuffling polynucleotides using templates
US20060242731A1 (en) * 1998-08-12 2006-10-26 Venkiteswaran Subramanian DNA shuffling to produce herbicide selective crops
WO2000009682A1 (en) 1998-08-12 2000-02-24 Maxygen, Inc. Dna shuffling of monooxygenase genes for production of industrial chemicals
DE69941193D1 (en) 1998-08-17 2009-09-10 Bristol Myers Squibb Co IDENTIFICATION OF COMPOUND PROTEIN INTERACTIONS WITH LIBRARIES OF COUPLED PROTEIN NUCLEIC ACID MOLECULES
JP2002524087A (en) * 1998-09-03 2002-08-06 ローマ リンダ ユニバーシティー How to study protein interactions in vivo
US20070178475A1 (en) * 1998-09-17 2007-08-02 Nehls Michael C Novel human polynucleotides and polypeptides encoded thereby
US7897843B2 (en) 1999-03-23 2011-03-01 Mendel Biotechnology, Inc. Transcriptional regulation of plant biomass and abiotic stress tolerance
US20050086718A1 (en) 1999-03-23 2005-04-21 Mendel Biotechnology, Inc. Plant transcriptional regulators of abiotic stress
KR100618495B1 (en) * 1998-10-06 2006-08-31 마크 아론 에말파브 Transformation system in the field of filamentous fungal hosts: in chrysosporium
JP2002526107A (en) 1998-10-07 2002-08-20 マキシジェン, インコーポレイテッド DNA shuffling to generate nucleic acids for mycotoxin detoxification
EP1157093A1 (en) * 1998-10-16 2001-11-28 Xencor, Inc. Protein design automation for protein libraries
US6403312B1 (en) 1998-10-16 2002-06-11 Xencor Protein design automatic for protein libraries
US7315786B2 (en) 1998-10-16 2008-01-01 Xencor Protein design automation for protein libraries
US20020048772A1 (en) 2000-02-10 2002-04-25 Dahiyat Bassil I. Protein design automation for protein libraries
AU1318400A (en) 1998-10-19 2000-05-08 Board Of Trustees Of The Leland Stanford Junior University Dna-templated combinatorial library chemistry
DE69932345T2 (en) 1998-10-26 2007-07-19 Novozymes A/S PREPARATION AND SCALING OF INTERESTING DNA BANKS IN CELLS OF FILAMENTOUS MUSHROOMS
GB9823468D0 (en) * 1998-10-28 1998-12-23 Secr Defence Novel enzyme
JP3267576B2 (en) * 1998-11-09 2002-03-18 三光純薬株式会社 Method for forming probe polymer
WO2000028018A1 (en) 1998-11-10 2000-05-18 Maxygen, Inc. Modified adp-glucose pyrophosphorylase for improvement and optimization of plant phenotypes
US6438561B1 (en) * 1998-11-19 2002-08-20 Navigation Technologies Corp. Method and system for using real-time traffic broadcasts with navigation systems
CA2350189A1 (en) * 1998-11-20 2000-06-02 Kosan Biosciences, Inc. Recombinant methods and materials for producing epothilone and epothilone derivatives
US6410301B1 (en) 1998-11-20 2002-06-25 Kosan Biosciences, Inc. Myxococcus host cells for the production of epothilones
US20040009535A1 (en) 1998-11-27 2004-01-15 Celltech R&D, Inc. Compositions and methods for increasing bone mineralization
ES2350454T3 (en) * 1998-11-27 2011-01-24 Ucb Pharma S.A. COMPOSITIONS AND METHODS TO INCREASE THE MINERALIZATION OF THE BONE SUBSTANCE.
EP1803817B1 (en) 1998-12-18 2011-04-06 Novozymes A/S Subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino acid residue in an active site loop region
US20020061556A1 (en) * 2000-04-27 2002-05-23 Walke D. Wade Novel membrane proteins and polynucleotides encoding the same
US6570003B1 (en) * 2001-01-09 2003-05-27 Lexion Genetics Incorporated Human 7TM proteins and polynucleotides encoding the same
US20020031802A1 (en) * 2000-05-12 2002-03-14 Yi Hu Novel seven transmembrane proteins and polynucleotides encoding the same
US6358712B1 (en) 1999-01-05 2002-03-19 Trustee Of Boston University Ordered gene assembly
US20040005673A1 (en) * 2001-06-29 2004-01-08 Kevin Jarrell System for manipulating nucleic acids
DE60042730D1 (en) * 1999-01-05 2009-09-24 Univ Boston IMPROVED CLONING PROCESS
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7013219B2 (en) 1999-01-12 2006-03-14 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7070934B2 (en) 1999-01-12 2006-07-04 Sangamo Biosciences, Inc. Ligand-controlled regulation of endogenous gene expression
WO2000042559A1 (en) * 1999-01-18 2000-07-20 Maxygen, Inc. Methods of populating data structures for use in evolutionary simulations
US6368861B1 (en) * 1999-01-19 2002-04-09 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
US6436675B1 (en) 1999-09-28 2002-08-20 Maxygen, Inc. Use of codon-varied oligonucleotide synthesis for synthetic shuffling
US20030054390A1 (en) * 1999-01-19 2003-03-20 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
US6917882B2 (en) * 1999-01-19 2005-07-12 Maxygen, Inc. Methods for making character strings, polynucleotides and polypeptides having desired characteristics
US6376246B1 (en) 1999-02-05 2002-04-23 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
US7873477B1 (en) * 2001-08-21 2011-01-18 Codexis Mayflower Holdings, Llc Method and system using systematically varied data libraries
US6961664B2 (en) 1999-01-19 2005-11-01 Maxygen Methods of populating data structures for use in evolutionary simulations
US7702464B1 (en) 2001-08-21 2010-04-20 Maxygen, Inc. Method and apparatus for codon determining
ATE465459T1 (en) 1999-01-19 2010-05-15 Maxygen Inc THROUGH OLIGONUCLEOTIDE-MEDIATED NUCLEIC ACID RECOMBINATION
US8457903B1 (en) 1999-01-19 2013-06-04 Codexis Mayflower Holdings, Llc Method and/or apparatus for determining codons
US20070065838A1 (en) * 1999-01-19 2007-03-22 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
US7024312B1 (en) * 1999-01-19 2006-04-04 Maxygen, Inc. Methods for making character strings, polynucleotides and polypeptides having desired characteristics
US20090130718A1 (en) * 1999-02-04 2009-05-21 Diversa Corporation Gene site saturation mutagenesis
JP2003524394A (en) 1999-02-11 2003-08-19 マキシジェン, インコーポレイテッド High-throughput mass spectrometry
CA2363779A1 (en) 1999-02-26 2000-08-31 Human Genome Sciences, Inc. Human endokine alpha and methods of use
NZ514569A (en) 1999-03-02 2004-02-27 Invitrogen Corp Compositions and methods for use in recombinational cloning of nucleic acids
JP3399518B2 (en) * 1999-03-03 2003-04-21 インターナショナル・ビジネス・マシーンズ・コーポレーション Semiconductor structure and method of manufacturing the same
US6531316B1 (en) 1999-03-05 2003-03-11 Maxyag, Inc. Encryption of traits using split gene sequences and engineered genetic elements
CA2362737A1 (en) * 1999-03-05 2000-09-08 Maxygen, Inc. Recombination of insertion modified nucleic acids
EP1159285B1 (en) * 1999-03-08 2005-05-25 Metrigen, Inc. Methods and compositions for economically synthesizing and assembling long dna sequences
AU3879300A (en) * 1999-03-09 2000-09-28 Diversa Corporation End selection in directed evolution
KR100312070B1 (en) * 1999-03-15 2001-11-03 박호군 Method of Selecting Enzyme Variants Including High Throughput Screening
US8148110B2 (en) * 1999-03-15 2012-04-03 The Board Of Trustees Of The Leland Stanford Junior University Detection of molecular interactions by β-lactamase reporter fragment complementation
US7105315B2 (en) * 1999-06-16 2006-09-12 Incyte Genomics, Inc. Transmembrane protein differentially expressed in cancer
US7030215B2 (en) * 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US20030104526A1 (en) * 1999-03-24 2003-06-05 Qiang Liu Position dependent recognition of GNN nucleotide triplets by zinc fingers
ES2618608T3 (en) 1999-03-30 2017-06-21 Novozymes A/S Alpha-amylase variants
MXPA01009706A (en) 1999-03-31 2002-05-14 Novozymes As Polypeptides having alkaline alpha-amylase activity and nucleic acids encoding same.
AU3419200A (en) 1999-03-31 2000-10-23 Novozymes A/S Polypeptides having alkaline alpha-amylase activity and nucleic acids encoding same
US6703240B1 (en) 1999-04-13 2004-03-09 Maxygar, Inc. Modified starch metabolism enzymes and encoding genes for improvement and optimization of plant phenotypes
US20020110809A1 (en) * 1999-04-30 2002-08-15 Nehls Michael C. Novel human polynucleotides and polypeptides encoded thereby
US20020095031A1 (en) * 1999-05-04 2002-07-18 Nehls Michael C. Novel human polynucleotides and polypeptides encoded thereby
ATE402996T1 (en) 1999-05-20 2008-08-15 Novozymes As SUBTILASE ENZYMES OF THE I-S1 AND I-S2 SUBGROUPS WITH AT LEAST ONE ADDITIONAL AMINO ACID RESIDUE BETWEEN POSITIONS 125 AND 126
AU4392700A (en) 1999-05-20 2000-12-12 Novozymes A/S Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additionalamino acid residue between positions 129 and 130
DE60040287D1 (en) 1999-05-20 2008-10-30 Novozymes As SUBTILASE ENZYMS OF I-S1 AND I-S2 SUB-GROUPS WITH AT LEAST ONE ADDITIONAL AMINO ACID BETWEEN POSITIONS 128 AND 129
EP1183341B2 (en) 1999-05-20 2012-05-02 Novozymes A/S Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additional amino acid residue between positions 127 and 128
WO2000071685A1 (en) 1999-05-20 2000-11-30 Novozymes A/S Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additional amino acid residue between positions 132 and 133
AU4392800A (en) 1999-05-20 2000-12-12 Novozymes A/S Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additionalamino acid residue between positions 126 and 127
US7332308B1 (en) 1999-05-21 2008-02-19 The Penn State Research Foundation Incrementally truncated nucleic acids and methods of making same
JP2003509010A (en) * 1999-05-31 2003-03-11 ノボザイムス アクティーゼルスカブ Peptide screening method
EP1067181A1 (en) * 1999-06-10 2001-01-10 Rijksuniversiteit te Groningen Enzyme selection
WO2000078968A2 (en) 1999-06-18 2000-12-28 Elitra Pharmaceuticals, Inc. Nucleotide sequences of moraxella catarrhalis genome
WO2001004287A1 (en) * 1999-07-07 2001-01-18 Maxygen Aps A method for preparing modified polypeptides
US6421613B1 (en) * 1999-07-09 2002-07-16 Pioneer Hi-Bred International, Inc. Data processing of the maize prolifera genetic sequence
CA2374009A1 (en) 1999-07-09 2001-01-18 Novozymes A/S Glucoamylase variant
US6387620B1 (en) * 1999-07-28 2002-05-14 Gilead Sciences, Inc. Transcription-free selex
US6653151B2 (en) 1999-07-30 2003-11-25 Large Scale Proteomics Corporation Dry deposition of materials for microarrays using matrix displacement
US6713309B1 (en) 1999-07-30 2004-03-30 Large Scale Proteomics Corporation Microarrays and their manufacture
US7179638B2 (en) 1999-07-30 2007-02-20 Large Scale Biology Corporation Microarrays and their manufacture by slicing
GB9918154D0 (en) * 1999-08-02 1999-10-06 Zeneca Ltd Expression system
EA200200056A1 (en) * 1999-08-12 2002-06-27 Пфайзер Продактс Инк. STREPTOMYCES AVERMITILIS GENE, GUIDES TO B2: B1 RELATIONSHIP
US6251604B1 (en) 1999-08-13 2001-06-26 Genopsys, Inc. Random mutagenesis and amplification of nucleic acid
US6720142B1 (en) * 1999-08-19 2004-04-13 University Of Rochester Method of determining evolutionary potential of mutant resistance genes and use thereof to screen for drug efficacy
US20050153323A1 (en) * 2000-07-28 2005-07-14 Yi Hu Novel human proteases and polynucleotides encoding the same
US6448388B1 (en) 2000-08-16 2002-09-10 Lexicon Genetics Incorporated Human proteases and polynucleotides encoding the same
US20030050464A1 (en) * 2000-07-28 2003-03-13 Yi Hu Novel human proteases and polynucleotides encoding the same
US20080003673A1 (en) * 1999-09-02 2008-01-03 Alejandro Abuin Novel human proteases and polynucleotides encoding the same
US6716614B1 (en) 1999-09-02 2004-04-06 Lexicon Genetics Incorporated Human calcium dependent proteases, polynucleotides encoding the same, and uses thereof
US6472588B1 (en) * 1999-09-10 2002-10-29 Texas Tech University Transgenic cotton plants with altered fiber characteristics transformed with a sucrose phosphate synthase nucleic acid
US7063837B2 (en) * 1999-09-14 2006-06-20 New Horizons Diagnostics Corp Syrup composition containing phage associated lytic enzymes
US20020127215A1 (en) * 1999-09-14 2002-09-12 Lawrence Loomis Parenteral use of bacterial phage associated lysing enzymes for the therapeutic treatment of bacterial infections
US6867291B1 (en) * 2000-09-15 2005-03-15 Lexicon Genetics Incorporated Human hemicentin proteins and polynucleotides encoding the same
US20020107382A1 (en) * 2000-09-27 2002-08-08 Friddle Carl Johan Novel human protease inhibitor proteins and polynucleotides encoding the same
US6790660B1 (en) * 2001-09-18 2004-09-14 Lexicon Genetics Incorporated Human kielin-like proteins and polynucleotides encoding the same
US6541252B1 (en) 2000-05-19 2003-04-01 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
US6602698B2 (en) 1999-12-07 2003-08-05 Lexicon Genetics Incorporated Human kinase proteins and polynucleotides encoding the same
US6444456B1 (en) 2000-03-10 2002-09-03 Lexicon Genetics Incorporated Human G-coupled protein receptor kinases and polynucleotides encoding the same
US6841377B1 (en) * 2001-06-13 2005-01-11 Lexicon Genetics Incorporated Human kinase and polynucleotides encoding the same
US6864079B2 (en) 2001-04-06 2005-03-08 Lexicon Genetics Incorporated Human kinase and polynucleotides encoding the same
US6797510B1 (en) * 2001-05-24 2004-09-28 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
US6777545B2 (en) * 2001-04-06 2004-08-17 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
US6716616B1 (en) 1999-09-28 2004-04-06 Lexicon Genetics Incorporated Human kinase proteins and polynucleotides encoding the same
US6476210B2 (en) 2000-10-12 2002-11-05 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
US6586230B1 (en) * 2000-10-27 2003-07-01 Lexicon Genetics Incorporated Human kinase and polynucleotides encoding the same
US20080050809A1 (en) * 1999-09-28 2008-02-28 Alejandro Abuin Novel human kinases and polynucleotides encoding the same
US6511840B1 (en) 2000-06-15 2003-01-28 Lexicon Genetics Incorporated Human kinase proteins and polynucleotides encoding the same
US20040002474A1 (en) * 1999-10-07 2004-01-01 Maxygen Inc. IFN-alpha homologues
AU782326B2 (en) 1999-10-12 2005-07-21 Lexicon Pharmaceuticals, Inc. Human LDL receptor family proteins and polynucleotides encoding the same
US7430477B2 (en) * 1999-10-12 2008-09-30 Maxygen, Inc. Methods of populating data structures for use in evolutionary simulations
WO2001027275A1 (en) * 1999-10-13 2001-04-19 Lexicon Genetics Incorporated Novel human membrane proteins
ES2335385T3 (en) 1999-10-13 2010-03-26 The Board Of Trustees Of The Leland Stanford Junior University BIOSYNTHESIS OF SUBSTRATES OF POLYETHYDE SYNTHEASE.
US20080213878A1 (en) * 1999-10-19 2008-09-04 Gregory Donoho Novel human membrane proteins and polynucleotides encoding the same
US6750054B2 (en) 2000-05-18 2004-06-15 Lexicon Genetics Incorporated Human semaphorin homologs and polynucleotides encoding the same
US6346249B1 (en) * 1999-10-22 2002-02-12 Ludwig Institute For Cancer Research Methods for reducing the effects of cancers that express A33 antigen using A33 antigen specific immunoglobulin products
GB9925161D0 (en) 1999-10-26 1999-12-22 Secr Defence Novel enzyme
US6635438B2 (en) * 1999-11-02 2003-10-21 Catch, Inc. Enzymatic cycling assays for homocysteine and cystathionine
WO2001032712A2 (en) 1999-11-03 2001-05-10 Maxygen, Inc. Antibody diversity generation
DE19953854C2 (en) 1999-11-09 2002-01-17 Max Planck Gesellschaft Process for the production of biopolymers with modified properties
CN1654641A (en) 1999-11-10 2005-08-17 诺维信公司 Fungamyl-like alpha-amylase variants
US6686515B1 (en) 1999-11-23 2004-02-03 Maxygen, Inc. Homologous recombination in plants
JP2003517823A (en) * 1999-11-26 2003-06-03 ビーエーエスエフ プランド サイエンス ゲーエムベーハー Methods for mutagenesis of plant, algal or fungal nucleotide sequences
US7115712B1 (en) 1999-12-02 2006-10-03 Maxygen, Inc. Cytokine polypeptides
AU2070701A (en) * 1999-12-07 2001-06-18 Lexicon Genetics Incorporated Novel human membrane proteins and polynucleotides encoding the same
CA2393723A1 (en) * 1999-12-09 2001-06-14 Lexicon Genetics Incorporated Human adam-ts proteases and polynucleotides encoding the same
CA2392753A1 (en) 1999-12-10 2001-06-14 Invitrogen Corporation Use of multiple recombination sites with unique specificity in recombinational cloning
US20020042055A1 (en) * 1999-12-23 2002-04-11 Affholter Joseph A. Alteration of hydrolase genes and screening of the resulting libraries for the ability to catalyze specific reactions
EP1242823B1 (en) * 1999-12-27 2007-07-04 Crucell Holland B.V. Selecting library members capable of binding to epitopes
US6927046B1 (en) 1999-12-30 2005-08-09 Archer-Daniels-Midland Company Increased lysine production by gene amplification using coryneform bacteria
CA2396320A1 (en) * 2000-01-11 2001-07-19 Maxygen, Inc. Integrated systems and methods for diversity generation and screening
US6395485B1 (en) 2000-01-11 2002-05-28 Aventis Cropscience N.V. Methods and kits for identifying elite event GAT-ZM1 in biological samples
WO2001053493A2 (en) * 2000-01-18 2001-07-26 Lexicon Genetics Incorporated Human kinase protein and polynucleotides encoding the same
US7022479B2 (en) * 2000-01-24 2006-04-04 Compound Therapeutics, Inc. Sensitive, multiplexed diagnostic assays for protein analysis
WO2001053539A1 (en) 2000-01-24 2001-07-26 Phylos, Inc. Sensitive, multiplexed diagnostic assays for protein analysis
AU782817B2 (en) * 2000-02-04 2005-09-01 Lexicon Pharmaceuticals, Inc. Novel human G protein coupled receptor proteins and polynucleotides encoding the same
JP2003521933A (en) * 2000-02-10 2003-07-22 ゼンコー Protein design automation for protein libraries
EP1621617A1 (en) * 2000-02-10 2006-02-01 Xencor, Inc. Protein design automation for protein libraries
WO2001064864A2 (en) * 2000-02-28 2001-09-07 Maxygen, Inc. Single-stranded nucleic acid template-mediated recombination and nucleic acid fragment isolation
WO2001064912A2 (en) * 2000-02-29 2001-09-07 Maxygen, Inc. Triazine degrading enzymes
US6436677B1 (en) * 2000-03-02 2002-08-20 Promega Corporation Method of reverse transcription
WO2001073000A2 (en) * 2000-03-24 2001-10-04 Maxygen, Inc. Methods for modulating cellular and organismal phenotypes
WO2001075108A1 (en) * 2000-04-03 2001-10-11 Lexicon Genetics Incorporated Human ion channel protein and polynucleotides encoding the same
JP2003532391A (en) * 2000-04-12 2003-11-05 レキシコン・ジェネティクス・インコーポレーテッド Human metalloprotease and polynucleotide encoding the protein
ES2529300T3 (en) 2000-04-12 2015-02-18 Novozymes Biopharma Dk A/S Albumin fusion proteins
US7129326B2 (en) * 2000-04-14 2006-10-31 Genencor International, Inc. Methods for selective targeting
DK1360500T3 (en) * 2000-04-14 2010-05-25 Genencor Int Selective targeting method
US20020187136A1 (en) * 2000-04-28 2002-12-12 Lawrence Loomis Use of bacterial phage associated lysing enzymes for treating various illnesses
US6534292B1 (en) 2000-05-08 2003-03-18 Genencor International, Inc. Methods for forming recombined nucleic acids
WO2001088134A2 (en) * 2000-05-12 2001-11-22 Lexicon Genetics Incorporated Novel human lipocalin homologs and polynucleotides encoding the same
US7115403B1 (en) 2000-05-16 2006-10-03 The California Institute Of Technology Directed evolution of galactose oxidase enzymes
US7244560B2 (en) * 2000-05-21 2007-07-17 Invitrogen Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
US20030032059A1 (en) * 2000-05-23 2003-02-13 Zhen-Gang Wang Gene recombination and hybrid protein development
AU2001274923A1 (en) * 2000-05-23 2001-12-03 Lexicon Genetics Incorporated Novel human thrombospondin-like proteins and polynucleotides encoding the same
US6790667B1 (en) * 2000-05-30 2004-09-14 Lexicon Genetics Incorporated Human mitochondrial proteins and polynucleotides encoding the same
US20030031675A1 (en) 2000-06-06 2003-02-13 Mikesell Glen E. B7-related nucleic acids and polypeptides useful for immunomodulation
WO2001094583A2 (en) * 2000-06-07 2001-12-13 Lexicon Genetics Incorporated Human transporter proteins and polynucleotides encoding the same
WO2001094417A2 (en) * 2000-06-09 2001-12-13 Lexicon Genetics Incorporated Novel human seven transmembrane proteins and polynucleotides encoding the same
US6465632B1 (en) * 2000-06-09 2002-10-15 Lexicon Genetics Incorporated Human phosphatases and polynucleotides encoding the same
FR2810339B1 (en) * 2000-06-14 2004-12-10 Hoechst Marion Roussel Inc COMBINATORIAL BANKS IMPROVED BY RECOMBINATION IN YEAST AND METHOD OF ANALYSIS
EP1294949A4 (en) 2000-06-15 2004-08-25 Human Genome Sciences Inc Human tumor necrosis factor delta and epsilon
KR101287395B1 (en) 2000-06-16 2014-11-04 휴먼 게놈 사이언시즈, 인코포레이티드 Antibodies that immunospecifically bind to BLyS
US7468475B2 (en) 2000-06-16 2008-12-23 Schmuelling Thomas Method for modifying plant morphology, biochemistry and physiology
US6410246B1 (en) 2000-06-23 2002-06-25 Genetastix Corporation Highly diverse library of yeast expression vectors
AU2001272978A1 (en) 2000-06-23 2002-01-08 Maxygen, Inc. Novel co-stimulatory molecules
US7074590B2 (en) * 2000-06-23 2006-07-11 Maxygen, Inc. Chimeric promoters
US6406863B1 (en) 2000-06-23 2002-06-18 Genetastix Corporation High throughput generation and screening of fully human antibody repertoire in yeast
US6410271B1 (en) 2000-06-23 2002-06-25 Genetastix Corporation Generation of highly diverse library of expression vectors via homologous recombination in yeast
WO2002000852A2 (en) 2000-06-26 2002-01-03 Novozymes A/S Lipolytic enzyme
US7846733B2 (en) 2000-06-26 2010-12-07 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
AU7151601A (en) * 2000-06-27 2002-01-08 Lexicon Genetics Inc Novel human gaba receptors and polynucleotides encoding the same
AU2001271912A1 (en) * 2000-07-07 2002-01-21 Maxygen, Inc. Molecular breeding of transposable elements
US6994963B1 (en) 2000-07-10 2006-02-07 Ambion, Inc. Methods for recombinatorial nucleic acid synthesis
US6858422B2 (en) * 2000-07-13 2005-02-22 Codexis, Inc. Lipase genes
WO2002006469A2 (en) * 2000-07-18 2002-01-24 Enchira Biotechnology Corporation Methods of ligation mediated chimeragenesis utilizing populations of scaffold and donor nucleic acids
US6878861B2 (en) * 2000-07-21 2005-04-12 Washington State University Research Foundation Acyl coenzyme A thioesterases
EP1315800A2 (en) * 2000-07-21 2003-06-04 Incyte Genomics, Inc. Human proteases
US7435562B2 (en) 2000-07-21 2008-10-14 Modular Genetics, Inc. Modular vector systems
US6887685B1 (en) 2000-07-25 2005-05-03 Lexicon Genetics Incorporated Human thymosin protein and polynucleotides encoding the same
WO2002010183A1 (en) * 2000-07-31 2002-02-07 Menzel, Rolf Compositions and methods for directed gene assembly
EP1354031A2 (en) * 2000-07-31 2003-10-22 Maxygen, Inc. Nucleotide incorporating enzymes
JP4855632B2 (en) 2000-08-01 2012-01-18 ノボザイムス アクティーゼルスカブ Α-Amylase mutants with altered properties
JP3859947B2 (en) * 2000-08-04 2006-12-20 独立行政法人理化学研究所 Mutation introduction method
DK1311661T3 (en) 2000-08-14 2012-11-26 Us Gov Health & Human Serv Increased homologous recombination mediated by lambda recombination proteins
US7198924B2 (en) 2000-12-11 2007-04-03 Invitrogen Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
EP1313846B1 (en) 2000-08-21 2013-10-16 Novozymes A/S Subtilase enzymes
DE60121777T2 (en) * 2000-08-22 2007-08-09 Lexicon Genetics Inc., The Woodlands HUMAN PROTEASES AND FOR THOSE CODING POLYNUCLEOTIDES
CA2420036A1 (en) * 2000-08-22 2002-02-28 Lexicon Genetics Incorporated Human 7tm proteins and polynucleotides encoding the same
WO2002015675A1 (en) 2000-08-22 2002-02-28 Mendel Biotechnology, Inc. Genes for modifying plant traits iv
WO2002016583A2 (en) * 2000-08-24 2002-02-28 Maxygen, Inc. Constructs and their use in metabolic pathway engineering
US7879540B1 (en) * 2000-08-24 2011-02-01 Promega Corporation Synthetic nucleic acid molecule compositions and methods of preparation
US20030157643A1 (en) * 2000-08-24 2003-08-21 Almond Brian D Synthetic nucleic acids from aquatic species
ZA200301579B (en) 2000-08-25 2004-06-21 Basf Plant Science Gmbh Plant polynucleotides encoding prenyl proteases.
FR2813314B1 (en) * 2000-08-25 2004-05-07 Biomethodes MASSIVE DIRECTED MUTAGENESIS PROCESS
AU2001285326A1 (en) * 2000-08-31 2002-03-13 Lexicon Genetics Incorporated Human kinase proteins and polynucleotides encoding the same
US7229790B2 (en) * 2000-09-01 2007-06-12 Lexicon Pharmaceuticals, Inc. Human GABA transporter protein and polynucleotides encoding the same
US6395504B1 (en) 2000-09-01 2002-05-28 New Horizons Diagnostics Corp. Use of phage associated lytic enzymes for the rapid detection of bacterial contaminants
US7125662B2 (en) * 2000-09-11 2006-10-24 University Of Rochester Method of identifying putative antibiotic resistance genes
JP2004509628A (en) * 2000-09-21 2004-04-02 メルク エンド カムパニー インコーポレーテッド Method for producing recombinant polynucleotide
US20020142324A1 (en) * 2000-09-22 2002-10-03 Xun Wang Fungal target genes and methods to identify those genes
EP2336339A3 (en) 2000-09-25 2011-09-14 The Regents of the University of Michigan Production of viral vectors
CA2423942A1 (en) 2000-09-27 2002-04-04 Lexicon Genetics Incorporated Human ion-exchanger proteins and polynucleotides encoding the same
WO2002026964A1 (en) * 2000-09-27 2002-04-04 Mitsubishi Chemical Corporation Method of constructing mutant dna library and utilization thereof
US20040091920A1 (en) * 2000-09-27 2004-05-13 Mitsubishi Chemical Corporation Method of constructing a mutant DNA library and use thereof
US6777232B1 (en) * 2000-10-02 2004-08-17 Lexicon Genetics Incorporated Human membrane proteins and polynucleotides encoding the same
WO2002033089A2 (en) * 2000-10-05 2002-04-25 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Ixodes scapularis tissue factor pathway inhibitor
AU2002211624A1 (en) * 2000-10-10 2002-04-22 Genencor International, Inc. Information rich libraries
JP3912595B2 (en) * 2000-10-11 2007-05-09 三光純薬株式会社 Method for producing probe self-assembly
CN101538563A (en) 2000-10-13 2009-09-23 诺维信公司 Subtilase variants
US20040091994A1 (en) 2000-10-13 2004-05-13 Carsten Andersen Alpha-amylase variant with altered properties
US7001763B1 (en) 2000-10-17 2006-02-21 Lexicon Genetics Incorporated Human semaphorin proteins and polynucleotides encoding the same
US7045283B2 (en) 2000-10-18 2006-05-16 The Regents Of The University Of California Methods of high-throughput screening for internalizing antibodies
WO2002070705A2 (en) * 2000-10-27 2002-09-12 Lexicon Genetics Incorporated Human 7tm proteins and polynucleotides encoding them
US7060442B2 (en) * 2000-10-30 2006-06-13 Regents Of The University Of Michigan Modulators on Nod2 signaling
AU2002232936A1 (en) * 2000-10-30 2002-05-15 Lexicon Genetics Incorporated Human 7TM proteins and polynucleotides encoding the same
US7202070B2 (en) * 2000-10-31 2007-04-10 Biocatalytics, Inc. Method for reductive amination of a ketone using a mutated enzyme
AU3941002A (en) 2000-10-31 2002-06-03 Zycos Inc Cyp1b1 nucleic acids and methods of use
CA2427828A1 (en) * 2000-11-01 2002-05-10 Lexicon Genetics Incorporated Novel human proteases and polynucleotides encoding the same
EP1333854A4 (en) * 2000-11-02 2005-10-05 New Horizons Diagnostics Corp The use of bacterial phage associated lytic enzymes to prevent food poisoning
AU2001274635A1 (en) * 2000-11-10 2002-05-21 Amicogen, Inc. Method for generating recombinant dna library using unidirectional single-stranded dna fragments
WO2002050278A2 (en) * 2000-11-15 2002-06-27 Lexicon Genetics Incorporated Novel human secreted proteins and polynucleotides encoding the same
US7858559B2 (en) * 2000-11-17 2010-12-28 University Of Rochester In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells
EP1830190A3 (en) * 2000-11-17 2008-07-09 University Of Rochester In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells
WO2002042438A2 (en) * 2000-11-20 2002-05-30 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
TWI327600B (en) 2000-11-28 2010-07-21 Medimmune Llc Methods of administering/dosing anti-rsv antibodies for prophylaxis and treatment
CA2431007A1 (en) 2000-12-11 2002-07-18 Lexicon Genetics Incorporated Novel human kinase and polynucleotides encoding the same
AU3264202A (en) * 2000-12-12 2002-06-24 Lexicon Genetics Inc Novel human kinases and polynucleotides encoding the same
US6958213B2 (en) * 2000-12-12 2005-10-25 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function
AU2002228974A1 (en) 2000-12-13 2002-06-24 Nugen Technologies, Inc Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
DE10062086A1 (en) * 2000-12-13 2002-07-04 Wella Ag Agent and method of dyeing keratin fibers
US6583269B1 (en) * 2000-12-18 2003-06-24 Lexicon Genetics Incorporated Human protease inhibitor and polynucleotides encoding the same
JP2005502307A (en) * 2000-12-20 2005-01-27 レキシコン・ジェネティクス・インコーポレーテッド Novel human ion channel protein and polynucleotide encoding the same
US6852844B1 (en) * 2000-12-20 2005-02-08 Lexicon Genetics Incorporated Human protocadherin proteins and polynucleotides encoding the same
US20020086292A1 (en) 2000-12-22 2002-07-04 Shigeaki Harayama Synthesis of hybrid polynucleotide molecules using single-stranded polynucleotide molecules
US20020150895A1 (en) * 2000-12-22 2002-10-17 Raymond Wheeler Method and apparatus for filtering and extending RNA alignment coverage
US7070973B2 (en) * 2000-12-26 2006-07-04 Board Of Regents Of The University Of Nebraska Butyrylcholinesterase variants and methods of use
WO2002059325A2 (en) * 2000-12-27 2002-08-01 Lexicon Genetics Incorporated Human kinases and polynucleotides encoding the same
AU2001297533A1 (en) * 2000-12-28 2002-09-12 Lexicon Genetics Incorporated Novel human ion channel-related proteins and polynucleotides encoding the same
US20020115844A1 (en) * 2001-01-05 2002-08-22 Yu Xuanchuan Sean Novel human lipase and polynucleotides encoding the same
CA2435394C (en) * 2001-01-22 2018-01-09 Sangamo Biosciences, Inc. Modified zinc finger binding proteins
WO2002086096A2 (en) * 2001-01-23 2002-10-31 University Of Rochester Medical Center Methods of producing or identifying intrabodies in eukaryotic cells
WO2002059287A2 (en) * 2001-01-23 2002-08-01 Lexicon Genetics Incorporated Novel human kinases and polynucleotides encoding the same
EP1356059A1 (en) * 2001-01-24 2003-10-29 Lexicon Genetics Incorporated Human lipase and polynucleotides encoding the same
EP1683865A3 (en) 2001-02-02 2006-10-25 Eli Lilly &amp; Company Mammalian proteins and in particular CD200
WO2002063002A2 (en) * 2001-02-02 2002-08-15 Lexicon Genetics Incorporated Novel human transporter protein and polynucleotides encoding the same
US20020192675A1 (en) * 2001-02-02 2002-12-19 The University Of Rochester Methods of identifying regulator molecules
CA2437811A1 (en) 2001-02-09 2002-08-22 Human Genome Sciences, Inc. Human g-protein chemokine receptor (ccr5) hdgnr10
JP2004529623A (en) * 2001-02-20 2004-09-30 レキシコン・ジェネティクス・インコーポレーテッド Novel protease and polynucleotide encoding it
US6849449B2 (en) * 2001-03-01 2005-02-01 Regents Of The University Of Michigan Orphanin FQ receptor nucleic acids
EP1366197B1 (en) 2001-03-09 2007-05-09 Nugen Technologies, Inc. Methods and compositions for amplification of rna sequences
AU2002245593A1 (en) * 2001-03-12 2002-09-24 Lexicon Genetics Incorporated Novel human dectin proteins and polynucleotides encoding the same
AU2002252296A1 (en) * 2001-03-12 2002-09-24 Lexicon Genetics Incorporated Novel human transporter proteins and polynucleotides encoding the same
JP2004535781A (en) * 2001-03-12 2004-12-02 レキシコン・ジェネティクス・インコーポレーテッド Novel human EFG family protein and polynucleotide encoding the same
US6994995B1 (en) * 2001-03-16 2006-02-07 Lexicon Genetics Incorporated Human synaptotagmin and polynucleotides encoding the same
US7678554B2 (en) 2001-03-19 2010-03-16 President And Fellows Of Harvard College Nucleic acid shuffling
AU2002306777C1 (en) 2001-03-19 2008-04-24 President And Fellows Of Harvard College Evolving new molecular function
US7807408B2 (en) * 2001-03-19 2010-10-05 President & Fellows Of Harvard College Directed evolution of proteins
WO2002074932A2 (en) 2001-03-20 2002-09-26 Lexicon Genetics Incorporated Novel human kinase and polynucleotides encoding the same
US20030049619A1 (en) * 2001-03-21 2003-03-13 Simon Delagrave Methods for the synthesis of polynucleotides and combinatorial libraries of polynucleotides
AR035799A1 (en) 2001-03-30 2004-07-14 Syngenta Participations Ag INSECTICIDE TOXINS ISOLATED FROM BACILLUS THURINGIENSIS AND ITS USES.
FR2823219B1 (en) * 2001-04-10 2003-07-04 Pasteur Institut MUTANTS OF DESOXYCYTIDINE KINASE WITH ENLARGED ENZYMATIC ACTIVITY
US6644173B2 (en) * 2001-04-11 2003-11-11 Keuring, Incorporated Beverage filter cartridge holder
CA2444632A1 (en) 2001-04-13 2002-10-24 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
US7465567B2 (en) * 2001-04-16 2008-12-16 California Institute Of Technology Peroxide-driven cytochrome P450 oxygenase variants
US6607895B2 (en) 2001-04-16 2003-08-19 Lexicon Genetics Incorporated Human adenylsuccinate synthetase and polynucleotides encoding the same
JP2004531259A (en) * 2001-04-19 2004-10-14 インヴィトロジェン コーポレーション Compositions and methods for recombinant cloning of nucleic acid molecules
AU2002338443B2 (en) * 2001-04-25 2007-07-19 Proteus Template-mediated, ligation-oriented method of nonrandomly shuffling polynucleotides
FR2824073B1 (en) * 2001-04-25 2003-12-19 Proteus PROCESS FOR THE IN VITRO CREATION OF RECOMBINANT POLYNUCLEOTIDE SEQUENCES BY ORIENTED LIGATION
WO2002088313A2 (en) * 2001-04-30 2002-11-07 Lexicon Genetics Incorporated Novel human nuclear transporters and polynucleotides encoding the same
EP1383887A4 (en) * 2001-05-03 2004-07-07 Rensselaer Polytech Inst Novel methods of directed evolution
CA2446967A1 (en) * 2001-05-09 2002-11-14 Lexicon Genetics Incorporated Novel human kinases and polynucleotides encoding the same
EP1950305A1 (en) 2001-05-09 2008-07-30 Monsanto Technology, LLC Tyr a genes and uses thereof
US7498158B2 (en) 2001-05-15 2009-03-03 Novozymes A/S Alpha-amylase variant with altered properties
WO2002092815A2 (en) * 2001-05-17 2002-11-21 Proteus Method of preparing polynucleotide fragments for use in shuffling, and shuffling of same
US6723537B2 (en) 2001-05-18 2004-04-20 Rigel Pharmaceuticals, Incorporated Directed evolution of protein in mammalian cells
BR0209154A (en) 2001-05-18 2004-07-20 Danisco Process of preparing a dough with an enzyme
CA2448505A1 (en) * 2001-05-21 2002-11-28 Invitrogen Corporation Compositions and methods for use in isolation of nucleic acid molecules
US20020193585A1 (en) * 2001-05-25 2002-12-19 Walke D. Wade Novel human transporter proteins and polynucleotides encoding the same
EP1572874B1 (en) 2001-05-25 2013-09-18 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to trail receptors
EP1461425B1 (en) 2001-05-29 2008-02-13 Lexicon Pharmaceuticals, Inc. Novel human hydroxylases and polynucleotides encoding the same
WO2002099080A2 (en) * 2001-06-05 2002-12-12 Gorilla Genomics, Inc. Methods for low background cloning of dna using long oligonucleotides
AU2002311012A1 (en) 2001-06-06 2002-12-16 Novozymes A/S Endo-beta-1,4-glucanase from bacillus
EP1406645A4 (en) * 2001-06-14 2005-02-16 Lexicon Genetics Inc Novel human transporter proteins and polynucleotides encoding the same
US7727713B2 (en) 2001-06-20 2010-06-01 Nuevolution A/S Templated molecules and methods for using such molecules
CA2451517C (en) 2001-06-22 2011-05-03 Pioneer Hi-Bred International, Inc. Defensin polynucleotides and methods of use
EP1925672A1 (en) 2001-06-22 2008-05-28 Syngeta Participations AG Abiotic stress responsive polynucleotides and polypeptides
US7785853B2 (en) 2001-06-26 2010-08-31 Novozymes A/S Polypeptides having cellobiohydrolase I activity and polynucleotides encoding same
WO2003002736A2 (en) * 2001-06-27 2003-01-09 Roche Diagnostics Gmbh A walk-through technique for in vitro recombination of polynucleotide sequences
GB0115841D0 (en) 2001-06-28 2001-08-22 Medical Res Council Ligand
WO2003004609A2 (en) * 2001-07-03 2003-01-16 Lexicon Genetics Incorporated Novel human kielin-like proteins and polynucleotides encoding the same
DK200101090A (en) 2001-07-12 2001-08-16 Novozymes As Subtilase variants
US20040213765A1 (en) * 2001-07-13 2004-10-28 Vincent Fischetti Use of bacterial phage associated lytic enzymes to prevent food poisoning
CA2492220C (en) * 2001-07-15 2014-03-18 Keck Graduate Institute Nucleic acid amplification using nicking agents
EP1277835A1 (en) * 2001-07-19 2003-01-22 Libragen Methods of creating genetic diversity
US7226768B2 (en) * 2001-07-20 2007-06-05 The California Institute Of Technology Cytochrome P450 oxygenases
US7402383B2 (en) 2001-07-23 2008-07-22 Dsm Ip Assets B.V. Process for preparing variant polynucleotides
US6867189B2 (en) * 2001-07-26 2005-03-15 Genset S.A. Use of adipsin/complement factor D in the treatment of metabolic related disorders
ATE432981T1 (en) * 2001-07-26 2009-06-15 Stratagene California MULTI-SITE MUTAGENesis
US6833441B2 (en) 2001-08-01 2004-12-21 Abmaxis, Inc. Compositions and methods for generating chimeric heteromultimers
US6943001B2 (en) * 2001-08-03 2005-09-13 Diversa Corporation Epoxide hydrolases, nucleic acids encoding them and methods for making and using them
WO2003012126A2 (en) * 2001-08-03 2003-02-13 Diversa Corporation Epoxide hydrolases, nucleic acids encoding them and methods for making and using them
US6987079B2 (en) * 2001-08-14 2006-01-17 W.R. Grace & Co.-Conn. Supported catalyst systems
EP1425382A4 (en) * 2001-08-14 2004-10-06 Lexicon Genetics Inc Novel human collagen proteins and polynucleotides encoding the same
DE60220804D1 (en) * 2001-08-20 2007-08-02 Regenesis Bioremediation Produ BIOSENSOR FOR SMALL MOLECULAR ANALYTES
US7647184B2 (en) * 2001-08-27 2010-01-12 Hanall Pharmaceuticals, Co. Ltd High throughput directed evolution by rational mutagenesis
KR101008785B1 (en) 2001-08-29 2011-01-14 제넨테크, 인크. Bv8 Nucleic Acids and Polypeptides with Mitogenic Activity
US7635798B2 (en) * 2001-08-31 2009-12-22 Dow Agrosciences, Llc Nucleic acid compositions conferring altered metabolic characteristics
KR100428998B1 (en) * 2001-09-10 2004-04-28 한국과학기술원 A Method for Manufacturing Mutant Library of Proteins with Variable Sequences and Sizes
EP1438428A4 (en) * 2001-09-14 2005-11-16 Univ Queensland Detection of dna methylation
JP2005508630A (en) * 2001-09-14 2005-04-07 インヴィトロジェン コーポレーション DNA polymerase and its variants
US7582425B2 (en) * 2001-09-21 2009-09-01 The Regents Of The University Of Michigan Atlastin
US7108975B2 (en) * 2001-09-21 2006-09-19 Regents Of The University Of Michigan Atlastin
DE10147074A1 (en) * 2001-09-25 2003-05-08 Beru Ag Method for operating a multi-stage electric heater consisting of several heating elements
US20030104445A1 (en) * 2001-09-25 2003-06-05 Hayes Robert J. RNA dependent RNA polymerase mediated protein evolution
DE60232672D1 (en) 2001-10-01 2009-07-30 Dyax Corp MULTILACKED EUKARYONTIC DISPLAY VECTORS AND THEIR USES
EP2279755B1 (en) 2001-10-10 2014-02-26 ratiopharm GmbH Remodelling and glycoconjugation of Fibroblast Growth Factor (FGF)
US8008252B2 (en) 2001-10-10 2011-08-30 Novo Nordisk A/S Factor VII: remodeling and glycoconjugation of Factor VII
US7214660B2 (en) 2001-10-10 2007-05-08 Neose Technologies, Inc. Erythropoietin: remodeling and glycoconjugation of erythropoietin
WO2004099231A2 (en) 2003-04-09 2004-11-18 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
EP2322229B1 (en) 2001-10-10 2016-12-21 Novo Nordisk A/S Remodeling and glycoconjugation of Factor IX
US7157277B2 (en) 2001-11-28 2007-01-02 Neose Technologies, Inc. Factor VIII remodeling and glycoconjugation of Factor VIII
US7173003B2 (en) * 2001-10-10 2007-02-06 Neose Technologies, Inc. Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF
US20040005709A1 (en) * 2001-10-24 2004-01-08 Hoogenboom Henricus Renerus Jacobus Mattheus Hybridization control of sequence variation
DK1440083T3 (en) 2001-10-25 2013-03-25 Medical Res Council MOLECULES
EP1840211A1 (en) 2001-10-31 2007-10-03 Danisco A/S Pyranosone dehydratase from phanerochaete chrysosporium
US7175983B2 (en) * 2001-11-02 2007-02-13 Abmaxis, Inc. Adapter-directed display systems
WO2003050914A1 (en) * 2001-12-05 2003-06-19 E-Tenna Corporation Capacitively-loaded bent-wire monopole on an artificial magnetic conductor
US7049121B2 (en) * 2001-12-20 2006-05-23 Applied Molecular Evolution Butyrylcholinesterase variant polypeptides with increased catalytic efficiency and methods of use
US6989261B2 (en) * 2001-12-20 2006-01-24 Eli Lilly And Company Butyrylcholinesterase variant polypeptides with increased catalytic efficiency and methods of use
MY139056A (en) * 2001-12-28 2009-08-28 Ab Enzymes Gmbh Microbially-expressed thermotolerant phytase for animal feed
KR100475305B1 (en) * 2002-01-08 2005-03-10 주식회사 마이크로아이디 Method for constructing chimeric dna library using exonuclease ⅶ
US20040009498A1 (en) * 2002-01-14 2004-01-15 Diversa Corporation Chimeric antigen binding molecules and methods for making and using them
US20060046249A1 (en) * 2002-01-18 2006-03-02 Fei Huang Identification of polynucleotides and polypetide for predicting activity of compounds that interact with protein tyrosine kinase and or protein tyrosine kinase pathways
US7262054B2 (en) * 2002-01-22 2007-08-28 Sangamo Biosciences, Inc. Zinc finger proteins for DNA binding and gene regulation in plants
DE20200926U1 (en) * 2002-01-23 2002-04-18 Hegenscheidt Mfd Gmbh & Co Kg Deep rolling device of a deep rolling machine for crankshafts
US20040096826A1 (en) * 2002-01-30 2004-05-20 Evans Glen A. Methods for creating recombination products between nucleotide sequences
US7255986B2 (en) * 2002-01-31 2007-08-14 The Board Of Trustees Operating Michigan State University Compositions for the diagnosis and treatment of epizootic catarrhal enteritis in ferrets
JP4426307B2 (en) 2002-02-08 2010-03-03 ノボザイムス アクティーゼルスカブ Phytase mutant
DE60326824D1 (en) * 2002-02-12 2009-05-07 Pfizer Prod Inc STREPTOMYCES AVERMITILIS GEN FOR REGULATING THE RELATIONSHIP OF THE B2: B1 AVERMECTINE
AU2002306484A1 (en) 2002-02-13 2003-09-04 Dow Global Technologies Inc. Over-expression of extremozyme genes in pseudomonads and closely related bacteria
WO2003072054A2 (en) * 2002-02-25 2003-09-04 Cabot Corporation Custom ligand design for biomolecular filtration and purification for bioseperation
US20030224404A1 (en) * 2002-02-25 2003-12-04 Manuel Vega High throughput directed evolution of nucleic acids by rational mutagenesis
CA2475547A1 (en) * 2002-02-26 2003-09-04 E.I. Du Pont De Nemours And Company Method for the recombination of genetic elements
EP1485404A4 (en) * 2002-02-27 2005-03-30 California Inst Of Techn Novel glucose 6-oxidases
DK2390803T3 (en) 2002-03-01 2014-01-27 Codexis Mayflower Holdings Llc Methods, systems and software for identifying functional biomolecules
BR0308134A (en) * 2002-03-01 2006-04-11 Ravgen Inc quick analysis of variations in a genome
US20040132101A1 (en) 2002-09-27 2004-07-08 Xencor Optimized Fc variants and methods for their generation
US6977162B2 (en) 2002-03-01 2005-12-20 Ravgen, Inc. Rapid analysis of variations in a genome
US7244592B2 (en) 2002-03-07 2007-07-17 Dyax Corp. Ligand screening and discovery
AU2003213846A1 (en) 2002-03-09 2003-09-29 Maxygen, Inc. Optimization of crossover points for directed evolution
ATE405676T1 (en) * 2002-03-11 2008-09-15 Nugen Technologies Inc METHOD FOR GENERATING DOUBLE STRANDED DNA WITH A 3' SINGLE STRAND PORTION AND USES OF THESE COMPLEXES FOR RECOMBINATION
US20050247001A1 (en) * 2002-03-15 2005-11-10 Nuevolution A/S Building block forming a c-c or a c-hetero atom bond uponreaction
AU2003253069A1 (en) * 2002-03-15 2003-09-29 Nuevolution A/S A building block forming a c-c bond upon reaction
EP1487851A2 (en) * 2002-03-15 2004-12-22 Nuevolution A/S A building block capable of functional entity transfer to nucleophil
EP2186897B1 (en) * 2002-03-15 2016-02-17 Nuevolution A/S An improved method for synthesising templated molecules
US20050034188A1 (en) * 2002-03-20 2005-02-10 J. R. Simplot Company Refined plant transformation
WO2003079765A2 (en) * 2002-03-20 2003-10-02 J.R. Simplot Company Refined plant transformation
CN100497378C (en) 2002-03-22 2009-06-10 拜尔生物科学公司 Novel bacillus thuringiensis insecticidal proteins
US7452666B2 (en) * 2002-03-25 2008-11-18 Lawrence Livermore National Security, Llc Synthesis of DNA
US20030186301A1 (en) * 2002-03-25 2003-10-02 The Regents Of The University Of California Constructing user-defined, long DNA sequences
US20030228598A1 (en) * 2002-03-25 2003-12-11 The Regents Of The University Of California DNA/RNA as a write/read medium
US20030180781A1 (en) * 2002-03-25 2003-09-25 The Regents Of The University Of California Constructing very long DNA sequences from synthetic DNA molecules
AU2003218456A1 (en) * 2002-04-01 2003-10-20 Human Genome Sciences, Inc. Antibodies that specifically bind to gmad
WO2003087763A2 (en) 2002-04-03 2003-10-23 Celltech R & D, Inc. Association of polymorphisms in the sost gene region with bone mineral density
WO2003086458A1 (en) 2002-04-12 2003-10-23 Medimmune, Inc. Recombinant anti-interleukin-9 antibodies
EP1497418B1 (en) 2002-04-19 2012-10-17 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
US7226771B2 (en) 2002-04-19 2007-06-05 Diversa Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
US6776751B2 (en) * 2002-04-22 2004-08-17 Kendor Laboratory Products, Lp Rotor cover attachment apparatus
US7442506B2 (en) 2002-05-08 2008-10-28 Ravgen, Inc. Methods for detection of genetic disorders
US20070178478A1 (en) * 2002-05-08 2007-08-02 Dhallan Ravinder S Methods for detection of genetic disorders
US7727720B2 (en) 2002-05-08 2010-06-01 Ravgen, Inc. Methods for detection of genetic disorders
DE60333285D1 (en) 2002-05-14 2010-08-19 Martek Biosciences Corp CAROTIN SYNTHASE GENE AND ITS USE
DK1504098T4 (en) * 2002-05-17 2011-05-23 Alligator Bioscience Ab Process for molecular evolution in vitro of protein function
DE10222858A1 (en) 2002-05-23 2003-12-04 Basf Ag Process for the fermentative production of sulfur-containing fine chemicals
US7132100B2 (en) 2002-06-14 2006-11-07 Medimmune, Inc. Stabilized liquid anti-RSV antibody formulations
US7425618B2 (en) 2002-06-14 2008-09-16 Medimmune, Inc. Stabilized anti-respiratory syncytial virus (RSV) antibody formulations
DE60319259T2 (en) * 2002-06-14 2009-03-05 Dyax Corp., Cambridge Recombination of nucleic acid library members
US7667099B2 (en) * 2002-06-20 2010-02-23 Board Of Trustees Of Michigan State University Plastid division and related genes and proteins, and methods of use
MXPA04012496A (en) * 2002-06-21 2005-09-12 Novo Nordisk Healthcare Ag Pegylated factor vii glycoforms.
CN1684974A (en) 2002-06-28 2005-10-19 美国陶氏益农公司 Pesticidally active proteins and polynucleotides obtainable from paenibacillus species
US20060002935A1 (en) 2002-06-28 2006-01-05 Domantis Limited Tumor Necrosis Factor Receptor 1 antagonists and methods of use therefor
EP2366718A3 (en) * 2002-06-28 2012-05-02 Domantis Limited Ligand
US9321832B2 (en) 2002-06-28 2016-04-26 Domantis Limited Ligand
US7615678B2 (en) 2002-07-18 2009-11-10 Monsanto Technology Llc Methods for using artificial polynucleotides and compositions thereof to reduce transgene silencing
JP2005532829A (en) * 2002-07-18 2005-11-04 インヴィトロジェン コーポレーション Viral vectors containing recombination sites
US7560260B2 (en) 2002-07-25 2009-07-14 Bio-Rad Laboratories, Inc. Compositions with polymerase activity
EP2264155A1 (en) 2002-07-25 2010-12-22 Bio-Rad Laboratories, Inc. Hybrid polymerase methods and compositions
AU2003247266A1 (en) 2002-08-01 2004-02-23 Nuevolution A/S Multi-step synthesis of templated molecules
CA2494798A1 (en) 2002-08-06 2005-01-13 Verdia, Inc. Ap1 amine oxidase variants
US20040067532A1 (en) 2002-08-12 2004-04-08 Genetastix Corporation High throughput generation and affinity maturation of humanized antibody
PT1534335E (en) 2002-08-14 2012-02-28 Macrogenics Inc Fcgammariib-specific antibodies and methods of use thereof
WO2004016767A2 (en) * 2002-08-19 2004-02-26 The President And Fellows Of Harvard College Evolving new molecular function
US7361635B2 (en) 2002-08-29 2008-04-22 Sangamo Biosciences, Inc. Simultaneous modulation of multiple genes
US20050202438A1 (en) * 2002-09-09 2005-09-15 Rene Gantier Rational directed protein evolution using two-dimensional rational mutagenesis scanning
ATE466085T1 (en) * 2002-09-09 2010-05-15 Hanall Pharmaceutical Co Ltd PROTEASE-RESISTANT MODIFIED INTERFERON ALPHA POLYPEPTIDES
US20060020396A1 (en) * 2002-09-09 2006-01-26 Rene Gantier Rational directed protein evolution using two-dimensional rational mutagenesis scanning
US7563600B2 (en) 2002-09-12 2009-07-21 Combimatrix Corporation Microarray synthesis and assembly of gene-length polynucleotides
WO2004031349A2 (en) 2002-09-18 2004-04-15 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
EP2042517B1 (en) 2002-09-27 2012-11-14 Xencor, Inc. Optimized FC variants and methods for their generation
GB0222846D0 (en) 2002-10-03 2002-11-06 Choo Yen Cell culture
AU2002951899A0 (en) * 2002-10-04 2002-10-24 Macquarie University Random drift mutagenesis
US6863731B2 (en) * 2002-10-18 2005-03-08 Controls Corporation Of America System for deposition of inert barrier coating to increase corrosion resistance
JP3447009B1 (en) * 2002-10-29 2003-09-16 實 平垣 Construct structure and method for producing the same
PT2348125T (en) 2002-10-30 2017-08-29 Nuevolution As Method for the synthesis of a bifunctional complex
EP1558745A4 (en) * 2002-11-01 2006-08-30 Evogenix Pty Ltd Mutagenesis methods using ribavirin and/or rna replicases
TWI319007B (en) 2002-11-06 2010-01-01 Novozymes As Subtilase variants
US7365186B2 (en) * 2002-11-22 2008-04-29 Arborgen, Llc Vascular-preferred promoter sequences and uses thereof
CN102584951A (en) 2002-11-25 2012-07-18 金克克国际有限公司 Skin or hair binding peptides
AU2003298920A1 (en) * 2002-12-04 2004-06-23 Applied Molecular Evolution, Inc. Butyrylcholinesterase variants that alter the activity of chemotherapeutic agents
DE60330406D1 (en) 2002-12-19 2010-01-14 Nuevolution As THROUGH QUASIZATIONAL STRUCTURES AND FUNCTIONS OF SYNTHESIS METHOD
DE60328715D1 (en) 2002-12-20 2009-09-17 Novozymes As POLYPEPTIDES USING CELLOBIOHYDROLASE II ACTIVITY AND POLYNUCLEOTIDES THAT CODE
US7537921B2 (en) 2002-12-20 2009-05-26 Novozymes A/S Galactanase variants
US20050221443A1 (en) * 2003-01-06 2005-10-06 Xencor, Inc. Tumor necrosis factor super family agonists
US20050130892A1 (en) * 2003-03-07 2005-06-16 Xencor, Inc. BAFF variants and methods thereof
US7553930B2 (en) * 2003-01-06 2009-06-30 Xencor, Inc. BAFF variants and methods thereof
US20060014248A1 (en) * 2003-01-06 2006-01-19 Xencor, Inc. TNF super family members with altered immunogenicity
CA2512729C (en) 2003-01-09 2014-09-16 Macrogenics, Inc. Identification and engineering of antibodies with variant fc regions and methods of using same
CN102021206A (en) 2003-01-17 2011-04-20 丹尼斯科公司 Method of producing a carbohydrate ester
US7955814B2 (en) 2003-01-17 2011-06-07 Danisco A/S Method
DE602004030000D1 (en) 2003-01-17 2010-12-23 Danisco PROCESS FOR IN-SITU-PRODUCTION OF AN EMULSIFIER IN A FOODSTUFF
US20050196766A1 (en) 2003-12-24 2005-09-08 Soe Jorn B. Proteins
JP2006518997A (en) * 2003-01-21 2006-08-24 ブリストル−マイヤーズ スクイブ カンパニー Novel acyl coenzyme A: polynucleotide encoding monoacylglycerol acyltransferase-3 (MGAT3) and uses thereof
US8709435B2 (en) 2003-01-21 2014-04-29 Biomay Ag Hypallergenic mosaic antigens and methods of making same
NZ567340A (en) 2003-02-20 2008-10-31 Athenix Corp Delta-endotoxin genes of bacillus thuringiensis and methods for their use as pesticide
EP1597395A2 (en) 2003-02-21 2005-11-23 Nuevolution A/S Method for producing second-generation library
EP1597394A2 (en) * 2003-02-21 2005-11-23 Nuevolution A/S A method for obtaining structural information about an encoded molecule
ES2713024T3 (en) 2003-03-06 2019-05-17 Basf Enzymes Llc Amylases, nucleic acids that encode them and methods for their manufacture and use
EP1601332A4 (en) 2003-03-07 2012-05-02 Verenium Corp Hydrolases, nucleic acids encoding them and mehods for making and using them
EP1615659B1 (en) 2003-03-12 2014-04-16 Genentech, Inc. Use of bv8 and/or eg-vegf to promote hematopoiesis
JP2006523211A (en) * 2003-03-14 2006-10-12 ネオス テクノロジーズ インコーポレイテッド Branched water-soluble polymers and their composites
DK1608748T3 (en) * 2003-03-20 2009-06-29 Nuevolution As Ligation coding of small molecules
EP1610808A4 (en) 2003-03-26 2011-04-06 Sudhir Paul Covalent attachment of ligands to nucleophilic proteins guided by non-covalent binding
US8980646B2 (en) * 2003-03-26 2015-03-17 Sudhir Paul Proteolytic and covalent antibodies
WO2004087882A2 (en) * 2003-03-26 2004-10-14 Washington University Neuregulin protein regulation of synaptic proteins
US8017323B2 (en) * 2003-03-26 2011-09-13 President And Fellows Of Harvard College Free reactant use in nucleic acid-templated synthesis
US8246957B2 (en) * 2003-03-27 2012-08-21 Sudhir Paul Lupus antibodies for passive immunotherapy of HIV/AIDS
ES2545639T3 (en) * 2003-04-04 2015-09-14 Basf Enzymes Llc Pectate liases, nucleic acids that encode them and methods for their preparation and use
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
KR20110094361A (en) 2003-04-11 2011-08-23 메디뮨 엘엘씨 Recombinant il-9 antibodies and uses thereof
WO2004092418A2 (en) 2003-04-14 2004-10-28 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
US20040214194A1 (en) * 2003-04-17 2004-10-28 Mj Bioworks, Inc. Methods of making hybrid proteins
AU2004235346A1 (en) 2003-04-25 2004-11-11 North Carolina State University Lactobacillus acidophilus nucleic acid sequences encoding cell surface protein homologues and uses therefore
US7834147B2 (en) * 2003-04-28 2010-11-16 Childrens Hospital Medical Center Saposin C-DOPS: a novel anti-tumor agent
CN1863914B (en) 2003-04-29 2011-03-09 先锋高级育种国际公司 Novel glyphosate-n-acetyltransferase (GAT) genes
EP1622921B1 (en) 2003-05-02 2010-06-16 Novozymes Inc. Variants of beta-glucosidases
US20050009772A1 (en) * 2003-05-06 2005-01-13 The Regents Of The University Of California Methods and compositions for the treatment of glaucoma and other retinal diseases
DE602004016314D1 (en) 2003-05-07 2008-10-16 Maxygen Inc ENZYM VARIANTS OF SUBTILISIN (SUBTILASES)
EP1624847B1 (en) * 2003-05-09 2012-01-04 BioGeneriX AG Compositions and methods for the preparation of human growth hormone glycosylation mutants
US7521242B2 (en) 2003-05-09 2009-04-21 The United States Of America As Represented By The Department Of Health And Human Services Host cells deficient for mismatch repair and their use in methods for inducing homologous recombination using single-stranded nucleic acids
US20070073048A1 (en) 2003-05-15 2007-03-29 Ying Lian Hiv polynucleotides and polypeptides derived from botswana mj4
US7173010B2 (en) * 2003-05-19 2007-02-06 Regents Of The University Of Michigan Orphanin FQ receptor
US7655833B2 (en) * 2003-05-29 2010-02-02 Brookhaven Science Associates, Llc ADS genes for reducing saturated fatty acid levels in seed oils
WO2005001051A2 (en) * 2003-06-06 2005-01-06 Arborgen Llc. Compositions and methods for regulating polysaccharides of a plant cell
CA2528536A1 (en) * 2003-06-06 2005-01-06 Arborgen, Llc. Transcription factors
CN1835974A (en) 2003-06-16 2006-09-20 细胞技术研究与发展公司 Antibodies specific for sclerostin and methods for increasing bone mineralization
WO2005017106A2 (en) * 2003-06-17 2005-02-24 California Institute Of Technology Libraries of optimized cytochrome p450 enzymes and the optimized p450 enzymes
WO2005017105A2 (en) 2003-06-17 2005-02-24 California University Of Technology Regio- and enantioselective alkane hydroxylation with modified cytochrome p450
EP1633865B1 (en) 2003-06-18 2011-09-28 Bayer Pharma Aktiengesellschaft New biological entities and the use thereof
EP1639106B1 (en) 2003-06-19 2010-05-26 Novozymes A/S Proteases
AU2004252528B8 (en) 2003-06-23 2010-08-05 North Carolina State University Lactobacillus acidophilus nucleic acids encoding fructo-oligosaccharide utilization compounds and uses thereof
US20050095615A1 (en) * 2003-06-26 2005-05-05 Welch Peter J. Methods and compositions for detecting promoter activity and expressing fusion proteins
US7960148B2 (en) 2003-07-02 2011-06-14 Verenium Corporation Glucanases, nucleic acids encoding them and methods for making and using them
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
CA2535526C (en) 2003-08-11 2015-09-29 Diversa Corporation Laccases, nucleic acids encoding them and methods for making and using them
DK1660646T3 (en) * 2003-08-11 2015-03-09 California Inst Of Techn Thermostable peroxide-driven cytochrome P450 oxygenase variants and methods of use
JP4934426B2 (en) 2003-08-18 2012-05-16 メディミューン,エルエルシー Antibody humanization
US20060228350A1 (en) * 2003-08-18 2006-10-12 Medimmune, Inc. Framework-shuffling of antibodies
WO2005021586A2 (en) * 2003-08-21 2005-03-10 Wisconsin Alumni Research Foundation Metabolic engineering of viomycin biosynthesis
EP2617826B1 (en) 2003-08-25 2015-08-12 Novozymes Inc. Variants of glycoside hydrolases
US7354734B2 (en) * 2003-08-25 2008-04-08 Funzyme Biotechnologies Sa Fungal proteins and nucleic acids encoding same
WO2005023993A2 (en) 2003-09-09 2005-03-17 Integrigen, Inc. Methods and compositions for generation of germline human antibody genes
DK1670939T3 (en) 2003-09-18 2010-03-01 Nuevolution As Method for obtaining structural information on a coded molecule and method for selecting compounds
EP1689430B1 (en) * 2003-10-07 2009-07-29 United Therapeutics Corporation Tyrosinase mutant and methods of use thereof
EP2308966A1 (en) 2003-10-10 2011-04-13 Novozymes A/S Protease variants
WO2005035564A2 (en) 2003-10-10 2005-04-21 Xencor, Inc. Protein based tnf-alpha variants for the treatment of tnf-alpha related disorders
US20050084868A1 (en) * 2003-10-16 2005-04-21 Wolfgang Aehle Generation of stabilized proteins by combinatorial consensus mutagenesis
EP1678296B1 (en) 2003-10-23 2011-07-13 Novozymes A/S Protease with improved stability in detergents
US7244605B2 (en) 2003-10-28 2007-07-17 Novozymes, Inc. Polypeptides having beta-glucosidase activity and polynucleotides encoding same
CA2542574C (en) 2003-11-12 2014-03-18 E. I. Du Pont De Nemours And Company Delta-15 desaturases suitable for altering levels of polyunsaturated fatty acids in oleaginous plants and yeast
WO2005052161A2 (en) 2003-11-19 2005-06-09 Genencor International, Inc. Serine proteases, nucleic acids encoding serine enzymes and vectors and host cells incorporating same
US7985569B2 (en) 2003-11-19 2011-07-26 Danisco Us Inc. Cellulomonas 69B4 serine protease variants
AU2003283943A1 (en) * 2003-11-20 2005-06-08 Agency For Science Technology And Research Method
EP1694347B1 (en) * 2003-11-24 2013-11-20 BioGeneriX AG Glycopegylated erythropoietin
US20080305992A1 (en) 2003-11-24 2008-12-11 Neose Technologies, Inc. Glycopegylated erythropoietin
US8633157B2 (en) * 2003-11-24 2014-01-21 Novo Nordisk A/S Glycopegylated erythropoietin
EP1697534B1 (en) * 2003-12-01 2010-06-02 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
US7956032B2 (en) 2003-12-03 2011-06-07 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
BRPI0417342A (en) * 2003-12-03 2007-04-17 Neose Technologies Inc glycopeguylated granulocyte colony stimulating factor
US20060040856A1 (en) * 2003-12-03 2006-02-23 Neose Technologies, Inc. Glycopegylated factor IX
ATE352616T1 (en) * 2003-12-04 2007-02-15 Hoffmann La Roche METHOD FOR PRODUCING CIRCULAR MUTATED AND/OR CHIMERIC POLYNUCLEOTIDES
US20070169227A1 (en) 2003-12-16 2007-07-19 Pioneer Hi-Bred International Inc. Dominant Gene Suppression Transgenes and Methods of Using Same
GB0716126D0 (en) 2007-08-17 2007-09-26 Danisco Process
AU2004312215B2 (en) 2003-12-24 2009-03-05 Dupont Nutrition Biosciences Aps Proteins
US7718408B2 (en) 2003-12-24 2010-05-18 Danisco A/S Method
ES2560657T3 (en) * 2004-01-08 2016-02-22 Ratiopharm Gmbh O-linked glycosylation of G-CSF peptides
US7432057B2 (en) * 2004-01-30 2008-10-07 Michigan State University Genetic test for PSE-susceptible turkeys
EP2305703B1 (en) 2004-01-30 2014-03-12 Novozymes Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2006078256A2 (en) 2004-02-12 2006-07-27 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
AU2005215852B2 (en) 2004-02-25 2010-04-22 Novozymes A/S Fungal cell wall degrading enzyme
AU2005216528B2 (en) 2004-02-25 2009-06-04 Pioneer Hi-Bred International, Inc. Novel Bacillus thuringiensis crystal polypeptides, polynucleotides, and compositions thereof
US20060127920A1 (en) * 2004-02-27 2006-06-15 President And Fellows Of Harvard College Polynucleotide synthesis
US7459289B2 (en) 2004-03-08 2008-12-02 North Carolina State University Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor
GB0405637D0 (en) 2004-03-12 2004-04-21 Danisco Protein
US7569223B2 (en) * 2004-03-22 2009-08-04 The Rockefeller University Phage-associated lytic enzymes for treatment of Streptococcus pneumoniae and related conditions
RU2381813C2 (en) 2004-03-22 2010-02-20 Зольвай Фармасьютиклз Гмбх Oral pharmaceutical compositions based on products containing lipases, first of all pancreatine, and surfactants
WO2005090566A2 (en) * 2004-03-22 2005-09-29 Nuevolution A/S Ligational encoding using building block oligonucleotides
US7973139B2 (en) * 2004-03-26 2011-07-05 Human Genome Sciences, Inc. Antibodies against nogo receptor
EP1732650A4 (en) * 2004-03-27 2008-06-11 Univ Arizona Composition and method for cancer treatment
RU2409938C2 (en) 2004-04-02 2011-01-27 КРОПДИЗАЙН Н.Фи. Method for increase of plant seeds yield, method for production of transgenic plant characterised by increased seeds yield, gene structure for expression in plant and transgenic plant
WO2005115477A2 (en) * 2004-04-13 2005-12-08 Quintessence Biosciences, Inc. Non-natural ribonuclease conjugates as cytotoxic agents
BRPI0509460B8 (en) 2004-04-30 2022-06-28 Dow Agrosciences Llc herbicide resistance genes
US7622125B2 (en) * 2004-05-05 2009-11-24 Novartis Vaccines And Diagnostics, Inc. Polycistronic HIV vector constructs
KR101083595B1 (en) * 2004-05-14 2011-11-16 가톨릭대학교 산학협력단 Recombinant baculoviral vector containing protein transduction domain gene
US7674621B2 (en) 2004-05-21 2010-03-09 The United States Of America As Represented By The Department Of Health And Human Services Plasmids and phages for homologous recombination and methods of use
CN1993475A (en) 2004-05-27 2007-07-04 诺维信股份有限公司 Methods for transforming and expression screening of filamentous fungal cells with a DNA library
US7825293B2 (en) 2004-05-28 2010-11-02 Cropdesign N.V. Plants having improved growth characteristics and a method for making the same
BRPI0511868A (en) 2004-06-09 2008-01-15 Pioneer Hi Bred Internacional isolated peptide, fusion polypeptide, isolated nucleic acid molecules, vectors, polypeptide targeting methods, and peptide identification method
EA013993B1 (en) 2004-06-16 2010-08-30 Верениум Корпорейшн Compositions and methods for enzymatic de-colorization of chlorophyll
US20060008844A1 (en) 2004-06-17 2006-01-12 Avidia Research Institute c-Met kinase binding proteins
CA2591858C (en) 2004-06-21 2015-05-05 Novozymes A/S Proteases derived from norcardiopsis
US7985892B1 (en) 2004-06-29 2011-07-26 Dow Agrosciences Llc Truncated Cry35 proteins
WO2006005032A2 (en) 2004-06-29 2006-01-12 Novozymes, Inc. Polypeptides having alpha-glucosidase activity and polynucleotides encoding same
EP1781795B1 (en) 2004-06-30 2011-01-19 Pioneer-Hi-Bred International, Inc. Methods of protecting plants from pathogenic fungi
BRPI0512920B1 (en) 2004-07-02 2017-05-16 Du Pont expression cassette, transformed microorganism, method for inducing plant pathogen resistance in a plant, anti-pathogenic composition and method for protecting a plant against a plant pathogen
US20080193999A1 (en) 2004-07-05 2008-08-14 Novozymes A/S Alpha-Amylase Variants With Altered Properties
BRPI0513329A (en) 2004-07-12 2008-05-13 Cropdesign Nv method for improving plant growth characteristics, plants, construction, method for producing a transgenic plant, transgenic plant, collectible parts of a plant, products, and use of a yippee-like gene or nucleic acid or variant thereof or use of a yippee-like or homologous polypeptide thereof
US20080300173A1 (en) 2004-07-13 2008-12-04 Defrees Shawn Branched Peg Remodeling and Glycosylation of Glucagon-Like Peptides-1 [Glp-1]
PL1791933T3 (en) 2004-07-16 2011-12-30 Dupont Nutrition Biosci Aps Enzymatic oil-degumming method
US20090053249A1 (en) 2004-07-20 2009-02-26 Yan Qi Specific Inhibition of Autoimmunity and Diseases Associated With Autoantigens
WO2006020372A2 (en) * 2004-07-23 2006-02-23 Neose Technologies, Inc. Enzymatic modification of glycopeptides
CA2575878A1 (en) 2004-08-02 2006-02-09 Novozymes A/S Creation of diversity in polypeptides
WO2006017694A1 (en) * 2004-08-05 2006-02-16 Biosite Incorporated Compositions and methods for phage display of polypeptides
US7597884B2 (en) 2004-08-09 2009-10-06 Alios Biopharma, Inc. Hyperglycosylated polypeptide variants and methods of use
US7148051B2 (en) * 2004-08-16 2006-12-12 E. I. Du Pont De Nemours And Company Production of 3-hydroxycarboxylic acid using nitrilase
FI20050753A (en) 2004-09-03 2006-03-04 Licentia Oy New peptides
WO2006031811A2 (en) * 2004-09-10 2006-03-23 Neose Technologies, Inc. Glycopegylated interferon alpha
US7728118B2 (en) 2004-09-17 2010-06-01 Promega Corporation Synthetic nucleic acid molecule compositions and methods of preparation
US7563443B2 (en) * 2004-09-17 2009-07-21 Domantis Limited Monovalent anti-CD40L antibody polypeptides and compositions thereof
JP2008513540A (en) 2004-09-21 2008-05-01 メディミューン,インコーポレーテッド Antibody to respiratory syncytial virus and method for producing vaccine for the virus
US7453025B2 (en) 2004-09-22 2008-11-18 Arborgen, Llc Reproductive ablation constructs
US7799906B1 (en) 2004-09-22 2010-09-21 Arborgen, Llc Compositions and methods for modulating lignin of a plant
GB0421598D0 (en) 2004-09-29 2004-10-27 Cambridge Advanced Tech Modification of plant development and morphology
WO2006049777A2 (en) * 2004-09-30 2006-05-11 Trustees Of Dartmouth College Nucleic acid sequences encoding luciferase for expression in filamentous fungi
EP2298872A3 (en) 2004-09-30 2011-08-10 Novozymes A/S Polypeptides having lipase activity and polynucleotides encoding same
GB0422052D0 (en) 2004-10-04 2004-11-03 Dansico As Enzymes
ES2614744T3 (en) 2004-10-04 2017-06-01 Novozymes A/S Polypeptides with phytase activity and polynucleotides encoding them
AR050895A1 (en) 2004-10-04 2006-11-29 Novozymes As POLYPEPTIDES THAT HAVE FITASA ACTIVITY AND POLYUCLEOTIDES THAT CODE THEM
EP2166098B1 (en) 2004-10-05 2013-11-06 SunGene GmbH Constitutive expression cassettes for regulation of plant expression
RU2401842C2 (en) 2004-10-08 2010-10-20 Домантис Лимитед Antagonists and method of using said antagonists
MX2007004231A (en) 2004-10-08 2007-06-14 Dow Agrosciences Llc Certain plants with no saturate or reduced saturate levels of fatty acids in seeds, and oil derived from the seeds.
GB0423139D0 (en) 2004-10-18 2004-11-17 Danisco Enzymes
JP2008518023A (en) 2004-10-27 2008-05-29 メディミューン,インコーポレーテッド Regulation of antibody specificity by altering affinity for cognate antigens
WO2006047791A2 (en) * 2004-10-27 2006-05-04 The Board Of Trustees Of The Leland Stanford Junior University Dna-templated combinatorial library device and method for use
WO2006047669A2 (en) * 2004-10-27 2006-05-04 Monsanto Technology Llc Non-random method of gene shuffling
WO2006045829A1 (en) 2004-10-29 2006-05-04 Cropdesign N.V. Plants having improved growth characteristics and method for making the same
EP2586456B1 (en) * 2004-10-29 2016-01-20 ratiopharm GmbH Remodeling and glycopegylation of fibroblast growth factor (FGF)
EP1655364A3 (en) 2004-11-05 2006-08-02 BASF Plant Science GmbH Expression cassettes for seed-preferential expression in plants
CA2590245A1 (en) 2004-11-11 2006-05-18 Modular Genetics, Inc. Ladder assembly and system for generating diversity
EP1814996A2 (en) 2004-11-19 2007-08-08 Novozymes A/S Polypeptides having antimicrobial activity and polynucleotides encoding same
EP2163631B1 (en) 2004-11-25 2013-05-22 SunGene GmbH Expression cassettes for guard cell-preferential expression in plants
EP1666599A3 (en) 2004-12-04 2006-07-12 SunGene GmbH Expression cassettes for mesophyll- and/or epidermis-preferential expression in plants
EP2072620B1 (en) 2004-12-08 2013-05-08 SunGene GmbH Expression casstettes for vascular tissue-preferential expression in plants
EP1669456A3 (en) 2004-12-11 2006-07-12 SunGene GmbH Expression cassettes for meristem-preferential expression in plants
DK2365068T3 (en) 2004-12-22 2017-05-15 Novozymes As ENZYMER FOR PROCESSING STARCH
US7198927B2 (en) * 2004-12-22 2007-04-03 E. I. Du Pont De Nemours And Company Enzymatic production of glycolic acid
WO2006071219A1 (en) 2004-12-28 2006-07-06 Pioneer Hi-Bred International, Inc. Improved grain quality through altered expression of seed proteins
EP1838332A1 (en) * 2005-01-06 2007-10-03 Neose Technologies, Inc. Glycoconjugation using saccharyl fragments
CA2593682C (en) 2005-01-10 2016-03-22 Neose Technologies, Inc. Glycopegylated granulocyte colony stimulating factor
CA2594832A1 (en) * 2005-01-13 2006-07-20 Codon Devices, Inc. Compositions and methods for protein design
CA2594728C (en) 2005-01-13 2013-03-19 Pioneer Hi-Bred International, Inc. Maize cyclo1 gene and promoter
EP1844151A2 (en) 2005-01-27 2007-10-17 CropDesign N.V. Plants having increased yield and a method for making the same
JP5651285B2 (en) 2005-02-15 2015-01-07 デューク ユニバーシティ Anti-CD19 antibodies and use in oncology
US20100029485A1 (en) 2005-03-02 2010-02-04 Instituto Nacional De Tecnologia Agropecuaria Herbicide-resistant rice plants, polynucleotides encoding herbicide-resistant acetohydroxyacid synthase large subunit proteins, and methods of use
WO2006099207A2 (en) 2005-03-10 2006-09-21 Diversa Corporation Lyase enzymes, nucleic acids encoding them and methods for making and using them
CA2861310A1 (en) 2005-03-15 2006-09-28 Bp Corporation North America Inc. Cellulases, nucleic acids encoding them and methods for making and using them
CN100577805C (en) * 2005-03-16 2010-01-06 中国科学院沈阳应用生态研究所 Method of modifying gene and obtained recombinant gene and encoded protin
AR052947A1 (en) 2005-03-18 2007-04-11 Novozymes As POLYPEPTIDES THAT HAVE ANTIMICROBIAL ACTIVITY AND POLINUCLEOTIDES THAT CODE FOR THE SAME
AU2006227377B2 (en) 2005-03-18 2013-01-31 Medimmune, Llc Framework-shuffling of antibodies
AU2006226192A1 (en) 2005-03-19 2006-09-28 Medical Research Council Improvements in or relating to treatment and prevention of viral infections
US8715988B2 (en) * 2005-03-28 2014-05-06 California Institute Of Technology Alkane oxidation by modified hydroxylases
US11214817B2 (en) 2005-03-28 2022-01-04 California Institute Of Technology Alkane oxidation by modified hydroxylases
ATE548378T1 (en) 2005-04-01 2012-03-15 Athenix Corp AXMI-036 A DELTA-ENDOTOXIN GENE AND METHOD OF USE THEREOF
US20070154992A1 (en) * 2005-04-08 2007-07-05 Neose Technologies, Inc. Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
AU2006236439B2 (en) 2005-04-15 2012-05-03 Macrogenics, Inc. Covalent diabodies and uses thereof
WO2012018687A1 (en) 2010-08-02 2012-02-09 Macrogenics, Inc. Covalent diabodies and uses thereof
BRPI0610609A2 (en) 2005-04-15 2010-07-06 Univ North Carolina State methods and compositions for modulating bacterial adhesion and stress tolerance
CA2606220A1 (en) 2005-04-19 2006-12-21 Basf Plant Science Gmbh Starchy-endosperm and/or germinating embryo-specific expression in mono-cotyledonous plants
WO2006116682A2 (en) 2005-04-27 2006-11-02 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
US8003108B2 (en) 2005-05-03 2011-08-23 Amgen Inc. Sclerostin epitopes
US7592429B2 (en) 2005-05-03 2009-09-22 Ucb Sa Sclerostin-binding antibody
EP2221316A1 (en) 2005-05-05 2010-08-25 Duke University Anti-CD19 antibody therapy for autoimmune disease
CN1861789B (en) * 2005-05-13 2010-04-28 中国科学院沈阳应用生态研究所 Integrated gene recombined process, recombined gene and coding protein obtained thereby
AR053269A1 (en) 2005-05-16 2007-04-25 Monsanto Technology Llc CORN PLANTS AND SEEDS WITH IMPROVEMENT OF ASPARAGIN AND PROTEIN
WO2006127423A2 (en) * 2005-05-18 2006-11-30 Codon Devices, Inc. Methods of producing polynucleotide libraries using scarless ligation
US20060263974A1 (en) * 2005-05-18 2006-11-23 Micron Technology, Inc. Methods of electrically interconnecting different elevation conductive structures, methods of forming capacitors, methods of forming an interconnect between a substrate bit line contact and a bit line in DRAM, and methods of forming DRAM memory cell
US20060271262A1 (en) * 2005-05-24 2006-11-30 Mclain Harry P Iii Wireless agricultural network
US20110003744A1 (en) * 2005-05-25 2011-01-06 Novo Nordisk A/S Glycopegylated Erythropoietin Formulations
US20080255026A1 (en) * 2005-05-25 2008-10-16 Glycopegylated Factor 1X Glycopegylated Factor Ix
WO2006127910A2 (en) * 2005-05-25 2006-11-30 Neose Technologies, Inc. Glycopegylated erythropoietin formulations
WO2006133013A2 (en) * 2005-06-03 2006-12-14 The Regents Of The University Of California Methods of generating protein variants with altered function
PL2314623T3 (en) 2005-06-21 2012-11-30 Xoma Us Llc IL-1beta binding antibodies and fragments thereof
KR20080025174A (en) 2005-06-23 2008-03-19 메디뮨 인코포레이티드 Antibody formulations having optimized aggregation and fragmentation profiles
US7582291B2 (en) * 2005-06-30 2009-09-01 The Rockefeller University Bacteriophage lysins for Enterococcus faecalis, Enterococcus faecium and other bacteria
EA201301103A1 (en) 2005-07-01 2014-02-28 Басф Се RESISTANT TO HERBICIDES OF A SUNFLOWER PLANT, POLYNUCLEOTES ARE RESOLVERS ARE RESISTANT TO A HERBICIDES LARGE SUB-UNIFICATIONS OF ACETOHYDROXY-SYLOTIC SYNTHASIS PROTEIN UNITS, AND CHARACTERISTICS, AND RECORDS AND RECORDS OF THE SURFACE, RESISTANCE TO THE SUNFLOWER PLANTS
EP2431472A1 (en) 2005-07-06 2012-03-21 CropDesign N.V. Plant yield improvement by STE20-like gene expression
MX2007016045A (en) 2005-07-08 2008-03-10 Novozymes As Subtilase variants.
AU2006267039A1 (en) * 2005-07-12 2007-01-18 Codon Devices, Inc. Compositions and methods for biocatalytic engineering
WO2007008951A1 (en) * 2005-07-12 2007-01-18 Codon Devices, Inc. Compositions and methods for design of non-immunogenic proteins
WO2007011722A2 (en) 2005-07-15 2007-01-25 President And Fellows Of Harvard College Reaction discovery system
CA2615797C (en) 2005-07-18 2014-04-29 Pioneer Hi-Bred International, Inc. Modified frt recombination sites and methods of use
ES2635308T3 (en) 2005-07-29 2017-10-03 Abbott Laboratories Gmbh Pancreatin with reduced viral content
UA97787C2 (en) 2005-07-29 2012-03-26 Таргитед Гроут, Инк. Dominant negative mutant krp-protein protects inhibition of active cyclin-cdk complex by wild type krp-protein
DE102005037351B3 (en) * 2005-08-08 2007-01-11 Geneart Ag In vitro method for directed evolution of proteins, useful e.g. in pharmaceutical development, uses expression system for performing translation, transcription and reverse transcription
CA2618681C (en) 2005-08-10 2015-10-27 Macrogenics, Inc. Identification and engineering of antibodies with variant fc regions and methods of using same
US20080193477A1 (en) * 2005-08-10 2008-08-14 Acambis Inc. Vaccination Against Dengue Virus Infection
US11266607B2 (en) 2005-08-15 2022-03-08 AbbVie Pharmaceuticals GmbH Process for the manufacture and use of pancreatin micropellet cores
US9198871B2 (en) 2005-08-15 2015-12-01 Abbott Products Gmbh Delayed release pancreatin compositions
US20070105755A1 (en) 2005-10-26 2007-05-10 Neose Technologies, Inc. One pot desialylation and glycopegylation of therapeutic peptides
DE602006018746D1 (en) 2005-08-24 2011-01-20 Univ Rockefeller PLY-GBS MUTANT LYSINE
PT1922333E (en) 2005-08-26 2010-07-08 Novozymes Adenium Biotech As Polypeptides having antimicrobial activity and polynucleotides encoding same
CA2621267A1 (en) 2005-09-07 2007-03-15 Nugen Technologies, Inc. Improved nucleic acid amplification procedure
US7541431B2 (en) * 2005-09-07 2009-06-02 Maine Medical Center Cristin/R-spondin ligands active in the Wnt signaling pathway and methods, compositions and kits relating thereto
AR055439A1 (en) 2005-09-15 2007-08-22 Cropdesign Nv PLANTS WITH INCREASED PERFORMANCE AND METHOD FOR PREPARING THEM
US8173613B2 (en) * 2005-09-22 2012-05-08 Maine Medical Center Modulation of mesenchymal and metastatic cell growth
EP1941023B1 (en) 2005-09-30 2017-04-05 Novozymes Inc. Methods for enhancing the degradation or conversion of cellulosic material
US7312079B1 (en) 2005-10-06 2007-12-25 Lexicon Pharmaceuticals, Inc. Variants of FAM3C
US7470532B2 (en) 2005-10-19 2008-12-30 E.I. Du Pont De Nemours And Company Mortierella alpina C16/18 fatty acid elongase
NZ595200A (en) 2005-10-28 2013-04-26 Dow Agrosciences Llc Novel herbicide resistance genes
WO2007056191A2 (en) * 2005-11-03 2007-05-18 Neose Technologies, Inc. Nucleotide sugar purification using membranes
WO2007051866A2 (en) 2005-11-07 2007-05-10 Cropdesign N.V. Plants having improved growth characteristics and a method for making the same
US8853492B2 (en) 2005-11-07 2014-10-07 Cropdesign N.V. Plants having improved growth characteristics and a method for making the same
AU2006311005B2 (en) 2005-11-08 2013-06-06 Cropdesign N.V. Plants having improved growth characteristics and a method for making the same
US20070118920A1 (en) 2005-11-09 2007-05-24 Basf Agrochemical Products B.V. Herbicide-resistant sunflower plants, polynucleotides encoding herbicide-resistant acetohydroxyacid synthase large subunit proteins, and methods of use
EP2112223A3 (en) 2005-11-10 2010-01-27 Pioneer Hi-Bred International Inc. DOF (DNA binding with one finger) sequences and method of use
US20070199096A1 (en) 2005-11-14 2007-08-23 E.I. Du Pont De Nemours And Company Compositions and Methods for Altering Alpha- and Beta-Tocotrienol Content
US20070129290A1 (en) * 2005-11-18 2007-06-07 Or Yat S Metabolite derivatives of the HDAC inhibitor FK228
GB2432366B (en) * 2005-11-19 2007-11-21 Alligator Bioscience Ab A method for in vitro molecular evolution of protein function
GB0526664D0 (en) * 2005-11-30 2006-02-08 Plasticell Ltd Method
DK3305900T3 (en) 2005-12-01 2021-10-25 Nuevolution As ENZYMATIC ENCODING METHODS FOR EFFICIENT SYNTHESIS OF LARGE LIBRARIES
AU2006320596B2 (en) 2005-12-01 2013-02-07 Cropdesign N.V. Plants having improved growth characteristics and methods for making the same
AR057205A1 (en) 2005-12-01 2007-11-21 Athenix Corp GRG23 AND GRG51 GENES THAT CONFERENCE RESISTANCE TO HERBICIDES
WO2007065035A2 (en) * 2005-12-02 2007-06-07 Synthetic Genomics, Inc. Synthesis of error-minimized nucleic acid molecules
KR100784478B1 (en) * 2005-12-05 2007-12-11 한국과학기술원 A Prepartion method for a protein with new function through simultaneous incorporation of functional elements
US20080313769A9 (en) 2006-01-12 2008-12-18 Athenix Corporation EPSP synthase domains conferring glyphosate resistance
EP1987142A4 (en) 2006-02-02 2009-07-15 Verenium Corp Esterases and related nucleic acids and methods
MY160772A (en) 2006-02-10 2017-03-15 Verenium Corp Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them
BRPI0707784B1 (en) 2006-02-14 2018-05-22 Verenium Corporation ISOLATED, SYNTHETIC OR RECOMBINANT NUCLEIC ACID, EXPRESSION CASSETTE, CLONING VECTOR OR VEHICLE, TRANSFORMED ISOLATED HOST CELL, AND METHOD FOR PRODUCTION OF A RECOMBINANT POLYPEPTIDE
AR059724A1 (en) 2006-03-02 2008-04-23 Athenix Corp METHODS AND COMPOSITIONS TO IMPROVE ENZYMATIC ACTIVITY IN TRANSGENIC PLANTS
WO2007103469A2 (en) 2006-03-06 2007-09-13 Aeres Biomedical Ltd. Humanized anti-cd22 antibodies and their use in treatment of oncology, transplantation and autoimmune disease
US8043837B2 (en) 2006-03-07 2011-10-25 Cargill, Incorporated Aldolases, nucleic acids encoding them and methods for making and using them
ATE548450T1 (en) 2006-03-07 2012-03-15 Verenium Corp ALDOLASES, NUCLEIC ACIDS FOR ENCODING THEM AND METHODS FOR THEIR PRODUCTION AND USE
US20070214515A1 (en) 2006-03-09 2007-09-13 E.I.Du Pont De Nemours And Company Polynucleotide encoding a maize herbicide resistance gene and methods for use
DK1999257T3 (en) 2006-03-20 2011-10-31 Novozymes Inc Polypeptides that have endo-glucosidase activity and polynucleotides encoding the same
US8703278B2 (en) * 2006-03-28 2014-04-22 Honeywell International Inc. Light weight printed wiring board
BRPI0710217A2 (en) 2006-03-30 2011-08-02 Novozymes Inc polypeptide, polynucleotide, nucleic acid construct, recombinant expression vector, recombinant host cell, methods for producing the polypeptide, for producing a mutant from a precursor cell, for producing a protein, for producing a polynucleotide, for degrading or converting a cellulosic material, to produce a substance and to inhibit the expression of a polynucleotide in a cell, mutant cell, transgenic plant, plant part or plant cell, and double stranded rna molecule
EP2441844A1 (en) 2006-03-31 2012-04-18 BASF Plant Science GmbH Plants having enhanced yield-related traits and a method for making the same
ES2387203T3 (en) 2006-04-04 2012-09-17 Novozymes A/S Phytase variants
EP1842915A1 (en) 2006-04-04 2007-10-10 Libragen Method of in vitro polynucleotide sequences shuffling by recursive circular DNA molecules fragmentation and ligation
US7557266B2 (en) 2006-04-19 2009-07-07 Pioneer Hi-Bred International, Inc. Isolated polynucleotide molecules corresponding to mutant and wild-type alleles of the maize D9 gene and methods of use
US9539303B2 (en) * 2006-04-24 2017-01-10 Celgene Corporation Treatment of Ras-expressing tumors
ATE497539T1 (en) 2006-05-16 2011-02-15 Pioneer Hi Bred Int ANTIFUNGAL POLYPEPTIDES
EP2308986B1 (en) 2006-05-17 2014-10-08 Pioneer Hi-Bred International Inc. Artificial plant minichromosomes
WO2007136834A2 (en) * 2006-05-19 2007-11-29 Codon Devices, Inc. Combined extension and ligation for nucleic acid assembly
US10072256B2 (en) 2006-05-22 2018-09-11 Abbott Products Gmbh Process for separating and determining the viral load in a pancreatin sample
EP2423317A3 (en) 2006-05-30 2012-05-30 CropDesign N.V. Plants with modulated expression of RAN binding protein (RANB) having enhanced yield-related traits and a method for making the same
US8957027B2 (en) * 2006-06-08 2015-02-17 Celgene Corporation Deacetylase inhibitor therapy
EP2035555A2 (en) 2006-06-08 2009-03-18 BASF Plant Science GmbH Plants having improved growth characteristics and method for making the same
AR061491A1 (en) 2006-06-15 2008-09-03 Athenix Corp A FAMILY OF PESTICIDE PROTEINS AND METHODS OF THE SAME USE
EP2599870A3 (en) 2006-06-15 2013-09-11 CropDesign N.V. Plants having enhanced yield-related traits and a method for making the same
WO2007144190A2 (en) 2006-06-15 2007-12-21 Cropdesign N.V. Plants with modulated expression of nac transcription factors having enhanced yield-related traits and a method for making the same
CA3189669A1 (en) 2006-06-19 2008-02-21 22Nd Century Limited, Llc Nucleic acid encoding n-methylputrescine oxidase and uses thereof
WO2007149594A2 (en) * 2006-06-23 2007-12-27 Quintessence Biosciences, Inc. Modified ribonucleases
EP2505209A1 (en) 2006-06-26 2012-10-03 MacroGenics, Inc. Fcgamma-RIIB-specific antibodies and methods of the use thereof
US7572618B2 (en) 2006-06-30 2009-08-11 Bristol-Myers Squibb Company Polynucleotides encoding novel PCSK9 variants
US20080004410A1 (en) * 2006-06-30 2008-01-03 Yu-Chin Lai Hydrophilic macromonomers having alpha,beta-conjugated carboxylic terminal group and medical devices incorporating same
US7977535B2 (en) 2006-07-12 2011-07-12 Board Of Trustees Of Michigan State University DNA encoding ring zinc-finger protein and the use of the DNA in vectors and bacteria and in plants
CN101489412B (en) 2006-07-13 2013-03-27 诺维信公司 Use of bacterial amylases in feed for bovine animals
JP2009543868A (en) 2006-07-17 2009-12-10 クインテセンス バイオサイエンシーズ インコーポレーティッド Methods and compositions for cancer treatment
US20080280818A1 (en) * 2006-07-21 2008-11-13 Neose Technologies, Inc. Glycosylation of peptides via o-linked glycosylation sequences
US20100167040A1 (en) * 2006-07-25 2010-07-01 Bayer Bioscience N.V. Identification of a novel type of sucrose synthase and use thereof in fiber modification
EP2546262A1 (en) 2006-08-02 2013-01-16 CropDesign N.V. Modifying the content of storage compounds in seeds by expression of a Class II HD-Zip transcription factor
CA2659414A1 (en) 2006-08-02 2008-02-07 Cropdesign N.V. Use of synovial sarcoma translocation (syt) polypeptide for increasing plant yield under abiotic stress
CN101528766A (en) 2006-08-04 2009-09-09 维莱尼姆公司 Glucanases, nucleic acids encoding them and methods for making and using them
US8252559B2 (en) * 2006-08-04 2012-08-28 The California Institute Of Technology Methods and systems for selective fluorination of organic molecules
US8026085B2 (en) * 2006-08-04 2011-09-27 California Institute Of Technology Methods and systems for selective fluorination of organic molecules
US7968691B2 (en) 2006-08-23 2011-06-28 Danisco Us Inc. Pullulanase variants with increased productivity
MY162024A (en) 2006-08-28 2017-05-31 La Jolla Inst Allergy & Immunology Antagonistic human light-specific human monoclonal antibodies
WO2008027558A2 (en) * 2006-08-31 2008-03-06 Codon Devices, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
PE20081216A1 (en) 2006-09-01 2008-09-04 Therapeutic Human Polyclonals Inc ENHANCED EXPRESSION OF HUMAN OR HUMANIZED IMMUNOGLOBULIN IN NON-HUMAN TRANSGENIC ANIMALS
WO2008030735A2 (en) 2006-09-06 2008-03-13 Janssen Pharmaceutical N.V. Biomarkers for assessing response to c-met treatment
ES2383710T3 (en) 2006-09-08 2012-06-25 Medimmune, Llc Humanized anti-CD19 antibodies and their use in the treatment of tumors, transplants and autoimmune diseases
EP2617729B1 (en) 2006-09-21 2016-03-16 BASF Enzymes LLC Phytases, nucleic acids encoding them and methods for making and using them
CA2663001A1 (en) 2006-09-21 2008-03-27 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
CA2663094C (en) 2006-09-29 2016-03-22 Novozymes A/S Xylanases for animal feed
WO2008057683A2 (en) * 2006-10-03 2008-05-15 Novo Nordisk A/S Methods for the purification of polypeptide conjugates
JP5457185B2 (en) * 2006-10-04 2014-04-02 ノヴォ ノルディスク アー/エス Glycerol-linked PEGylated sugars and glycopeptides
GB0620715D0 (en) 2006-10-18 2006-11-29 Univ Durham Ethanol production
US20100162433A1 (en) 2006-10-27 2010-06-24 Mclaren James Plants with improved nitrogen utilization and stress tolerance
US20100036091A1 (en) * 2006-11-10 2010-02-11 Amgen Inc. Antibody-based diagnostics and therapeutics
ES2397637T3 (en) 2006-11-10 2013-03-08 Massachusetts Institute Of Technology PAK inhibitors for use in the treatment of neurodevelopmental disorders
WO2008133722A2 (en) * 2006-11-10 2008-11-06 Ucb Pharma S.A. Anti human sclerostin antibodies
CA2664729A1 (en) 2006-11-16 2008-05-22 Basf Plant Science Gmbh Plants having enhanced yield-related traits and a method for making the same using consensus sequences from the yabby protein family
MX2009005280A (en) 2006-11-24 2009-08-12 Cropdesign Nv Transgenic plants comprising as transgene a class i tcp or clavata 1 (clv1) or cah3 polypeptide having increased seed yield and a method for making the same.
EP2099818A2 (en) 2006-11-29 2009-09-16 Novozymes Inc. Bacillus licheniformis chromosome
CN101595214B (en) 2006-11-29 2013-06-12 诺维信股份有限公司 Methods of improving the introduction of DNA into bacterial cells
MX2009006093A (en) * 2006-12-07 2009-06-17 Dow Agrosciences Llc Novel selectable marker genes.
EP2102366A4 (en) 2006-12-10 2010-01-27 Dyadic International Inc Expression and high-throughput screening of complex expressed dna libraries in filamentous fungi
US9862956B2 (en) 2006-12-10 2018-01-09 Danisco Us Inc. Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi
WO2008071767A1 (en) 2006-12-15 2008-06-19 Cropdesign N.V. Plants having enhanced seed yield-related traits and a method for making the same
WO2008074116A1 (en) 2006-12-20 2008-06-26 Alellyx S.A. Nucleic acid molecules encoding plant proteins in the c3hc4 family and methods for the alteration of plant cellulose and lignin content
EP2094852B1 (en) 2006-12-20 2014-01-22 Monsanto do Brasil LTDA Nucleic acid constructs and methods for altering plant fiber length and/or plant height
DK3101128T3 (en) 2006-12-21 2019-07-08 Basf Enzymes Llc AMYLASES AND GLUCOAMYLASES, NUCLEAR ACIDS CODING FOR THESE, AND METHODS OF MANUFACTURE AND USE THEREOF
EP2069505A2 (en) 2006-12-21 2009-06-17 BASF Plant Science GmbH Plants having enhanced yield-related traits and a method for method for making the same
EP2815761A1 (en) * 2006-12-29 2014-12-24 Celgene Corporation Romidepsin-based treatments for cancer
BRPI0720991A8 (en) * 2006-12-29 2016-03-15 Gloucester Pharmaceuticals Inc ROMIDEPSIN PREPARATION
DE102007003143A1 (en) 2007-01-16 2008-07-17 Henkel Kgaa New alkaline protease from Bacillus gibsonii and detergents and cleaners containing this novel alkaline protease
CN101652474B (en) 2007-01-25 2012-06-27 丹尼斯科有限公司 Production of a lipid acyltransferase from transformed bacillus licheniformis cells
WO2008095033A2 (en) 2007-01-30 2008-08-07 Verenium Corporation Enzymes for the treatment of lignocellulosics, nucleic acids encoding them and methods for making and using them
EP2548963A1 (en) 2007-01-30 2013-01-23 CropDesign N.V. Plants having enhanced yield-related traits and a method for making the same
CA2673413A1 (en) 2007-01-31 2008-08-07 Basf Plant Science Gmbh Plants having enhanced yield-related traits and/or increased abiotic stress resistance, and a method for making the same
WO2008121435A2 (en) * 2007-02-02 2008-10-09 The California Institute Of Technology Methods for generating novel stabilized proteins
US8143046B2 (en) 2007-02-07 2012-03-27 Danisco Us Inc., Genencor Division Variant Buttiauxella sp. phytases having altered properties
US20080268517A1 (en) * 2007-02-08 2008-10-30 The California Institute Of Technology Stable, functional chimeric cytochrome p450 holoenzymes
EP2115130B1 (en) 2007-02-08 2011-08-03 Codexis, Inc. Ketoreductases and uses thereof
WO2009008908A2 (en) 2007-02-12 2009-01-15 Codexis, Inc. Structure-activity relationships
AU2008214568B2 (en) 2007-02-16 2013-04-18 Basf Plant Science Gmbh Nucleic acid sequences for regulation of embryo-specific expression in monocotyledonous plants
MX2009008600A (en) 2007-02-28 2009-08-21 Cropdesign Nv Plants having enhanced yield-related traits and a method for making the same.
WO2008118445A1 (en) * 2007-03-26 2008-10-02 Promega Corporation Methods to quench light from optical reactions
US7923232B2 (en) 2007-03-26 2011-04-12 Novozymes A/S Hafnia phytase
DE102007016139A1 (en) 2007-03-30 2008-10-02 Jenabios Gmbh Method for regioselective oxygenation of N-heterocycles
JP5456658B2 (en) 2007-03-30 2014-04-02 メディミューン,エルエルシー Antibody preparation
BRPI0809670A8 (en) * 2007-04-03 2018-12-18 Biogenerix Ag methods to increase stem cell production, to increase the number of granulocytes in an individual, to prevent, treat and alleviate myelosuppression that results from cancer therapy, to treat a condition in an individual, to treat neutropenia and thrombocytopenia in a mammal, to expand hematopoietic stem cells in culture, to increase hematopoiesis in an individual, to increase the number of hematopoietic progenitor cells in an individual, and to provide stable bone marrow graft, and, oral dosage form.
CN103992986A (en) 2007-04-04 2014-08-20 巴斯夫欧洲公司 Herbicide-resistant brassica plants and methods of use
ES2526646T3 (en) 2007-04-09 2015-01-14 Novozymes A/S Enzymatic treatment for the degreasing of skins and skins
MX2009011716A (en) 2007-05-03 2009-12-11 Basf Plant Science Gmbh Plants having enhanced yield-related traits and a method for making the same.
SG194368A1 (en) 2007-05-04 2013-11-29 Technophage Investigacao E Desenvolvimento Em Biotecnologia Sa Engineered rabbit antibody variable domains and uses thereof
EP2703011A3 (en) 2007-05-07 2014-03-26 MedImmune, LLC Anti-icos antibodies and their use in treatment of oncology, transplantation and autoimmune disease
EP2155910A4 (en) * 2007-05-08 2010-06-02 Univ Duke Compositions and methods for characterizing and regulating olfactory sensation
EP2145007B1 (en) 2007-05-09 2017-01-25 Dow AgroSciences LLC Novel herbicide resistance genes
WO2008141190A1 (en) * 2007-05-11 2008-11-20 Wisconsin Alumni Research Foundation Heterologous production of capreomycin and generation of new capreomycin derivatives through metabolic engineering
CN103223167B (en) 2007-05-14 2015-06-17 米迪缪尼有限公司 Methods of reducing eosinophil levels
BRPI0811185A2 (en) 2007-05-23 2014-10-07 Cropdesign Nv METHOD FOR INTENSIFYING YIELD-RELATED PLANT CHARACTERISTICS IN RELATION TO PLANTS OF CONTROL, PLANT, PART OF PLANT OR PLANT CELL, CONSTRUCTION, USE OF A CONSTRUCTION PLANT WITHIN THE CARDENTIAL CONGRENDED PLANT CONTROL PLANTS, TRANSGENIC PLANT, HARVESTING PARTS OF A PLANT, PRODUCTS DERIVED FROM A PLANT, AND USE OF A NUCLEIC ACID SEQUENCE
EP2152733A2 (en) 2007-05-25 2010-02-17 CropDesign N.V. Yield enhancement in plants by modulation of maize alfins
CN110885835B (en) 2007-05-25 2023-08-11 22世纪有限责任公司 Nucleic acid sequences encoding transcription factors regulating alkaloid synthesis and their use in improving plant metabolism
CN101778937A (en) * 2007-06-04 2010-07-14 诺和诺德公司 o-linked glycosylation using n-acetylglucosaminyl transferases
MX2009013259A (en) 2007-06-12 2010-01-25 Novo Nordisk As Improved process for the production of nucleotide sugars.
US7625555B2 (en) 2007-06-18 2009-12-01 Novagen Holding Corporation Recombinant human interferon-like proteins
KR101799337B1 (en) 2007-06-21 2017-12-20 마크로제닉스, 인크. Covalent diabodies and uses thereof
US7968811B2 (en) * 2007-06-29 2011-06-28 Harley-Davidson Motor Company Group, Inc. Integrated ignition and key switch
US20100199380A1 (en) 2007-06-29 2010-08-05 Basf Plant Science Gmbh Plants having enhanced yield-related traits and a method for making the same
EP2676678A1 (en) 2007-07-17 2013-12-25 The General Hospital Corporation Methods to identify and enrich populations of ovarian cancer stem cells and somatic stem cells and uses thereof
AR067633A1 (en) 2007-07-20 2009-10-21 Basf Plant Science Gmbh PLANTS THAT HAVE INCREASED FEATURES RELATED TO PERFORMANCE AND A METHOD FOR PRODUCING
CN101952441B (en) 2007-07-31 2013-08-14 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
AR067748A1 (en) 2007-07-31 2009-10-21 Basf Plant Science Gmbh PLANTS THAT HAVE IMPROVED FEATURES RELATED TO PERFORMANCE AND A METHOD FOR OBTAINING THEM
US7923236B2 (en) 2007-08-02 2011-04-12 Dyadic International (Usa), Inc. Fungal enzymes
CN101842159B (en) * 2007-08-09 2014-09-24 赛路拉公司 Methods and devices for correlated, multi-parameter single cell measurements and recovery of remnant biological material
WO2009029554A2 (en) 2007-08-24 2009-03-05 Codexis, Inc. Improved ketoreductase polypeptides for the stereoselective production of (r)-3-hydroxythiolane
US8207112B2 (en) * 2007-08-29 2012-06-26 Biogenerix Ag Liquid formulation of G-CSF conjugate
EP2197893B1 (en) * 2007-09-07 2013-07-24 Dyadic International, Inc. Novel fungal enzymes
US7579155B2 (en) * 2007-09-12 2009-08-25 Transgenomic, Inc. Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule
US7749708B2 (en) * 2007-09-12 2010-07-06 Transgenomic, Inc. Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule
JP5933894B2 (en) 2007-09-14 2016-06-15 アディマブ, エルエルシー Rationally designed synthetic antibody libraries and their use
US8877688B2 (en) 2007-09-14 2014-11-04 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
CA2699066A1 (en) 2007-09-14 2009-03-19 Basf Plant Science Gmbh Plants having increased yield-related traits and a method for making the same comprising expression of a growth-regulating factor (grf) polypeptide
CL2008002775A1 (en) 2007-09-17 2008-11-07 Amgen Inc Use of a sclerostin binding agent to inhibit bone resorption.
US8609386B2 (en) 2007-09-18 2013-12-17 Novozymes A/S Polypeptides having tyrosinase activity and polynucleotides encoding same
CA2700294A1 (en) 2007-09-21 2009-03-26 Basf Plant Science Gmbh Plants having increased yield-related traits and a method for making the same
US8486680B2 (en) 2007-10-03 2013-07-16 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
CA2702043A1 (en) * 2007-10-08 2009-04-16 Quintessence Biosciences, Inc. Compositions and methods for ribonuclease-based therapies
AU2008310796B2 (en) 2007-10-10 2015-03-05 BASF Agricultural Solutions Seed US LLC Synthetic genes encoding Cry1Ac
EP3181579A1 (en) 2007-10-16 2017-06-21 Athenix Corporation Axmi-066 and axmi-076: delta-endotoxin proteins and methods for their use
DE102007051092A1 (en) 2007-10-24 2009-04-30 Henkel Ag & Co. Kgaa Subtilisin from Becillus pumilus and detergents and cleaners containing this new subtilisin
CN101842489B (en) 2007-10-29 2012-12-26 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
US7618801B2 (en) * 2007-10-30 2009-11-17 Danison US Inc. Streptomyces protease
US7871802B2 (en) * 2007-10-31 2011-01-18 E.I. Du Pont De Nemours And Company Process for enzymatically converting glycolonitrile to glycolic acid
US7741088B2 (en) * 2007-10-31 2010-06-22 E.I. Dupont De Nemours And Company Immobilized microbial nitrilase for production of glycolic acid
NZ584434A (en) 2007-11-05 2011-12-22 Danisco Us Inc VARIANTS OF BACILLUS sp. TS-23 ALPHA-AMYLASE WITH ALTERED PROPERTIES
JP2011505121A (en) 2007-11-05 2011-02-24 ダニスコ・ユーエス・インク Alpha-amylase with modified properties
EP2225378B1 (en) 2007-11-22 2013-05-08 CropDesign N.V. Plants having increased yield-related traits and a method for making the same
ES2553652T3 (en) 2007-11-26 2015-12-10 Basf Plant Science Gmbh Plants that have enhanced traits related to performance and a production procedure for them
CA2706644A1 (en) 2007-11-27 2009-06-04 Novozymes A/S Polypeptides having alpha-glucuronidase activity and polynucleotides encoding same
US20100267067A1 (en) 2007-11-29 2010-10-21 Novozymes A/S Synthase Inhibitor Screening Method
EP2222709B1 (en) 2007-11-30 2016-11-23 Glaxo Group Limited Antigen-binding constructs
ES2490608T3 (en) 2007-12-06 2014-09-04 Novozymes A/S Polypeptides with acetyloxylane esterase activity and polynucleotides encoding them
WO2009076196A1 (en) * 2007-12-07 2009-06-18 Dow Global Technologies Inc. High copy number self-replicating plasmids in pseudomonas
CN101896201A (en) * 2007-12-14 2010-11-24 安进公司 Method for treating bone fracture with anti-sclerostin antibodies
AU2008343105A1 (en) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
DE112008003316T5 (en) 2007-12-20 2011-05-05 Basf Plant Science Gmbh Plants with enhanced yield-related traits and methods of making the same
AU2008339660B2 (en) 2007-12-21 2013-09-26 Dupont Nutrition Biosciences Aps Process for edible oil refining using a lipid acyltransferase
EP2706122A3 (en) 2008-01-03 2014-06-18 Verenium Corporation Isomerases, nucleic acids encoding them and methods for making and using them
CN104651381A (en) 2008-01-03 2015-05-27 巴斯夫酶有限责任公司 Transferases and oxidoreductases, nucleic acids encoding them and methods for making and using them
JP5647899B2 (en) * 2008-01-08 2015-01-07 ラツィオファルム ゲーエムベーハーratiopharm GmbH Glycoconjugation of polypeptides using oligosaccharyltransferase
BRPI0907046A2 (en) 2008-01-18 2015-07-28 Medimmune Llc Engineered cysteine antibody, isolated nucleic acid, vector, host cell, antibody conjugate, pharmaceutical composition, methods of detecting cancer, autoimmune, inflammatory or infectious disorders in an individual and inhibiting proliferation of a target cell
EP2599872A3 (en) 2008-01-25 2013-11-13 BASF Plant Science GmbH Plants having enhanced yield-related traits and a method for making the same
US9029638B2 (en) 2008-01-31 2015-05-12 National Institute For Biological Sciences Plants having altered growth and/or development resulted from modulated expression of ubiquitin-specific proteases and a method for making the same
KR20100109945A (en) 2008-02-04 2010-10-11 다니스코 유에스 인크. Ts23 alpha-amylase variants with altered properties
WO2009100460A1 (en) * 2008-02-07 2009-08-13 Epic Advertising, Inc. Systems and methods for measuring the effectiveness of advertising
AU2009212273B2 (en) 2008-02-08 2014-07-31 Astrazeneca Ab Anti-IFNAR1 antibodies with reduced Fc ligand affinity
EP2088432A1 (en) * 2008-02-11 2009-08-12 MorphoSys AG Methods for identification of an antibody or a target
WO2009102899A1 (en) 2008-02-12 2009-08-20 Codexis, Inc. Method of generating an optimized diverse population of variants
US8034568B2 (en) 2008-02-12 2011-10-11 Nugen Technologies, Inc. Isothermal nucleic acid amplification methods and compositions
EP2250594B1 (en) 2008-02-12 2017-04-19 Codexis, Inc. Method of generating an optimized, diverse population of variants
DK2257311T3 (en) 2008-02-27 2014-06-30 Novo Nordisk As Conjugated Factor VIII Molecules
GB2470672B (en) 2008-03-21 2012-09-12 Nugen Technologies Inc Methods of RNA amplification in the presence of DNA
US20110020368A1 (en) 2008-03-25 2011-01-27 Nancy Hynes Treating cancer by down-regulating frizzled-4 and/or frizzled-1
US20110020830A1 (en) * 2008-03-31 2011-01-27 Schneider Jane C Design for rapidly cloning one or more polypeptide chains into an expression system
EP2268820A1 (en) 2008-04-16 2011-01-05 BASF Plant Science GmbH Plants having enhanced yield-related traits and a method for making the same
WO2009133461A1 (en) 2008-04-30 2009-11-05 Danisco A/S Composition
US9017970B2 (en) 2008-05-02 2015-04-28 Cellscript, Llc RNA polyphosphatase compositions, kits, and uses thereof
BRPI0913007A2 (en) 2008-05-02 2019-09-24 Novartis Ag enhanced fibronectin-based binding molecules and uses thereof
AU2009243552A1 (en) 2008-05-05 2009-11-12 Basf Plant Science Gmbh Plants having enhanced yield-related traits and a method for making the same
SI2283034T1 (en) 2008-05-08 2015-07-31 Monsanto Do Brasil Ltda. Genes and methods for increasing disease resistance in plants
SG157299A1 (en) 2008-05-09 2009-12-29 Agency Science Tech & Res Diagnosis and treatment of kawasaki disease
CA2990650C (en) 2008-05-23 2021-02-09 Pioneer Hi-Bred International, Inc. Novel dgat genes for increased seed storage lipid production and altered fatty acid profiles in oilseed plants
CN102056944A (en) 2008-06-04 2011-05-11 莫蒂克斯特私人有限公司 Anti-inflammatory agents
CN102057040A (en) 2008-06-06 2011-05-11 丹尼斯科美国公司 Geobacillus stearothermophilus alpha-amylase (AMYS) variants with improved properties
EP2288697B1 (en) 2008-06-06 2013-11-06 Novozymes A/S Variants of a family 44 xyloglucanase
ES2705694T3 (en) 2008-06-13 2019-03-26 Codexis Inc Method of synthesis of polynucleotide variants
US8383346B2 (en) 2008-06-13 2013-02-26 Codexis, Inc. Combined automated parallel synthesis of polynucleotide variants
US20090312196A1 (en) 2008-06-13 2009-12-17 Codexis, Inc. Method of synthesizing polynucleotide variants
AU2009259433A1 (en) 2008-06-20 2009-12-23 Basf Plant Science Gmbh Plants having enhanced yield-related traits and a method for making the same
CN102816106A (en) 2008-06-24 2012-12-12 默沙东公司 Biocatalytic processes for the preparation of substantially stereomerically pure fused bicyclic proline compounds
CA2729294C (en) 2008-06-25 2018-08-14 Athenix Corp. Toxin genes and methods for their use
BRPI0914689A2 (en) 2008-06-26 2016-08-09 Basf Plant Science Gmbh methods for enhancing plant yield characteristics in relation to control plants, and for producing a transgenic plant, construct, use of a construct, use of a nucleic acid, plant, part of the plant or plant cell, harvestable parts of a plant and products
PE20140867A1 (en) 2008-07-02 2014-07-19 Athenix Corp AXMI-115, AXMI-113, AXMI-005, AXMI-163 AND AXMI-184: INSECTICIDE PROTEINS AND METHODS FOR THEIR USE
US8748699B2 (en) 2008-07-04 2014-06-10 Basf Plant Science Gmbh Plants having enhanced yield-related traits and a method for making the same by overexpressing a polynucleotide encoding a TFL1-like protein
CN102099480A (en) 2008-07-17 2011-06-15 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
WO2010007166A2 (en) 2008-07-18 2010-01-21 Novozymes A/S Treatment of inflammatory bowel diseases with mammal beta defensins
HUE029982T2 (en) 2008-07-18 2017-04-28 Bristol Myers Squibb Co Compositions monovalent for cd28 binding and methods of use
WO2010011600A2 (en) * 2008-07-22 2010-01-28 Northwestern University Method for removal or inactivation of heparin
MX2011000778A (en) 2008-07-31 2011-03-15 Basf Plant Science Gmbh Plants having modified growth characteristics and a method for making the same.
CN102170906B (en) 2008-08-05 2014-07-30 诺华股份有限公司 Compositions and methods for antibodies targeting complement protein C5
CN104531751A (en) 2008-08-20 2015-04-22 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
US8288141B2 (en) 2008-08-27 2012-10-16 Codexis, Inc. Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine
US8288131B2 (en) 2008-08-27 2012-10-16 Codexis, Inc. Ketoreductase polypeptides and uses thereof
HUE026181T2 (en) 2008-08-27 2016-05-30 Codexis Inc Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine
US8198062B2 (en) 2008-08-29 2012-06-12 Dsm Ip Assets B.V. Hydrolases, nucleic acids encoding them and methods for making and using them
US8357503B2 (en) 2008-08-29 2013-01-22 Bunge Oils, Inc. Hydrolases, nucleic acids encoding them and methods for making and using them
US8153391B2 (en) 2008-08-29 2012-04-10 Bunge Oils, Inc. Hydrolases, nucleic acids encoding them and methods for making and using them
US8273554B2 (en) 2008-08-29 2012-09-25 Codexis, Inc. Ketoreductase polypeptides for the stereoselective production of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one
AU2009293640A1 (en) * 2008-09-22 2010-03-25 Calmune Corporation Methods and vectors for display of 2G12 -derived domain exchanged antibodies
US20100081575A1 (en) * 2008-09-22 2010-04-01 Robert Anthony Williamson Methods for creating diversity in libraries and libraries, display vectors and methods, and displayed molecules
EP2910639A1 (en) 2008-09-24 2015-08-26 BASF Plant Science GmbH Plants having enhanced yield-related traits and a method for making the same
US8507243B2 (en) 2008-09-25 2013-08-13 Danisco Us Inc. Alpha-amylase blends and methods for using said blends
EA027693B1 (en) 2008-09-26 2017-08-31 Токаджен Инк. Recombinant replication competent retrovirus having cytosine deaminase activity for gene therapy of cell proliferative disorders
EP2342323B1 (en) 2008-09-26 2013-06-05 Novozymes A/S Hafnia phytase variants
PL2733212T3 (en) 2008-09-26 2019-06-28 Basf Agrochemical Products, B.V. Herbicide-resistant ahas-mutants and methods of use
WO2010039985A1 (en) 2008-10-01 2010-04-08 Quintessence Biosciences, Inc. Therapeutic Ribonucleases
JP5665747B2 (en) 2008-10-03 2015-02-04 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニーE.I.Du Pont De Nemours And Company Improved perhydrolase for enzymatic peracid production
WO2010052244A1 (en) * 2008-11-05 2010-05-14 Morphosys Ag Deconvolution method
US9221902B2 (en) 2008-11-07 2015-12-29 Fabrus, Inc. Combinatorial antibody libraries and uses thereof
US20110247098A1 (en) 2008-11-12 2011-10-06 Basf Plant Science Gmbh Plants Having Enhanced Abiotic Stress Tolerance and/or Enhanced Yield-Related Traits and a Method for Making the Same
CN102438652B (en) 2008-11-12 2014-08-13 米迪缪尼有限公司 Antibody formulation
CN102292444A (en) 2008-11-20 2011-12-21 诺维信股份有限公司 Polypeptides having amylolytic enhancing activity and polynucleotides encoding same
CN104328137A (en) 2008-12-03 2015-02-04 巴斯夫植物科学有限公司 Plants having enhanced abiotic stress tolerance and/or enhanced yield-related traits and a method for making the same
WO2010065867A1 (en) 2008-12-04 2010-06-10 Pioneer Hi-Bred International, Inc. Methods and compositions for enhanced yield by targeted expression of knotted1
BRPI0922773B1 (en) 2008-12-04 2018-10-09 Novozymes As transgenic host microbial cell, methods for producing a polypeptide having cellulolytic enhancing activity and for degrading or converting a cellulosic material, nucleic acid construct, expression vector, and detergent composition.
WO2010068650A1 (en) 2008-12-12 2010-06-17 Novozymes, Inc. Polypeptides having lipase activity and polynucleotides encoding same
WO2010068800A1 (en) 2008-12-12 2010-06-17 Novozymes, Inc. Polypeptides having aspartic endopeptidase activity and polynucleotides encoding same
WO2010074972A1 (en) 2008-12-15 2010-07-01 Novozymes, Inc. Polypeptides having catalase activity and polynucleotides encoding same
EP2366025A1 (en) 2008-12-15 2011-09-21 Athenix Corporation Genes encoding nematode toxins
CA2746757A1 (en) 2008-12-16 2010-07-08 Novozymes, Inc. Polypeptides having alpha-mannosidase activity and polynucleotides encoding same
WO2010074955A1 (en) 2008-12-16 2010-07-01 Novozymes, Inc. Polypeptides having carboxypeptidase activity and polynucleotides encoding same
US20110252508A1 (en) 2008-12-17 2011-10-13 Basf Plant Science Gmbh Plants Having Enhanced Yield-Related Traits and/or Abiotic Stress Tolerance and a Method for Making the Same
MX2011006416A (en) 2008-12-19 2011-07-12 Macrogenics Inc Covalent diabodies and uses thereof.
EP2379729A2 (en) 2008-12-19 2011-10-26 Novozymes A/S Use of enzymes having silicase activity
EP2361307B1 (en) 2008-12-22 2014-09-24 Athenix Corporation Pesticidal genes from Brevibacillus and methods for their use
CA2747826A1 (en) 2008-12-23 2010-07-01 Athenix Corporation Axmi-150 delta-endotoxin gene and methods for its use
WO2010075574A2 (en) 2008-12-25 2010-07-01 Codexis, Inc. Enone reductases
EP2385983B1 (en) 2009-01-08 2017-12-20 Codexis, Inc. Transaminase polypeptides
EP2206723A1 (en) 2009-01-12 2010-07-14 Bonas, Ulla Modular DNA-binding domains
US20110239315A1 (en) 2009-01-12 2011-09-29 Ulla Bonas Modular dna-binding domains and methods of use
US8492133B2 (en) 2009-01-20 2013-07-23 Ramot At Tel Aviv University, Ltd. MIR-21 promoter driven targeted cancer therapy
ES2558698T3 (en) 2009-01-21 2016-02-08 Novozymes A/S Polypeptides with esterase activity and nucleic acids encoding them
WO2010088387A1 (en) 2009-01-28 2010-08-05 Novozymes, Inc. Polypeptides having beta-glucosidase activity and polynucleotides encoding same
EA201170947A1 (en) 2009-01-28 2012-07-30 Басф Плант Сайенс Компани Гмбх PLANTS HAVING IMPROVED YIELD CHARACTERISTICS AND METHOD OF OBTAINING THEM
WO2010088447A1 (en) 2009-01-30 2010-08-05 Novozymes A/S Polypeptides having alpha-amylase activity and polynucleotides encoding same
US8629324B2 (en) 2009-01-30 2014-01-14 Novozymes, Inc. Polypeptides having expansin activity and polynucleotides encoding same
WO2010087927A2 (en) 2009-02-02 2010-08-05 Medimmune, Llc Antibodies against and methods for producing vaccines for respiratory syncytial virus
BRPI1007915B1 (en) 2009-02-05 2019-05-14 Athenix Corporation AXMI-R1 VARIANT GENES DELTA-ENDOTOXIN, VECTOR, MICROBIAN HOST CELL, RECOMBINANT POLYPEPTIDE AND ITS METHOD OF PRODUCTION, COMPOSITION, AND METHODS FOR CONTROLING AND KILLING A CLETETETE CLETETE PANTOTTETE POPULATION
WO2010089302A1 (en) 2009-02-06 2010-08-12 University Of Chile Protein and dna sequence encoding a cold adapted xylanase
WO2010091221A1 (en) 2009-02-06 2010-08-12 Novozymes A/S Polypeptides having alpha-amylase activity and polynucleotides encoding same
ES2630253T3 (en) 2009-02-11 2017-08-18 Albumedix A/S Albumin variants and conjugates
CN102325876A (en) 2009-02-19 2012-01-18 诺维信公司 Brewing method
MY189620A (en) * 2009-02-20 2022-02-21 Malaysian Palm Oil Board A constitutive promoter from oil palm
JP2012518398A (en) 2009-02-24 2012-08-16 グラクソ グループ リミテッド Antigen binding construct
WO2010097394A1 (en) 2009-02-24 2010-09-02 Glaxo Group Limited Multivalent and/or multispecific rankl-binding constructs
WO2010097386A1 (en) 2009-02-24 2010-09-02 Glaxo Group Limited Antigen-binding constructs
BRPI1008725A2 (en) 2009-02-25 2016-03-08 Basf Plant Science Co Gmbh methods for enhancing and improving plant yield characteristics relative to control plants, and for the production of a transgenic plant, construct, construct uses, a nucleic acid, and a nucleic acid sequence, plant, part of the plant or plant cell, transgenic plant, harvestable parts, products, isolated nucleic acid molecule, and isolated polypeptide
CA2752818A1 (en) 2009-02-26 2010-09-02 Codexis, Inc. Beta-glucosidase variant enzymes and related polynucleotides
WO2010097436A1 (en) 2009-02-27 2010-09-02 Novozymes A/S Mutant cells having reduced expression of metallopeptidase, suitable for recombinant polypeptide production
MX2011008955A (en) 2009-02-27 2011-09-30 Athenix Corp Pesticidal proteins and methods for their use.
US20110311521A1 (en) 2009-03-06 2011-12-22 Pico Caroni Novel therapy for anxiety
AR075799A1 (en) 2009-03-06 2011-04-27 Athenix Corp METHODS AND COMPOSITIONS FOR CONTROLLING PLANT PESTS
CA2754845A1 (en) 2009-03-11 2010-12-09 Athenix Corporation Axmi-030 insecticidal protein from bacillus thuringiensis and methods for use
WO2010106068A1 (en) 2009-03-17 2010-09-23 Novozymes A/S Polypeptides having tyrosinase activity and polynucleotides encoding same
CA2754580C (en) 2009-03-17 2014-06-17 Codexis, Inc. Variant endoglucanases and related polynucleotides
US8658409B2 (en) 2009-03-24 2014-02-25 Novozymes A/S Polypeptides having acetyl xylan esterase activity and polynucleotides encoding same
US20120093775A1 (en) 2009-03-27 2012-04-19 Proyecto De Biomedicina Cima, S.L. Methods and compositions for the treatment of cirrhosis and liver fibrosis
WO2010120557A1 (en) 2009-03-31 2010-10-21 Codexis, Inc. Improved endoglucanases
CA2757347A1 (en) 2009-04-01 2010-10-07 Danisco Us Inc. Cleaning system comprising an alpha-amylase and a protease
EP2241323A1 (en) 2009-04-14 2010-10-20 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Tenascin-W and brain cancers
GB0908770D0 (en) 2009-04-24 2009-07-01 Danisco Method
KR20150046788A (en) 2009-04-29 2015-04-30 바스프 플랜트 사이언스 게엠베하 Plants having enhanced yield-related traits and a method for making the same
US20120090052A1 (en) 2009-04-29 2012-04-12 Ana Isabel Sanz Molinero Plants Having Enhanced Yield-Related Traits And A Method For Making The Same
US8779239B2 (en) 2009-05-04 2014-07-15 Pioneeri Hi-Bred International, Inc. Yield enhancement in plants by modulation of AP2 transcription factor
EP2248893A1 (en) 2009-05-06 2010-11-10 Novozymes A/S DFPase Enzymes from Octopus Vulgaris
CN102803291B (en) 2009-05-06 2015-11-25 巴斯夫植物科学有限公司 There is the plant of the Correlated Yield Characters of enhancing and/or the abiotic stress tolerance of enhancing and prepare its method
MY186558A (en) * 2009-05-13 2021-07-27 Malaysian Palm Oil Board A constitutive promoter from oil palm type 2
BRPI1011036A8 (en) 2009-05-19 2017-11-07 Danisco USE
BRPI1011160A8 (en) 2009-05-21 2018-01-02 Verenium Corp PHYTASES, PROTEIN PREPARATION INCLUDING THEM, AND THEIR USES
JP2012527248A (en) 2009-05-22 2012-11-08 クルナ・インコーポレーテッド Treatment of TFE3 and insulin receptor substrate 2 (IRS2) related diseases by inhibition of natural antisense transcripts against transcription factor E3 (TFE3)
US9989533B2 (en) 2009-05-28 2018-06-05 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Anti-TNF induced apoptosis (ATIA) diagnostic markers and therapies
AU2010252939B2 (en) 2009-05-29 2014-06-26 Morphosys Ag A collection and methods for its use
EP2435561B1 (en) 2009-05-29 2018-08-08 Novozymes Inc. Methods for enhancing the degradation or conversion of cellulosic material
DK2438163T3 (en) 2009-06-02 2015-04-20 Novozymes Inc Polypeptides having cellobiohydrolase activity and polynucleotides encoding them
EP2440663A1 (en) 2009-06-09 2012-04-18 Pioneer Hi-Bred International Inc. Early endosperm promoter and methods of use
US20100317539A1 (en) * 2009-06-12 2010-12-16 Guo-Liang Yu Library of Engineered-Antibody Producing Cells
AR077042A1 (en) 2009-06-12 2011-07-27 Danisco METHOD TO TREAT A COMPOSITION CONTAINING PYROPHIOPHYTIN
BRPI1012922A2 (en) 2009-06-16 2016-10-11 Codexis Inc b-glycosidase polypeptide variant, expression vector, host cell, production method of b-glycosidase variant polypeptide, enzymatic composition, method of converting a biomass substrate into fermentable sugar and alcohol production method
CN104762316A (en) 2009-06-19 2015-07-08 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
WO2011005527A2 (en) 2009-06-22 2011-01-13 Codexis, Inc. Ketoreductase-mediated stereoselective route to alpha chloroalcohols
AU2010270979B2 (en) 2009-06-22 2015-04-23 Medimmune, Llc Engineered Fc regions for site-specific conjugation
CN102712919B (en) 2009-06-26 2017-06-23 诺维信北美公司 Heat endurance carbonic anhydrase and application thereof
CN102648281B (en) 2009-07-02 2017-04-05 阿森尼克斯公司 205 killing genes of AXMI and its using method
WO2011000924A1 (en) 2009-07-03 2011-01-06 Abbott Products Gmbh Spray-dried amylase, pharmaceutical preparations comprising the same and use
EP2451947A1 (en) 2009-07-07 2012-05-16 Linda A. Castle Crystal structure of glyphosate acetyltransferase (glyat) and methods of use
EP2451957B1 (en) 2009-07-07 2017-11-15 Novozymes Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
US8614081B2 (en) 2009-07-23 2013-12-24 Codexis, Inc. Nitrilase biocatalysts
WO2011009182A2 (en) 2009-07-24 2011-01-27 Embrapa - Empresa Brasileira De Pesquisa Agropecuária Isolated nucleic acid molecule, genetic construct, vector, transgenic cell, method for producing a transgenic cell and plant, isolated and purified polypeptide, biodegradable pesticide composition, pest control method, method for producing transgenic strains resistant to insect pests
US20120100250A1 (en) 2009-07-24 2012-04-26 Novozymes A/S Carbohydrate Oxidases
WO2011014458A1 (en) 2009-07-28 2011-02-03 Novozymes, Inc. Polypeptides having phytase activity and polynucleotides encoding same
CN108484739B (en) 2009-07-31 2022-08-30 巴斯夫农业种子解决方案美国有限责任公司 AXMI-192 family of pesticidal genes and methods of using the same
US9133500B2 (en) 2009-08-10 2015-09-15 MorphoSys A6 Screening strategies for the identification of binders
WO2011020079A1 (en) * 2009-08-13 2011-02-17 Calmune Corporation Antibodies against human respiratory syncytial virus (rsv) and methods of use
CA2769822C (en) 2009-08-13 2019-02-19 The Johns Hopkins University Methods of modulating immune function
EP2949661A1 (en) 2009-08-19 2015-12-02 BASF Plant Science Company GmbH Plants having enhanced yield-related traits and a method for making the same
SG178456A1 (en) 2009-08-19 2012-04-27 Codexis Inc Ketoreductase polypeptides for the preparation of phenylephrine
MX2012002113A (en) 2009-08-20 2012-08-08 Pioneer Hi Bred Int Functional expression of shuffled yeast nitrate transporter (ynti) in maize to improve nitrate uptake under low nitrate environment.
MX2012002095A (en) 2009-08-21 2012-04-10 Novozymes As Polypeptides having isoamylase activity and polynucleotides encoding same.
EP2292266A1 (en) 2009-08-27 2011-03-09 Novartis Forschungsstiftung, Zweigniederlassung Treating cancer by modulating copine III
BR112012004594A2 (en) 2009-09-01 2016-06-21 Novozymes Inc filamentous fungal host cell, and method for producing a dicarboxylic acid
EP2473604B1 (en) 2009-09-04 2017-01-11 Codexis, Inc. Variant cbh2 cellulases and related polynucleotides
IN2012DN02878A (en) 2009-09-09 2015-07-24 Braskem Sa
US8293483B2 (en) 2009-09-11 2012-10-23 Epitomics, Inc. Method for identifying lineage-related antibodies
EP3269804B1 (en) 2009-09-17 2020-11-11 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011035029A1 (en) 2009-09-18 2011-03-24 Novozymes, Inc. Polypeptides having beta-glucosidase activity and polynucleotides encoding same
EP3301208B1 (en) 2009-09-18 2020-11-04 Codexis, Inc. Reduced codon mutagenesis
WO2011035205A2 (en) 2009-09-18 2011-03-24 Calmune Corporation Antibodies against candida, collections thereof and methods of use
US20120244170A1 (en) 2009-09-22 2012-09-27 Rafal Ciosk Treating cancer by modulating mex-3
AU2010299799B2 (en) 2009-09-25 2015-10-29 Novozymes A/S Subtilase variants
EP2480566A1 (en) 2009-09-25 2012-08-01 BASF Plant Science Company GmbH Plants having enhanced yield-related traits and a method for making the same
AR080340A1 (en) 2009-09-25 2012-04-04 Crop Functional Genomics Ct PLANTS THAT HAVE BETTER FEATURES RELATED TO PERFORMANCE AND A METHOD FOR PRODUCTION THROUGH THE MODULATION OF THE EXPRESSION OF SGT-1, CLCPKG OR AN HD-HYDROLASE TYPE POLYPEPTIDE
AU2010299800B2 (en) 2009-09-25 2014-08-07 Novozymes A/S Use of protease variants
DK2483403T3 (en) 2009-09-29 2018-02-12 Novozymes Inc POLYPEPTIDES WITH XYLANASE ACTIVITY AND POLYNUCLEOTIDES CODING THEM
EP2483295B1 (en) 2009-09-29 2015-11-25 Novozymes Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
ES2551141T3 (en) 2009-09-30 2015-11-16 Novozymes Inc. Thermoascus crustaceus derived polypeptides with cellulolytic augmentation activity and polynucleotides encoding them
WO2011039319A1 (en) 2009-09-30 2011-04-07 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
US8222012B2 (en) * 2009-10-01 2012-07-17 E. I. Du Pont De Nemours And Company Perhydrolase for enzymatic peracid production
EP2482639B1 (en) 2009-10-02 2017-08-02 Syngenta Participations AG Insecticidal proteins
PL2486141T3 (en) 2009-10-07 2018-07-31 Macrogenics, Inc. Fc region-containing polypeptides that exhibit improved effector function due to alterations of the extent of fucosylation, and methods for their use
WO2011045352A2 (en) 2009-10-15 2011-04-21 Novartis Forschungsstiftung Spleen tyrosine kinase and brain cancers
UA111708C2 (en) 2009-10-16 2016-06-10 Бандж Ойлз, Інк. METHOD OF OIL REFINING
UA109884C2 (en) 2009-10-16 2015-10-26 A POLYPEPTIDE THAT HAS THE ACTIVITY OF THE PHOSPHATIDYLINOSYTOL-SPECIFIC PHOSPHOLIPASE C, NUCLEIC ACID, AND METHOD OF METHOD
MX2012004659A (en) 2009-10-22 2012-07-30 Basf Plant Science Co Gmbh Plants having enhanced yield-related traits and a method for making the same.
WO2011050037A1 (en) 2009-10-23 2011-04-28 Novozymes, Inc. Cellobiohydrolase variants and polynucleotides encoding same
WO2011056544A1 (en) 2009-10-26 2011-05-12 Pioneer Hi-Bred International, Inc. Somatic ovule specific promoter and methods of use
US20120260371A1 (en) 2009-10-29 2012-10-11 Novozymes A/S Polypeptides Having Cellobiohydrolase Activity and Polynucleotides Encoding Same
CN102741280B (en) 2009-10-30 2015-12-02 诺维信生物制药丹麦公司 Albumin variants
US20120213801A1 (en) 2009-10-30 2012-08-23 Ekaterina Gresko Phosphorylated Twist1 and cancer
WO2011051327A2 (en) 2009-10-30 2011-05-05 Novartis Ag Small antibody-like single chain proteins
WO2011053764A2 (en) 2009-11-02 2011-05-05 Plant Sensory Systems, Llc Method for the biosynthesis of taurine or hypotaurine in cells
WO2011051466A1 (en) 2009-11-02 2011-05-05 Novartis Ag Anti-idiotypic fibronectin-based binding molecules and uses thereof
WO2011056872A2 (en) 2009-11-03 2011-05-12 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
US9267125B2 (en) 2009-11-06 2016-02-23 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
DK2496692T3 (en) 2009-11-06 2016-06-27 Novozymes Inc POLYPEPTIDES WITH xylanase AND POLYNUCLEOTIDES ENCODING THEM
US20120227133A1 (en) 2009-11-13 2012-09-06 Basf Plant Science Company Gmbh Plants Having Enhanced Yield-Related Traits and a Method for Making the Same
GB0920089D0 (en) 2009-11-17 2009-12-30 Danisco Method
WO2011063198A2 (en) 2009-11-20 2011-05-26 St. Jude Children's Research Hospital Methods and compositions for modulating the activity of the interleukin-35 receptor complex
US8993840B2 (en) 2009-11-23 2015-03-31 E I du Pont de Nemours and Compay Sucrose transporter genes for increasing plant seed lipids
EP3597771A1 (en) 2009-11-25 2020-01-22 Gen9, Inc. Methods and apparatuses for chip-based dna error reduction
WO2011066185A1 (en) 2009-11-25 2011-06-03 Gen9, Inc. Microfluidic devices and methods for gene synthesis
US8652813B2 (en) 2009-11-25 2014-02-18 Codexis, Inc. Recombinant Thermoascus aurantiacus β-glucosidase variants
MX355841B (en) 2009-11-25 2018-05-02 Codexis Inc Recombinant beta-glucosidase variants for production of soluble sugars from cellulosic biomass.
CA2781835A1 (en) 2009-11-27 2011-06-03 Basf Plant Science Company Gmbh Chimeric endonucleases and uses thereof
JP5922029B2 (en) 2009-11-27 2016-05-24 ビーエーエスエフ プラント サイエンス カンパニー ゲーエムベーハー Chimeric endonuclease and use thereof
BR112012012588B1 (en) 2009-11-27 2019-03-26 Basf Plant Science Company Gmbh ENDONUCLEASE, METHOD FOR HOMOLOGICAL RECOMBINATION OF POLINUCLEOTIDES AND METHOD FOR DIRECTED POLINUCLEOTIDE MUTATION
CA2782154C (en) 2009-11-30 2018-10-16 Novozymes A/S Polypeptides having glucoamylase activity and polynucleotides encoding same
AU2010276470B2 (en) 2009-11-30 2015-05-14 Novozymes A/S Polypeptides having glucoamylase activity and polynucleotides encoding same
MX2012006176A (en) 2009-12-01 2012-06-25 Novozymes North America Inc Polypeptides having glucoamylase activity and polynucleotides encoding same.
TW201125577A (en) 2009-12-02 2011-08-01 Novozymes As Use of defensins for treatment of infective endocarditis
CA2782814A1 (en) 2009-12-02 2011-06-09 Amgen Inc. Binding proteins that bind to human fgfr1c, human .beta.-klotho and both human fgfr1c and human .beta.-klotho
EP2507369A1 (en) 2009-12-03 2012-10-10 Novozymes A/S Variants of a polypeptide with lipolytic activity and improved stability
SI2510089T1 (en) 2009-12-08 2015-12-31 Codexis, Inc. Synthesis of prazole compounds
CN106929494B (en) 2009-12-09 2021-05-14 诺维信公司 Methods of producing GH8 xylanase variants
US8653024B2 (en) 2009-12-11 2014-02-18 Adenium Biotech Aps Use of AMPs for treatment of UTI/cystitis
AU2010328033B2 (en) 2009-12-11 2014-12-04 Novozymes A/S Protease variants
EP2501792A2 (en) 2009-12-29 2012-09-26 Novozymes A/S Gh61 polypeptides having detergency enhancing effect
MX2012007681A (en) 2009-12-31 2013-01-29 Pioneer Hi Bred Int Engineering plant resistance to diseases caused by pathogens.
WO2011082429A1 (en) 2010-01-04 2011-07-07 Novozymes A/S Alpha-amylases
WO2011085075A2 (en) 2010-01-07 2011-07-14 Gen9, Inc. Assembly of high fidelity polynucleotides
US20120295793A1 (en) 2010-01-22 2012-11-22 Proteus Methods of generating modified polynucleotide libraries and methods of using the same for directed protein evolution
BR112012018443A8 (en) 2010-01-25 2019-01-29 Bayer Cropscience Nv methods for making plant cell walls comprising chitin
WO2011092233A1 (en) 2010-01-29 2011-08-04 Novartis Ag Yeast mating to produce high-affinity combinations of fibronectin-based binders
US9896673B2 (en) 2010-02-10 2018-02-20 Novozymes A/S Compositions of high stability alpha amylase variants
EP2357220A1 (en) 2010-02-10 2011-08-17 The Procter & Gamble Company Cleaning composition comprising amylase variants with high stability in the presence of a chelating agent
US9080192B2 (en) 2010-02-10 2015-07-14 Codexis, Inc. Processes using amino acid dehydrogenases and ketoreductase-based cofactor regenerating system
CA2789629A1 (en) 2010-02-10 2011-08-18 Immunogen, Inc. Cd20 antibodies and uses thereof
CA2790029C (en) 2010-02-18 2019-09-03 Athenix Corp. Axmi221z, axmi222z, axmi223z, axmi224z, and axmi225z delta-endotoxin genes and methods for their use
AU2011218130B2 (en) 2010-02-18 2016-03-03 Athenix Corp. AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230, and AXMI231 delta-endotoxin genes and methods for their use
AU2011219927A1 (en) 2010-02-24 2012-08-23 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and a method for making the same
EP2578686A1 (en) 2010-02-24 2013-04-10 BASF Plant Science Company GmbH Plants having enhanced yield-related traits and a method for making the same
EP2539447B1 (en) 2010-02-25 2017-07-26 Novozymes A/S Variants of a lysozyme and polynucleotides encoding same
WO2011104284A1 (en) 2010-02-25 2011-09-01 Novozymes A/S Polypeptides having antimicrobial activity
EP2361985A1 (en) 2010-02-26 2011-08-31 BASF Plant Science Company GmbH Plants having enhanced yield-related traits and a method for making the same
EP2361927A1 (en) 2010-02-26 2011-08-31 BASF Plant Science Company GmbH Plants having enhanced yield-related traits and a method for making the same
CA2791353A1 (en) 2010-03-03 2011-09-09 Novozymes, Inc. Xylanase variants and polynucleotides encoding same
US20130004519A1 (en) 2010-03-05 2013-01-03 Ruth Chiquet-Ehrismann Smoci, tenascin-c and brain cancers
WO2011110967A1 (en) 2010-03-12 2011-09-15 Danisco A/S Process
WO2011114251A1 (en) 2010-03-18 2011-09-22 Danisco A/S Foodstuff
EA201290930A1 (en) 2010-03-18 2013-04-30 Басф Плант Сайенс Компани Гмбх PLANTS WITH IMPROVED PERFORMANCE CHARACTERISTICS AND METHOD FOR PRODUCING THEM
WO2011114279A1 (en) 2010-03-18 2011-09-22 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and method for making the same
CN102971427A (en) 2010-03-19 2013-03-13 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and method for making same
WO2011116396A2 (en) 2010-03-19 2011-09-22 The Board Of Trustees Of The Leland Stanford Junior University Hepatocyte growth factor fragments that function as potent met receptor agonists and antagonists
EP2547775A4 (en) 2010-03-19 2014-01-22 Basf Plant Science Co Gmbh Plants having enhanced yield-related traits and method for making the same
EP2371845A1 (en) 2010-03-22 2011-10-05 BASF Plant Science Company GmbH Plants having enhanced yield-related traits and a method for making the same
EP2552232B1 (en) 2010-03-26 2016-07-06 Novozymes A/S Thermostable phytase variants
US9222109B2 (en) 2010-03-28 2015-12-29 Novozymes A/S Enzymatic hydroxylation of aliphatic hydrocarbon
CN102834521B (en) 2010-03-30 2018-02-27 诺维信公司 For strengthening the method from zymotechnique accessory substance
EP2553108A4 (en) 2010-03-31 2015-01-28 Codexis Inc Production of monoterpenes
MX2012011153A (en) 2010-03-31 2012-11-29 Novozymes Inc Cellobiohydrolase variants and polynucleotides encoding same.
US10233228B2 (en) 2010-04-09 2019-03-19 Albumedix Ltd Albumin derivatives and variants
DK2558484T3 (en) 2010-04-14 2016-03-29 Novozymes As Polypeptides having glucoamylase activity and polynucleotides encoding them
US8383793B2 (en) 2010-04-15 2013-02-26 St. Jude Children's Research Hospital Methods and compositions for the diagnosis and treatment of cancer resistant to anaplastic lymphoma kinase (ALK) kinase inhibitors
EP3540059A1 (en) 2010-04-16 2019-09-18 Nuevolution A/S Bi-functional complexes and methods for making and using such complexes
EP2561076A1 (en) 2010-04-19 2013-02-27 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Modulating xrn1
US9040262B2 (en) 2010-05-04 2015-05-26 Codexis, Inc. Biocatalysts for ezetimibe synthesis
CA2797910A1 (en) 2010-05-06 2011-11-10 Pioneer Hi-Bred International, Inc. Maize acc synthase 3 gene and protein and uses thereof
SG185415A1 (en) 2010-05-06 2012-12-28 Novartis Ag Compositions and methods of use for therapeutic low density lipoprotein - related protein 6 (lrp6) multivalent antibodies
NZ603829A (en) 2010-05-06 2015-03-27 Novartis Ag Compositions and methods of use for therapeutic low density lipoprotein -related protein 6 (lrp6) antibodies
EP2569331A1 (en) 2010-05-10 2013-03-20 Perseid Therapeutics LLC Polypeptide inhibitors of vla4
RS56210B1 (en) 2010-05-14 2017-11-30 Amgen Inc High concentration antibody formulations
WO2011147863A1 (en) 2010-05-25 2011-12-01 INSERM (Institut National de la Santé et de la Recherche Médicale) Combination of anti-envelope antibodies and anti-receptor antibodies for the treatment and prevention of hcv infection
WO2011150313A1 (en) 2010-05-28 2011-12-01 Codexis, Inc. Pentose fermentation by a recombinant microorganism
DK2576606T3 (en) 2010-06-04 2015-02-23 Novozymes Inc Preparation of C4 dicarboxylic acid in filamentous fungi
US20130089538A1 (en) 2010-06-10 2013-04-11 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute forBiomedical Researh Treating cancer by modulating mammalian sterile 20-like kinase 3
CN102984961A (en) 2010-06-11 2013-03-20 诺维信公司 A method to reduce biogenic amine content in food
US8835604B2 (en) 2010-06-12 2014-09-16 Adenium Biotech Aos Antimicrobial peptide variants and polynucleotides encoding same
WO2011159910A2 (en) 2010-06-17 2011-12-22 Codexis, Inc. Biocatalysts and methods for the synthesis of (s)-3-(1-aminoethyl)-phenol
EA201291270A1 (en) 2010-06-17 2013-09-30 ДюПон НЬЮТРИШН БАЙОСАЙЕНСИЗ АпС METHOD OF MANUFACTURE OF REFINED PLANT OIL USING CHLOROPHYLASE
CN102947458B (en) 2010-06-21 2016-05-04 诺维信股份有限公司 Be used for the method for the generation of the improvement of filamentous fungi C4-dicarboxylic acids
WO2011163269A1 (en) 2010-06-21 2011-12-29 Novozymes, Inc. Aspergillus aculeatus derived polypeptides having c4-dicarboxylic acid transporter activity and polynucleotides encoding same
CN105063063A (en) 2010-06-24 2015-11-18 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and method for making the same
WO2011163590A1 (en) 2010-06-25 2011-12-29 E. I. Du Pont De Nemours And Company Compositions and methods for enhancing resistance to northern leaf blight in maize
DE112011102151T5 (en) 2010-06-25 2013-07-11 Basf Plant Science Company Gmbh Plants with enhanced yield-related traits and methods for their production
BR112012033361A2 (en) 2010-06-28 2020-10-20 Codexis, Inc. fatty alcohols-forming reductases and their modes of use.
AU2011272878B2 (en) 2010-06-30 2015-04-23 Codexis, Inc. Highly stable beta-class carbonic anhydrases useful in carbon capture systems
CN103108951A (en) 2010-06-30 2013-05-15 诺维信公司 Polypeptides having beta-glucosidase activity and polynucleotides encoding same
GB201011513D0 (en) 2010-07-08 2010-08-25 Danisco Method
NZ603488A (en) 2010-07-09 2015-02-27 Crucell Holland Bv Anti-human respiratory syncytial virus (rsv) antibodies and methods of use
WO2012009336A1 (en) 2010-07-12 2012-01-19 Gloucester Pharmaceuticals, Inc. Romidepsin solid forms and uses thereof
US9354228B2 (en) 2010-07-16 2016-05-31 Adimab, Llc Antibody libraries
MX2013000576A (en) 2010-07-16 2013-02-27 Basf Plant Science Co Gmbh Plants having enhanced yield-related traits and method for making the same.
WO2012016245A2 (en) 2010-07-30 2012-02-02 Novartis Ag Fibronectin cradle molecules and libraries thereof
WO2012013197A2 (en) 2010-07-30 2012-02-02 Aalborg Universitet Aspergillus encoding beta-glucosidases and nucleic acids encoding same
US9057086B2 (en) 2010-08-12 2015-06-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a bicycle compound and uses thereof
CA2805803A1 (en) 2010-08-13 2012-02-16 Pioneer Hi-Bred International, Inc. Compositions and methods comprising sequences having hydroxyphenylpyruvate dioxygenase (hppd) activity
EP2605772B1 (en) 2010-08-16 2016-06-22 Cardiome International AG Process for preparing aminocyclohexyl ether compounds
WO2012022734A2 (en) 2010-08-16 2012-02-23 Medimmune Limited Anti-icam-1 antibodies and methods of use
WO2012024698A1 (en) 2010-08-20 2012-02-23 Codexis, Inc. Use of glycoside hydrolase 61 family proteins in processing of cellulose
WO2012024662A2 (en) 2010-08-20 2012-02-23 Codexis, Inc. Expression constructs comprising fungal promoters
SI2606070T1 (en) 2010-08-20 2017-04-26 Novartis Ag Antibodies for epidermal growth factor receptor 3 (her3)
CN103154252B (en) 2010-08-23 2017-05-24 先锋国际良种公司 Novel defensin variants and methods of use
CN107299095B (en) 2010-08-24 2021-05-11 诺维信公司 Thermostable Persephonella carbonic anhydrase and uses thereof
BR112013004183A2 (en) 2010-08-24 2016-05-10 Basf Plant Science Co Gmbh plants with better yield characteristics and method for their production
BR112013004356A2 (en) 2010-08-26 2017-06-27 Du Pont isolated polynucleotide, mutant polypeptide, recombinant construct, transformed cell, method for producing a polyunsaturated fatty acid, microbial oil and recombinant microbial host cell.
AU2011293189B2 (en) 2010-08-26 2017-02-16 E. I. Du Pont De Nemours And Company Mutant HPGG motif and HDASH motif delta-5 desaturases and their use in making polyunsaturated fatty acids
WO2012030849A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2012030844A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
US8629325B2 (en) 2010-08-30 2014-01-14 Novozymes A/S Polypeptides having beta-glucosidase activity and polynucleotides encoding same
US20130212746A1 (en) 2010-08-30 2013-08-15 Novoyzmes A/S Polypeptides Having Hemicellulolytic Activity And Polynucleotides Encoding Same
WO2012030811A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
EP2735611B1 (en) 2010-08-30 2018-11-21 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012032143A1 (en) 2010-09-10 2012-03-15 Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute For Biomedical Research Phosphorylated twist1 and metastasis
US8859502B2 (en) 2010-09-13 2014-10-14 Celgene Corporation Therapy for MLL-rearranged leukemia
WO2012035103A1 (en) 2010-09-16 2012-03-22 Novozymes A/S Lysozymes
RU2610088C2 (en) 2010-09-22 2017-02-07 Байер Интеллектуэль Проперти Гмбх Use of active ingredients agains nematodes in agricultural plants, resistant to nematodes
US9816082B2 (en) 2010-09-30 2017-11-14 Novozymes, Inc. Variants of polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
DK2622068T3 (en) 2010-09-30 2016-10-17 Novozymes Inc Variants of polypeptides having cellulolytic enhancing ACTIVITY AND POLYNUCLEOTIDES ENCODING THEM
WO2012042037A1 (en) 2010-10-01 2012-04-05 Novozymes A/S Polypeptides having endopeptidase activity and polynucleotides encoding same
DK3023492T3 (en) 2010-10-01 2018-03-05 Novozymes Inc Beta-glucosidase variants and polynucleotides encoding them
BR112013008347A2 (en) 2010-10-06 2016-06-14 Bp Corp North America Inc cbh variant polypeptides i
US20130260423A1 (en) 2010-10-26 2013-10-03 Novozymes North America, Inc. Methods of Saccharifying Sugar Cane Trash
EP2632943A1 (en) 2010-10-29 2013-09-04 Novozymes A/S Polypeptides having succinyl-coa:aceto acetate transferase activity and polynucleotides encoding same
CN103314100A (en) 2010-10-29 2013-09-18 诺维信公司 Recombinant n-propanol and isopropanol production
CN110079558A (en) 2010-11-02 2019-08-02 诺维信股份有限公司 With the method for 61 polypeptide pre-treating cellulosic material of family
EP2635594B1 (en) 2010-11-04 2017-01-11 Novozymes Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012058768A1 (en) 2010-11-05 2012-05-10 Zymeworks Inc. Stable heterodimeric antibody design with mutations in the fc domain
WO2012064351A1 (en) 2010-11-08 2012-05-18 Novozymes A/S Polypeptides having glucoamylase activity and polynucleotides encoding same
ES2649555T3 (en) 2010-11-08 2018-01-12 Novozymes A/S Polypeptides with glucoamylase activity and polynucleotides that encode them
MX2013005236A (en) 2010-11-10 2013-06-28 Basf Plant Science Co Gmbh Plants having enhanced yield-related traits and method for making the same.
DK2638153T3 (en) 2010-11-12 2017-10-16 Novozymes Inc POLYPEPTIDES WITH ENDOGLUCANASE ACTIVITY AND POLYNUCLEOTIDES CODING THEM
EP2638135B1 (en) 2010-11-12 2017-01-11 Novozymes A/S Polypeptides having phospholipase c activity and polynucleotides encoding same
WO2012064975A1 (en) 2010-11-12 2012-05-18 Gen9, Inc. Protein arrays and methods of using and making the same
WO2012078312A2 (en) 2010-11-12 2012-06-14 Gen9, Inc. Methods and devices for nucleic acids synthesis
US20140093506A1 (en) 2010-11-15 2014-04-03 Marc Buehler Anti-fungal-agents
CN103339252A (en) 2010-11-18 2013-10-02 诺维信股份有限公司 Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
PL2640742T3 (en) 2010-11-19 2019-01-31 Morphosys Ag A collection of antibody sequences its use
US20130245233A1 (en) 2010-11-24 2013-09-19 Ming Lei Multispecific Molecules
KR20130121886A (en) 2010-11-24 2013-11-06 글락소 그룹 리미티드 Multispecific antigen binding proteins targeting hgf
US8927235B2 (en) 2010-12-06 2015-01-06 Novozymes A/S Methods of hydrolyzing oligomers in hemicellulosic liquor
WO2012078800A2 (en) 2010-12-08 2012-06-14 Codexis, Inc. Biocatalysts and methods for the synthesis of armodafinil
BR112013014698A2 (en) 2010-12-22 2017-03-07 E I Du Point De Nemours & Company dna construct, vector, plant cell, plant, transgenic plant-derived seed, method for expression of a nucleotide sequence in a plant, method for expression of a nucleotide sequence in a plant cell, method for selectively expressing a nucleotide sequence in corn root, stem, grain and tassel tissues
EP2655635A1 (en) 2010-12-22 2013-10-30 Pioneer Hi-Bred International, Inc. Viral promoter, truncations thereof, and methods of use
US8822762B2 (en) 2010-12-28 2014-09-02 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity
MX2013007852A (en) 2011-01-14 2013-09-26 Univ Florida Citrus trees with resistance to citrus canker.
EP2665819A4 (en) 2011-01-20 2014-09-10 Basf Plant Science Co Gmbh Plants having enhanced yield-related traits and method for making the same
US9080161B2 (en) 2011-01-26 2015-07-14 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
DK2668268T3 (en) 2011-01-26 2017-09-11 Novozymes Inc POLYPEPTIDES WITH CELLOBIO HYDROLASE ACTIVITY AND POLYNUCLEOTIDES CODING THEM
WO2012103322A1 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
CN110628803A (en) 2011-01-26 2019-12-31 诺维信公司 Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
MX2013007720A (en) 2011-01-26 2013-08-09 Novozymes As Polypeptides having cellobiohydrolase activity and polynucleotides encoding same.
WO2012103165A2 (en) 2011-01-26 2012-08-02 Kolltan Pharmaceuticals, Inc. Anti-kit antibodies and uses thereof
BR112013019324B1 (en) 2011-01-31 2021-06-08 Novozymes North America, Inc processes for enzymatic refinement of a pretreated cellulosic material
WO2012106579A1 (en) 2011-02-03 2012-08-09 Bristol-Myers Squibb Company Amino acid dehydrogenase and its use in preparing amino acids from keto acids
EP2675900B1 (en) 2011-02-15 2017-09-06 Pioneer Hi-Bred International, Inc. Root-preferred promoter and methods of use
WO2012110563A1 (en) 2011-02-16 2012-08-23 Novozymes A/S Detergent compositions comprising metalloproteases
US20140038876A1 (en) 2011-02-16 2014-02-06 Novozymes A/S Detergent Compositions Comprising Mettaloproteases
WO2012110562A2 (en) 2011-02-16 2012-08-23 Novozymes A/S Detergent compositions comprising metalloproteases
AR085251A1 (en) 2011-02-23 2013-09-18 Danisco PROCESS TO TREAT VEGETABLE OIL
US20140051146A1 (en) 2011-02-23 2014-02-20 Dupont Nutrition Biosciences Aps Method
WO2012113340A1 (en) 2011-02-23 2012-08-30 Novozymes Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
CN103459606B (en) 2011-02-23 2017-06-16 杜邦营养生物科学有限公司 Processing method
EP2995622A1 (en) 2011-02-28 2016-03-16 BASF Plant Science Company GmbH Plants having enhanced yield-related traits and a method for making the same
US20140033368A1 (en) 2011-02-28 2014-01-30 Valerie Frankard Plants Having Enhanced Yield-Related Traits and Producing Methods Thereof
US8722387B2 (en) 2011-02-28 2014-05-13 Novozymes, Inc. Microorganisms for C4-dicarboxylic acid production
EP2681242B1 (en) 2011-03-01 2018-01-24 Amgen Inc. Sclerostin and dkk-1 bispecific binding agents
WO2012117368A1 (en) 2011-03-01 2012-09-07 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and producing methods thereof
EP2683830A1 (en) 2011-03-09 2014-01-15 Novozymes A/S Methods of increasing the cellulolytic enhancing activity of a polypeptide
US9409958B2 (en) 2011-03-10 2016-08-09 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
US8878007B2 (en) 2011-03-10 2014-11-04 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity
WO2012127002A1 (en) 2011-03-23 2012-09-27 Novozymes A/S Sweet-tasting polypeptide from gram-positive bacteria
CN103443278B (en) 2011-03-23 2017-07-04 诺维信公司 The method for producing secreted polypeptides
EP2689011B1 (en) 2011-03-25 2017-10-25 Novozymes A/S Method for degrading or converting cellulosic material
CN103517920B (en) 2011-03-25 2018-04-17 安进公司 Anti- hardened proteins (SCLEROSTIN) antibody crystals and its preparation
EP2691517A4 (en) 2011-03-30 2015-04-22 Codexis Inc Pentose fermentation by a recombinant microorganism
US9695440B2 (en) 2011-03-30 2017-07-04 Athenix Corp. AXMI232, AXMI233, and AXMI249 toxin genes and methods for their use
WO2012135436A1 (en) 2011-03-30 2012-10-04 Athenix Corp. Axmi238 toxin gene and methods for its use
EP2691519A1 (en) 2011-03-31 2014-02-05 Novozymes, Inc. Cellulose binding domain variants and polynucleotides encoding same
WO2012135659A2 (en) 2011-03-31 2012-10-04 Novozymes A/S Methods for enhancing the degradation or conversion of cellulosic material
US20130097728A1 (en) 2011-04-05 2013-04-18 Volker Heinrichs Axmi115 variant insecticidal gene and methods for its use
MX2013011617A (en) 2011-04-08 2013-11-21 Danisco Us Inc Compositions.
WO2012142302A2 (en) 2011-04-13 2012-10-18 Codexis, Inc. Biocatalytic process for preparing eslicarbazepine and analogs thereof
US20140157443A1 (en) 2011-04-14 2014-06-05 St. Jude Children's Research Hospital Methods and compositions for detecting and modulating a novel mtor complex
EP3252155A1 (en) 2011-04-21 2017-12-06 The Rockefeller University Streptococcus bacteriophage lysins for detection and treatment of gram positive bacteria
BR112013027333A2 (en) 2011-04-28 2016-11-29 Novozymes As transgenic microbial host cell, nucleic acid construct, expression vector, isolated polypeptide having endoglucanase activity, isolated polynucleotide, methods of producing an endoglucanase activity polypeptide, to produce a precursor cell mutant, to inhibit expression of a polypeptide with endoglucanase activity in a cell, to produce a protein, to degrade or convert a cellulosic material, to produce a fermentation product, and to ferment a cellulosic material.
EP2702162B1 (en) 2011-04-29 2020-02-26 Novozymes, Inc. Methods for enhancing the degradation or conversion of cellulosic material
EP2705051A1 (en) 2011-05-05 2014-03-12 Novozymes Biopharma DK A/S Albumin variants
EP2710132A1 (en) 2011-05-19 2014-03-26 Novozymes, Inc. Methods for enhancing the degradation of cellulosic material with chitin binding proteins
WO2012159009A1 (en) 2011-05-19 2012-11-22 Novozymes, Inc. Methods for enhancing the degradation of cellulosic material with chitin binding proteins
NZ618016A (en) 2011-05-21 2015-05-29 Macrogenics Inc Deimmunized serum-binding domains and their use for extending serum half-life
US9150625B2 (en) 2011-05-23 2015-10-06 E I Du Pont De Nemours And Company Chloroplast transit peptides and methods of their use
EP2527432A1 (en) 2011-05-23 2012-11-28 Novozymes A/S Bi-directional cytosine deaminase-encoding selection marker
EP2527448A1 (en) 2011-05-23 2012-11-28 Novozymes A/S Simultaneous site-specific integrations of multiple gene-copies in filamentous fungi
DK2714738T3 (en) 2011-05-24 2019-01-28 Zyngenia Inc MULTIVALENT AND MONOVALENT MULTISPECIFIC COMPLEXES AND THEIR APPLICATIONS
WO2012168259A1 (en) 2011-06-06 2012-12-13 Novartis Forschungsstiftung, Zweigniederlassung Protein tyrosine phosphatase, non-receptor type 11 (ptpn11) and triple-negative breast cancer
WO2012170742A2 (en) 2011-06-07 2012-12-13 University Of Hawaii Treatment and prevention of cancer with hmgb1 antagonists
WO2012170740A2 (en) 2011-06-07 2012-12-13 University Of Hawaii Biomarker of asbestos exposure and mesothelioma
WO2012172495A1 (en) 2011-06-14 2012-12-20 Novartis Ag Compositions and methods for antibodies targeting tem8
US20140216118A1 (en) 2011-06-14 2014-08-07 Synthon Biopharmaceuticals B.V. Compositions and Methods for Making and Biocontaining Auxotrophic Transgenic Plants
EP2723874A2 (en) 2011-06-21 2014-04-30 Pioneer Hi-Bred International, Inc. Methods and compositions for producing male sterile plants
EP2723858B1 (en) 2011-06-24 2017-04-12 Novozymes A/S Polypeptides having protease activity and polynucleotides encoding same
EP2726651B1 (en) 2011-06-28 2018-11-07 Codexis, Inc. Protein variant generation by region shuffling
PL3543333T3 (en) 2011-06-30 2022-06-13 Novozymes A/S Method for screening alpha-amylases
US8778652B2 (en) 2011-06-30 2014-07-15 Codexis, Inc. Pentose fermentation by a recombinant microorganism
KR20140056237A (en) 2011-06-30 2014-05-09 노보자임스 에이/에스 Alpha-amylase variants
EP2540824A1 (en) 2011-06-30 2013-01-02 The Procter & Gamble Company Cleaning compositions comprising amylase variants reference to a sequence listing
WO2013006756A2 (en) 2011-07-06 2013-01-10 Novozymes A/S Alpha amylase variants and polynucleotides encoding same
CN103703136A (en) 2011-07-07 2014-04-02 诺维信公司 Enzymatic preparation of diols
US9217021B2 (en) 2011-07-08 2015-12-22 Defensin Therapeutics Aps Oral treatment of inflammatory bowel disease
US20130097734A1 (en) 2011-07-12 2013-04-18 Two Blades Foundation Late blight resistance genes
WO2013010783A1 (en) 2011-07-15 2013-01-24 Novozymes A/S Lipase variants and polynucleotides encoding same
MX347810B (en) 2011-07-15 2017-05-15 Syngenta Participations Ag Methods of increasing yield and stress tolerance in a plant.
WO2013012643A1 (en) 2011-07-15 2013-01-24 Syngenta Participations Ag Polynucleotides encoding trehalose-6-phosphate phosphatase and methods of use thereof
CN103703139A (en) 2011-07-22 2014-04-02 诺维信北美公司 Processes for pretreating cellulosic material and improving hydrolysis thereof
US9322007B2 (en) 2011-07-22 2016-04-26 The California Institute Of Technology Stable fungal Cel6 enzyme variants
AR087367A1 (en) 2011-07-28 2014-03-19 Athenix Corp AXMI 270 TOXIN GEN AND ITS METHODS OF USE
US9862965B2 (en) 2011-07-28 2018-01-09 Athenix Corp. AXMI205 variant proteins and methods of use
WO2013015993A1 (en) 2011-07-28 2013-01-31 Syngenta Participations Ag Methods and compositions for controlling nematode pests
MX2014001070A (en) 2011-07-29 2014-04-14 Athenix Corp Axmi279 pesticidal gene and methods for its use.
US9951346B2 (en) 2011-08-03 2018-04-24 Pioneer Hi-Bred International, Inc. Methods and compositions for targeted integration in a plant
EP3382016B1 (en) 2011-08-04 2019-10-09 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
MX355816B (en) 2011-08-04 2018-05-02 Amgen Inc Method for treating bone gap defects.
CN103703125B (en) 2011-08-04 2016-09-07 诺维信公司 There is polypeptide and the coded polynucleotide thereof of endoglucanase activity
WO2013021065A1 (en) 2011-08-10 2013-02-14 Novozymes A/S Polypeptides having peroxygenase activity and polynucleotides encoding same
US9506044B2 (en) 2011-08-10 2016-11-29 Novozymes A/S Polypeptides having peroxygenase activity and polynucleotides encoding same
WO2013021064A1 (en) 2011-08-10 2013-02-14 Novozymes A/S Polypeptides having peroxygenase activity and polynucleotides encoding same
WO2013021062A1 (en) 2011-08-10 2013-02-14 Novozymes A/S Polypeptides having peroxygenase activity and polynucleotides encoding same
WO2013021059A1 (en) 2011-08-10 2013-02-14 Novozymes A/S Polypeptides having peroxygenase activity and polynucleotides encoding same
EP2742129B1 (en) 2011-08-10 2017-08-02 Novozymes A/S Polypeptides having peroxygenase activity and polynucleotides encoding same
CN103732740B (en) 2011-08-10 2018-07-31 诺维信公司 Polypeptide with peroxidase activity and the polynucleotides for encoding the polypeptide
US9000138B2 (en) 2011-08-15 2015-04-07 Novozymes A/S Expression constructs comprising a Terebella lapidaria nucleic acid encoding a cellulase, host cells, and methods of making the cellulase
WO2013024157A2 (en) 2011-08-17 2013-02-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Combinations of host targeting agents for the treatment and the prevention of hcv infection
WO2013024155A1 (en) 2011-08-17 2013-02-21 Inserm (Institut National De La Sante Et De La Recherche Medicale) Combinations of anti-hcv-entry factor antibodies and direct acting antivirals for the treatment and the prevention of hcv infection
WO2013024156A2 (en) 2011-08-17 2013-02-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Combinations of anti-hcv-entry factor antibodies and interferons for the treatment and the prevention of hcv infection
ES2579706T3 (en) 2011-08-19 2016-08-16 Novozymes, Inc. Recombinant microorganisms for the production of C4-dicarboxylic acids
ES2641039T3 (en) 2011-08-19 2017-11-07 Novozymes A/S Polypeptides with protease activity
US20130052213A1 (en) 2011-08-19 2013-02-28 Novozymes A/S Novel immunomodulatory peptide
WO2013028701A1 (en) 2011-08-22 2013-02-28 Codexis, Inc. Gh61 glycoside hydrolase protein variants and cofactors that enhance gh61 activity
EP2748188A4 (en) 2011-08-26 2015-03-18 Novozymes As Polypeptides having glucoamylase activity and polynucleotides encoding same
US20130059296A1 (en) 2011-08-26 2013-03-07 Gen9, Inc. Compositions and Methods For High Fidelity Assembly of Nucleic Acids
US11377663B1 (en) 2011-08-30 2022-07-05 Monsanto Technology Llc Genetic regulatory elements
CA2847057C (en) 2011-08-31 2019-06-11 St. Jude Children's Research Hospital Methods and compositions to detect the level of lysosomal exocytosis activity and methods of use
MX351154B (en) 2011-09-06 2017-10-04 Novozymes As Glucoamylase variants and polynucleotides encoding same.
EP2753640B1 (en) 2011-09-08 2016-03-09 Codexis, Inc. Biocatalysts and methods for the synthesis of substituted lactams
US9994834B2 (en) 2011-09-09 2018-06-12 Novozymes A/S Polynucleotides encoding polypeptides having alpha-amylase activity and methods of making the same
PL2753749T3 (en) 2011-09-09 2019-10-31 Novozymes As Improving properties of paper materials
WO2013039776A1 (en) 2011-09-13 2013-03-21 Novozymes North America, Inc. Methods of hydrolyzing and fermenting cellulosic material
US20140308705A1 (en) 2011-09-20 2014-10-16 Novozymes A/S Polypeptides Having Cellulolytic Enhancing Activity And Polynucleotides Encoding Same
JP2014530598A (en) 2011-09-22 2014-11-20 ノボザイムスアクティーゼルスカブ Polypeptide having protease activity and polynucleotide encoding the same
MX363042B (en) 2011-09-23 2019-03-06 Novozymes As Color modification of textile.
EP2760997A4 (en) 2011-09-30 2015-02-11 Codexis Inc Fungal proteases
CN103857791B (en) 2011-09-30 2016-11-23 诺维信股份有限公司 Dehydrogenase modification and their polynucleotide of coding
US20150031091A1 (en) 2011-09-30 2015-01-29 Novozymes A/S Polypeptides having alpha-amylase activity and polynucleotides encoding same
AU2012318609B2 (en) 2011-10-05 2018-02-08 The Rockefeller University Dimeric bacteriophage lysins
US9353362B2 (en) 2011-10-11 2016-05-31 Novozymes A/S Glucoamylase variants and polynucleotides encoding same
CN116063509A (en) 2011-10-11 2023-05-05 维埃拉生物股份有限公司 CD 40L-specific TN 3-derived scaffolds and methods of use thereof
WO2013057141A2 (en) 2011-10-17 2013-04-25 Novozymes A/S Alpha-amylase variants and polynucleotides encoding same
MX354704B (en) 2011-10-17 2018-03-16 Novozymes As Alpha-amylase variants and polynucleotides encoding same.
CN103890179A (en) 2011-10-28 2014-06-25 纳幕尔杜邦公司 Methods and compositions for silencing genes using artificial microRNAs
US10308921B2 (en) 2011-10-31 2019-06-04 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP2773373B1 (en) 2011-11-01 2018-08-22 Bionomics, Inc. Methods of blocking cancer stem cell growth
WO2013067057A1 (en) 2011-11-01 2013-05-10 Bionomics, Inc. Anti-gpr49 antibodies
CN104053671A (en) 2011-11-01 2014-09-17 生态学有限公司 Antibodies and methods of treating cancer
AU2012332593B2 (en) 2011-11-01 2016-11-17 Bionomics, Inc. Anti-GPR49 antibodies
CN109897103A (en) 2011-11-04 2019-06-18 酵活有限公司 There is the antibody design of the stabilization heterodimeric of mutation in Fc structural domain
WO2013064195A1 (en) 2011-11-04 2013-05-10 Enel Ingegneria E Ricerca S.P.A. A new heat-stable carbonic anhydrase and uses thereof
CN103930544A (en) 2011-11-08 2014-07-16 诺维信公司 Methods for production of archeae protease in yeast
US20140314787A1 (en) 2011-11-08 2014-10-23 Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute Treatment for neurodegenerative diseases
US20140294732A1 (en) 2011-11-08 2014-10-02 Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute Early diagnostic of neurodegenerative diseases
US20140315817A1 (en) 2011-11-18 2014-10-23 Eleven Biotherapeutics, Inc. Variant serum albumin with improved half-life and other properties
EP2780449B1 (en) 2011-11-18 2018-04-11 Novozymes, Inc. Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same
CN106479990B (en) 2011-11-18 2020-09-18 科德克希思公司 Biocatalysts for the preparation of hydroxy-substituted carbamates
US10351834B2 (en) 2011-11-21 2019-07-16 Novozymes, Inc. GH61 polypeptide variants and polynucleotides encoding same
DK2782998T3 (en) 2011-11-22 2018-04-16 Novozymes Inc POLYPEPTIDES WITH BETA-XYLOSIDASE ACTIVITY AND POLYNUCLEOTIDES CODING THEM
US20140342433A1 (en) 2011-11-25 2014-11-20 Novozymes A/S Subtilase Variants and Polynucleotides Encoding Same
ES2631605T3 (en) 2011-11-25 2017-09-01 Novozymes A/S Polypeptides with lysozyme activity and polynucleotides encoding them
BR112014012417A2 (en) 2011-12-01 2017-06-06 Novozymes Inc isolated polypeptide and polynucleotide, recombinant host cell, methods for producing a polypeptide, a mutant of a source cell, and a protein, and for inhibiting expression of a polypeptide, transgenic plant, part of the plant or cell of plant, rna molecule, processes for degrading or converting a cellulosic or xylan-containing material, to produce a fermentation product, and fermentation of a cellulosic or xylan-containing material, and integral broth formulation or cell culture composition
WO2013079533A1 (en) 2011-12-02 2013-06-06 Novozymes A/S Polypeptides having peroxygenase activity and polynucleotides encoding same
US9169469B2 (en) 2011-12-02 2015-10-27 Novozymes A/S Polypeptides having peroxygenase activity and polynucleotides encoding same
ES2758433T3 (en) 2011-12-05 2020-05-05 Novartis Ag Antibodies to epidermal growth factor receptor 3 (HER3)
TW201328707A (en) 2011-12-05 2013-07-16 Novartis Ag Antibodies for epidermal growth factor receptor 3 (HER3) directed to domain II of HER3
DK2791330T3 (en) 2011-12-16 2017-11-06 Novozymes Inc Polypeptides with laccase activity and polynucleotides encoding them
BR112014014697A2 (en) 2011-12-19 2020-10-27 Novozymes, Inc. isolated polypeptide, composition, isolated polynucleotide, nucleic acid construct or expression vector, recombinant host cell, methods for producing a polypeptide and a protein, for generating molecular oxygen, and for removing hydrogen peroxide from tissue, processes for degrading or converting a cellulosic material, and to produce a fermentation product, and, integral broth formulation or cell culture composition
US9611462B2 (en) 2011-12-20 2017-04-04 Codexis, Inc. Endoglucanase 1B (EG1B) variants
BR112014015228B1 (en) 2011-12-20 2022-07-05 Novozymes, Inc. GENITOR CELOBIOHIDROLASE VARIANT, PROCESSES FOR DEGRADING OR CONVERTING A CELLULOSIC MATERIAL, FOR PRODUCTION OF A FERMENTATION PRODUCT, AND FOR FERMENTATION OF A CELLULOSIC MATERIAL, WHOLE BROTH FORMULATION OR CELL CULTURE COMPOSITION, AND, TRANSGENIC MICROBIAN HOST CELL
CN104011204A (en) 2011-12-20 2014-08-27 诺维信公司 Subtilase Variants And Polynucleotides Encoding Same
EP2607468A1 (en) 2011-12-20 2013-06-26 Henkel AG & Co. KGaA Detergent compositions comprising subtilase variants
MX2014007665A (en) 2011-12-21 2015-03-05 Novartis Ag Compositions and methods for antibodies targeting factor p.
WO2013096562A1 (en) 2011-12-22 2013-06-27 E. I. Du Pont De Nemours And Company Use of the soybean sucrose synthase promoter to increase plant seed lipid content
EP3539982A3 (en) 2011-12-23 2020-01-15 Pfizer Inc Engineered antibody constant regions for site-specific conjugation and methods and uses therefor
IN2014CN04634A (en) 2011-12-28 2015-09-18 Amgen Inc
ES2667318T3 (en) 2011-12-28 2018-05-10 Novozymes A/S Polypeptides with protease activity
WO2013102825A1 (en) 2012-01-02 2013-07-11 Novartis Ag Cdcp1 and breast cancer
MX355636B (en) 2012-01-06 2018-04-25 Pioneer Hi Bred Int Compositions and methods for the expression of a sequence in a reproductive tissue of a plant.
CA2860783A1 (en) 2012-01-06 2013-07-11 Pioneer Hi-Bred International, Inc. Ovule specific promoter and methods of use
WO2013103365A1 (en) 2012-01-06 2013-07-11 Pioneer Hi-Bred International, Inc. Pollen preferred promoters and methods of use
US20130180005A1 (en) 2012-01-06 2013-07-11 Pioneer Hi Bred International Inc Method to Screen Plants for Genetic Elements Inducing Parthenogenesis in Plants
WO2013104659A2 (en) 2012-01-13 2013-07-18 Dupont Nutrition Biosciences Aps Process
WO2013104660A1 (en) 2012-01-13 2013-07-18 Dupont Nutrition Biosciences Aps Process for treating a plant oil comprising hydrolysing chlorophyll or a chlorophyll derivative and involving partial caustic neutralisation
WO2013110766A1 (en) 2012-01-26 2013-08-01 Novozymes A/S Use of polypeptides having protease activity in animal feed and detergents
WO2013111018A1 (en) 2012-01-26 2013-08-01 Norfolk Plant Sciences, Ltd. Methods for increasing the anthocyanin content of citrus fruit
AR089793A1 (en) 2012-01-27 2014-09-17 Du Pont METHODS AND COMPOSITIONS TO GENERATE COMPOSITE TRANSGENIC RISK LOCUS
WO2013116773A1 (en) 2012-02-01 2013-08-08 Dow Agrosciences Llc Chloroplast transit peptide
EP3696265A3 (en) 2012-02-03 2020-10-07 Novozymes A/S Lipase variants and polynucleotides encoding same
PL2623586T3 (en) 2012-02-03 2018-01-31 Procter & Gamble Compositions and methods for surface treatment with lipases
EP2814956B1 (en) 2012-02-17 2017-05-10 Novozymes A/S Subtilisin variants and polynucleotides encoding same
EP2628785B1 (en) 2012-02-17 2016-05-18 Henkel AG & Co. KGaA Detergent compositions comprising subtilase variants
WO2013123871A1 (en) 2012-02-20 2013-08-29 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
GB201203374D0 (en) 2012-02-28 2012-04-11 Univ Sheffield Alkane production
MX362455B (en) 2012-03-08 2019-01-18 Athenix Corp Bacillus thuringiensis toxin gene axmi335 and methods for its use.
UA115236C2 (en) 2012-03-08 2017-10-10 Атенікс Корп. Axmi345 delta-endotoxin gene and methods for its use
CA2866626A1 (en) 2012-03-14 2013-09-19 E. I. Du Pont De Nemours And Company Improving agronomic characteristics of plants through abph2
EP2825025A1 (en) 2012-03-14 2015-01-21 E. I. Du Pont de Nemours and Company Nucleotide sequences encoding fasciated ear4 (fea4) and methods of use thereof
IN2014DN06887A (en) 2012-03-14 2015-05-15 Du Pont
EP2825556B1 (en) 2012-03-16 2018-01-03 Albumedix A/S Albumin variants
US9150853B2 (en) 2012-03-21 2015-10-06 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
SG11201405990PA (en) 2012-03-23 2014-11-27 Codexis Inc Biocatalysts and methods for synthesizing derivatives of tryptamine and tryptamine analogs
US20150266961A1 (en) 2012-03-29 2015-09-24 Novartis Forschungsstiftung, Zweigniederlassung, Fridrich Miescher Institute Inhibition of interleukin-8 and/or its receptor cxcr1 in the treatment of her2/her3-overexpressing breast cancer
WO2013144105A1 (en) 2012-03-31 2013-10-03 Novozymes A/S Epoxidation using peroxygenase
WO2013149858A1 (en) 2012-04-02 2013-10-10 Novozymes A/S Lipase variants and polynucleotides encoding same
CN104245929A (en) 2012-04-23 2014-12-24 诺维信公司 Polypeptides having alpha-glucuronidase activity and polynucleotides encoding same
BR112014024804A2 (en) 2012-04-23 2017-07-11 Novozymes As isolated polypeptide having glucuronyl esterase activity, composition, isolated polynucleotide, nucleic acid construct or expression vector, recombinant host cell, methods for producing a polypeptide, for degrading or converting a cellulosic material and for producing a product fermentation
US10081807B2 (en) 2012-04-24 2018-09-25 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US20150140168A1 (en) 2012-04-25 2015-05-21 Novozymes A/S Method of Baking
WO2013160372A1 (en) 2012-04-27 2013-10-31 Dupont Nutrition Biosciences Aps Process for treating plant oil involving addition of serial doses of chlorophyll or chlorophyll derivative degrading enzyme
WO2013160374A1 (en) 2012-04-27 2013-10-31 Dupont Nutrition Biosciences Aps Process for refining crude plant oil involving enzymatic hydrolysis and gum recycling
BR112014026625A2 (en) 2012-04-27 2017-07-18 Novozymes Inc E Novozymes As polypeptide variant, isolated polynucleotide, recombinant host cell, methods for producing a polypeptide variant and for obtaining a transgenic polypeptide variant, plant, plant part or cell, processes for degradation or conversion of a cellulosic material to production of a fermentation product and for the fermentation of a cellulosic material, composition, and whole broth cell culture formulation or composition
WO2013166113A1 (en) 2012-05-04 2013-11-07 E. I. Du Pont De Nemours And Company Compositions and methods comprising sequences having meganuclease activity
CN104271723B (en) 2012-05-07 2021-04-06 诺维信公司 Polypeptides having xanthan degrading activity and nucleotides encoding same
DK2847327T3 (en) 2012-05-08 2019-03-11 Codexis Inc BIO-CATALYSTS AND METHODS FOR HYDROXYLATION OF CHEMICAL COMPOUNDS
EP2847230B1 (en) 2012-05-10 2020-08-12 Zymeworks Inc. Heteromultimer constructs of immunoglobulin heavy chains with mutations in the fc domain
DK2847214T3 (en) 2012-05-11 2018-03-12 Codexis Inc CONSTRUCTED IMINE REDUCTIONS AND PROCEDURES FOR THE REDUCED AMINING OF KETON AND AMINE COMPOUNDS
BR112014028764A2 (en) 2012-05-18 2017-06-27 Novozymes As isolated mutant, methods for obtaining the isolated mutant and a lactobacillus transformant, and lactobacillus transformant.
WO2013178699A1 (en) 2012-05-31 2013-12-05 Novozymes A/S Isopropanol production by bacterial hosts
WO2013178808A2 (en) 2012-05-31 2013-12-05 Novozymes A/S Polypeptides having organophosphorous hydrolase activity
US9585970B2 (en) 2012-06-04 2017-03-07 Novartis Ag Site-specific labeling methods and molecules produced thereby
WO2013188305A2 (en) 2012-06-11 2013-12-19 Codexis, Inc. Fungal beta-xylosidase variants
CA2876426A1 (en) 2012-06-15 2013-12-19 E. I. Du Pont De Nemours And Company Methods and compositions involving als variants with native substrate preference
US20130337442A1 (en) 2012-06-15 2013-12-19 Pioneer Hi-Bred International, Inc. Genetic loci associated with soybean cyst nematode resistance and methods of use
US20150184208A1 (en) 2012-06-19 2015-07-02 Novozymes A/S Enzymatic reduction of hydroperoxides
WO2013189972A2 (en) 2012-06-20 2013-12-27 Novozymes A/S Use of polypeptides having protease activity in animal feed and detergents
DK2864491T3 (en) 2012-06-22 2018-12-17 Phytogene Inc Enzymes and methods for styrene synthesis
EP2677035A1 (en) 2012-06-22 2013-12-25 BASF Plant Science Company GmbH Plants having enhanced yield-related traits and a method for making the same
CA2877823A1 (en) 2012-06-25 2014-01-03 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
WO2014001482A1 (en) 2012-06-29 2014-01-03 Novartis Forschungsstiftung, Zweigniererlassung, Friedrich Miescher Institute For Biomedical Research Treating diseases by modulating a specific isoform of mkl1
UA117731C2 (en) 2012-06-29 2018-09-25 Атенікс Корп. AXMI277 TOXIN AGAINST NEMATODS AND ITS APPLICATION
WO2014006114A1 (en) 2012-07-05 2014-01-09 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research New treatment for neurodegenerative diseases
WO2014006100A1 (en) 2012-07-05 2014-01-09 Ucb Pharma S.A. Treatment for bone diseases
JP2015523078A (en) 2012-07-12 2015-08-13 ノボザイムス アクティーゼルスカブ Polypeptide having lipase activity and polynucleotide encoding the same
WO2014012506A1 (en) 2012-07-18 2014-01-23 Novozymes A/S Method of treating polyester textile
EP2875142A2 (en) 2012-07-20 2015-05-27 Novozymes A/S Enzymatic oxidation of 5-hydroxymethylfurfural and derivatives thereof
RU2681730C2 (en) 2012-07-25 2019-03-12 Селлдекс Терапьютикс Инк. Anti-kit antibodies and uses thereof
WO2014022434A1 (en) 2012-07-30 2014-02-06 Allylix, Inc. Sclareol and labdenediol diphosphate synthase polypeptides encoding nucleic acid molecules and uses thereof
US10752949B2 (en) 2012-08-14 2020-08-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9951386B2 (en) 2014-06-26 2018-04-24 10X Genomics, Inc. Methods and systems for processing polynucleotides
MX364957B (en) 2012-08-14 2019-05-15 10X Genomics Inc Microcapsule compositions and methods.
US10273541B2 (en) 2012-08-14 2019-04-30 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10323279B2 (en) 2012-08-14 2019-06-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10221442B2 (en) 2012-08-14 2019-03-05 10X Genomics, Inc. Compositions and methods for sample processing
US10400280B2 (en) 2012-08-14 2019-09-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9701998B2 (en) 2012-12-14 2017-07-11 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11591637B2 (en) 2012-08-14 2023-02-28 10X Genomics, Inc. Compositions and methods for sample processing
PT3553172T (en) 2012-08-16 2023-01-27 Novozymes As Method for treating textile with endoglucanase
MY175956A (en) 2012-08-16 2020-07-16 Bangladesh Jute Res Institute Cellulose and/or hemicelluloses degrading enzymes from macrophomina phaseolina and uses thereof
AU2013302535B2 (en) 2012-08-16 2018-12-06 Bangladesh Jute Research Institute Pectin degrading enzymes from Macrophomina phaseolina and uses thereof
MX2015001969A (en) 2012-08-17 2015-05-15 Novozymes As Thermostable asparaginase variants and polynucleotides encoding same.
WO2014029820A1 (en) 2012-08-22 2014-02-27 Novozymes A/S Detergent compositions comprising metalloproteases
WO2014029819A1 (en) 2012-08-22 2014-02-27 Novozymes A/S Metalloprotease from exiguobacterium
US9315791B2 (en) 2012-08-22 2016-04-19 Novozymes A/S Metalloproteases from alicyclobacillus
US9758793B2 (en) 2012-08-30 2017-09-12 Athenix Corp. AXMI-234 and AXMI-235 delta-endotoxin genes and methods for their use
WO2014033266A1 (en) 2012-08-31 2014-03-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Anti-sr-bi antibodies for the inhibition of hepatitis c virus infection
ES2694765T3 (en) 2012-09-05 2018-12-27 Novozymes A/S Polypeptides with protease activity
AU2013202506B2 (en) 2012-09-07 2015-06-18 Celgene Corporation Resistance biomarkers for hdac inhibitors
US9816102B2 (en) 2012-09-13 2017-11-14 Indiana University Research And Technology Corporation Compositions and systems for conferring disease resistance in plants and methods of use thereof
CN104685052A (en) 2012-09-19 2015-06-03 诺维信股份有限公司 Methods for enhancing the degradation or conversion of cellulosic material
WO2014047453A2 (en) 2012-09-20 2014-03-27 Novozymes A/S Screening polynucleotide libraries for variants that encode functional proteins
CN104619721A (en) 2012-09-27 2015-05-13 诺维信股份有限公司 Bacterial mutants with improved transformation efficiency
US10005988B2 (en) 2012-10-05 2018-06-26 Novozymes A/S Reducing adhesion of bacteria to a surface or releasing bacteria from a surface to which they adhere using endo-beta-A,4-glucanases
EP3586610A1 (en) 2012-10-08 2020-01-01 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
JP2015533832A (en) 2012-10-09 2015-11-26 アイジェニカ バイオセラピューティクス インコーポレイテッド Anti-C16orf54 antibody and method of use thereof
CA2887571A1 (en) 2012-10-11 2014-04-17 Pioneer Hi-Bred International, Inc. Guard cell promoters and uses thereof
CN104704115B (en) 2012-10-12 2018-11-02 诺维信公司 With the active polypeptide of peroxygenases
WO2014056927A2 (en) 2012-10-12 2014-04-17 Novozymes A/S Polypeptides having peroxygenase activity
WO2014056916A2 (en) 2012-10-12 2014-04-17 Novozymes A/S Polypeptides having peroxygenase activity
WO2014056920A2 (en) 2012-10-12 2014-04-17 Novozymes A/S Polypeptides having peroxygenase activity
EP2906688B1 (en) 2012-10-12 2018-08-29 Novozymes A/S Polypeptides having peroxygenase activity
WO2014056922A2 (en) 2012-10-12 2014-04-17 Novozymes A/S Polypeptides having peroxygenase activity
US9404094B2 (en) 2012-10-12 2016-08-02 Novozymes A/S Polypeptides having peroxygenase activity
US9783820B2 (en) 2012-10-15 2017-10-10 Pioneer Hi-Bred International, Inc. Methods and compositions to enhance activity of Cry endotoxins
WO2014063097A1 (en) 2012-10-19 2014-04-24 Danisco Us Inc. Stabilization of biomimetic membranes
CA2888157A1 (en) 2012-10-23 2014-05-01 Montana State University Production of high quality durum wheat having increased amylose content
US20150275194A1 (en) 2012-10-24 2015-10-01 Novozymes A/S Polypeptides Having Cellulolytic Enhancing Activity And Polynucleotides Encoding Same
US9663532B2 (en) 2012-10-29 2017-05-30 University Of Rochester Artemisinin derivatives, methods for their preparation and their use as antimalarial agents
WO2014068010A1 (en) 2012-10-31 2014-05-08 Novozymes A/S Isopropanol production by bacterial hosts
EP2914729B1 (en) 2012-11-01 2022-01-26 The University of British Columbia Cytochrome p450 and cytochrome p450 reductase polypeptides, encoding nucleic acid molecules and uses thereof
WO2014072481A1 (en) 2012-11-08 2014-05-15 Novozymes Biopharma Dk A/S Albumin variants
AU2013202507B9 (en) 2012-11-14 2015-08-13 Celgene Corporation Inhibition of drug resistant cancer cells
WO2014076232A2 (en) 2012-11-19 2014-05-22 Novozymes A/S Isopropanol production by recombinant hosts using an hmg-coa intermediate
EP2922953A4 (en) 2012-11-20 2016-11-09 Shell Int Research Pentose fermentation by a recombinant microorganism
UY35148A (en) 2012-11-21 2014-05-30 Amgen Inc HETERODIMERIC IMMUNOGLOBULINS
EP2925873B1 (en) 2012-11-30 2017-07-12 Novozymes, Inc. 3-hydroxypropionic acid production by recombinant yeasts
WO2014084859A1 (en) 2012-11-30 2014-06-05 Novartis Ag Molecules and methods for modulating tmem16a activities
RS61648B1 (en) 2012-12-05 2021-04-29 Novartis Ag Compositions and methods for antibodies targeting epo
WO2014088693A1 (en) 2012-12-06 2014-06-12 Agilent Technologies, Inc. Molecular fabrication
CN104837993A (en) 2012-12-07 2015-08-12 诺维信公司 Method for generating site-specific mutations in filamentous fungi
EP2740840A1 (en) 2012-12-07 2014-06-11 Novozymes A/S Improving drainage of paper pulp
BR112015012982A2 (en) 2012-12-07 2017-09-12 Novozymes As detergent composition, washing method for textile, washed textile, and use of a deoxyribonuclease
PT2928865T (en) 2012-12-07 2018-06-11 Merck Sharp & Dohme Biocatalytic transamination process
CN112852777A (en) 2012-12-11 2021-05-28 诺维信公司 Polypeptides having phospholipase C activity and polynucleotides encoding same
WO2014090765A1 (en) 2012-12-12 2014-06-19 Bayer Cropscience Ag Use of 1-[2-fluoro-4-methyl-5-(2,2,2-trifluoroethylsulfinyl)phenyl]-5-amino-3-trifluoromethyl)-1 h-1,2,4 tfia zole for controlling nematodes in nematode-resistant crops
US10533221B2 (en) 2012-12-14 2020-01-14 10X Genomics, Inc. Methods and systems for processing polynucleotides
CN104870639A (en) 2012-12-14 2015-08-26 诺维信公司 Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014090940A1 (en) 2012-12-14 2014-06-19 Novozymes A/S Removal of skin-derived body soils
EP3567116A1 (en) 2012-12-14 2019-11-13 10X Genomics, Inc. Methods and systems for processing polynucleotides
WO2014099653A1 (en) 2012-12-17 2014-06-26 Novozymes A/S Alpha-amylases and polynucleotides encoding same
SG11201503566QA (en) 2012-12-18 2015-09-29 Novartis Ag Compositions and methods that utilize a peptide tag that binds to hyaluronan
US20150337280A1 (en) 2012-12-19 2015-11-26 Novozymes A/S Polypeptides Having Cellulolytic Enhancing Activity And Polynucleotides Encoding Same
US9902943B2 (en) 2012-12-21 2018-02-27 Codexis, Inc. Engineered biocatalysts and methods for synthesizing chiral amines
BR112015014396B1 (en) 2012-12-21 2021-02-02 Novozymes A/S COMPOSITION, NUCLEIC ACID CONSTRUCTION OR EXPRESSION VECTOR, RECOMBINANT MICROORGANISM, METHODS OF IMPROVING THE NUTRITIONAL VALUE OF ANIMAL FEED, ANIMAL FEED ADDITIVE, AND USE OF ONE OR MORE PROTEASES
WO2014101753A1 (en) 2012-12-24 2014-07-03 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
CN103013938B (en) 2012-12-25 2014-12-17 北京大北农科技集团股份有限公司 Herbicide resistance protein, coding gene and application thereof
CN103060279B (en) 2012-12-25 2014-08-13 北京大北农科技集团股份有限公司 Herbicide resistance protein and encoding genes thereof and application thereof
CN104903443A (en) 2013-01-03 2015-09-09 诺维信公司 Alpha-amylase variants and polynucleotides encoding same
HUE044110T2 (en) 2013-01-18 2019-09-30 Codexis Inc Engineered biocatalysts useful for carbapenem synthesis
KR20150113166A (en) 2013-01-31 2015-10-07 코덱시스, 인코포레이티드 Methods, systems, and software for identifying bio-molecules with interacting components
WO2014120916A1 (en) 2013-02-01 2014-08-07 Bristol-Myers Squibb Company Pegylated domain antibodies monovalent for cd28 binding and methods of use
WO2014122109A1 (en) 2013-02-05 2014-08-14 Novozymes A/S Enzymatic preparation of indigo dyes and intermediates
CN104968211A (en) 2013-02-06 2015-10-07 诺维信公司 Polypeptides having protease activity
DK2953976T3 (en) 2013-02-08 2021-06-21 Novartis Ag SPECIFIC MODIFICATION PLACES IN ANTIBODIES FOR THE PRODUCTION OF IMMUNE CONJUGATES
WO2014124258A2 (en) 2013-02-08 2014-08-14 Irm Llc Specific sites for modifying antibodies to make immunoconjugates
WO2014124338A1 (en) * 2013-02-08 2014-08-14 10X Technologies, Inc. Polynucleotide barcode generation
ES2682351T3 (en) 2013-02-13 2018-09-20 Athenix Corp. Use of AXMI184 for root worm insect control
ES2900102T3 (en) 2013-02-20 2022-03-15 Univ Emory Compositions for Nucleic Acid Sequencing in Mixtures
SG11201506748WA (en) 2013-02-28 2015-09-29 Codexis Inc Engineered transaminase polypeptides for industrial biocatalysis
US9487773B2 (en) * 2013-03-01 2016-11-08 Technophage, Investigacao E Desenvolvimento Em Biotecnologia, Sa Cell-based methods for coupling protein interactions and binding molecule selection
DK2964767T3 (en) 2013-03-07 2020-03-23 BASF Agricultural Solutions Seed US LLC TOXICATIONS AND PROCEDURES FOR USE THEREOF
DK2964760T3 (en) 2013-03-08 2021-07-26 Novozymes Inc Cellobiohydrolase variants and polynucleotides encoding them
CA2905377A1 (en) 2013-03-11 2014-10-09 Pioneer Hi-Bred International, Inc. Methods and compositions to improve the spread of chemical signals in plants
CN105473721A (en) 2013-03-11 2016-04-06 先锋国际良种公司 Methods and compositions employing a sulfonylurea-dependent stabilization domain
GB201308828D0 (en) 2013-03-12 2013-07-03 Verenium Corp Phytase
US9803214B2 (en) 2013-03-12 2017-10-31 Pioneer Hi-Bred International, Inc. Breeding pair of wheat plants comprising an MS45 promoter inverted repeat that confers male sterility and a construct that restores fertility
US9243258B2 (en) 2013-03-12 2016-01-26 Pioneer Hi Bred International Inc Root-preferred promoter and methods of use
US9273322B2 (en) 2013-03-12 2016-03-01 Pioneer Hi Bred International Inc Root-preferred promoter and methods of use
US9498532B2 (en) 2013-03-13 2016-11-22 Novartis Ag Antibody drug conjugates
EP2970935A1 (en) 2013-03-14 2016-01-20 Pioneer Hi-Bred International, Inc. Compositions having dicamba decarboxylase activity and methods of use
GB201308843D0 (en) 2013-03-14 2013-07-03 Verenium Corp Phytase formulation
US20140289906A1 (en) 2013-03-14 2014-09-25 Pioneer Hi-Bred International, Inc. Compositions Having Dicamba Decarboxylase Activity and Methods of Use
EP3085778B1 (en) 2013-03-14 2021-07-07 Evolva, Inc. Valencene synthase polypeptides, encoding nucleic acid molecules and uses thereof
EP3611189A1 (en) 2013-03-14 2020-02-19 Novartis AG Antibodies against notch 3
CN105007950B (en) 2013-03-15 2019-01-15 诺华股份有限公司 Antibody drug conjugate
CN105473605A (en) 2013-03-15 2016-04-06 先锋国际良种公司 Phi-4 polypeptides and methods for their use
IL292498A (en) * 2013-03-15 2022-06-01 Gen9 Inc Compositions and methods for multiplex nucleic acids synthesis
CN105188738A (en) 2013-03-15 2015-12-23 奥普罗治疗学股份有限公司 Product and process for mucus viscosity normalization
EA201890895A1 (en) 2013-03-15 2019-02-28 Зинджения, Инк. MULTIVALENT AND MONOVALENT MULTIS-SPECIFIC COMPLEXES AND THEIR APPLICATION
EP2976423B1 (en) 2013-03-21 2018-11-28 Novozymes A/S Polypeptides having phospholipase a activity and polynucleotides encoding same
EP2976416B1 (en) 2013-03-21 2018-05-16 Novozymes A/S Polypeptides with lipase activity and polynucleotides encoding same
EA034034B1 (en) 2013-04-18 2019-12-20 Кодексис, Инк. Engineered phenylalanine ammonia-lyase polypeptides
EP2986701B1 (en) 2013-04-18 2018-11-14 Novozymes A/S Polypeptides having protease activity and polynucleotides encoding same
CN105164147B (en) 2013-04-23 2020-03-03 诺维信公司 Liquid automatic dishwashing detergent composition with stabilized subtilisin
WO2014177541A2 (en) 2013-04-30 2014-11-06 Novozymes A/S Glucoamylase variants and polynucleotides encoding same
CA2904022C (en) 2013-04-30 2022-04-19 Novozymes A/S Glucoamylase variants and polynucleotides encoding same
EP2992076B1 (en) 2013-05-03 2018-10-24 Novozymes A/S Microencapsulation of detergent enzymes
DK2994529T3 (en) 2013-05-10 2019-03-04 Novozymes As Polypeptides with xylanase activity and polynucleotides encoding them
MY192746A (en) 2013-05-14 2022-09-06 Novozymes As Detergent compositions
WO2014183921A1 (en) 2013-05-17 2014-11-20 Novozymes A/S Polypeptides having alpha amylase activity
ES2891755T3 (en) 2013-06-06 2022-01-31 Pf Medicament Anti-C10orf54 antibodies and uses thereof
EP3786269A1 (en) 2013-06-06 2021-03-03 Novozymes A/S Alpha-amylase variants and polynucleotides encoding same
EP2816115A1 (en) 2013-06-17 2014-12-24 BASF Plant Science Company GmbH Plants having one or more enhanced yield-related traits and a method for making the same
US20160122772A1 (en) 2013-06-21 2016-05-05 Novozymes A/S Production of Polypeptides Without Secretion Signal in Bacillus
US9562101B2 (en) 2013-06-21 2017-02-07 Novartis Ag Lectin-like oxidized LDL receptor 1 antibodies and methods of use
AR096601A1 (en) 2013-06-21 2016-01-20 Novartis Ag ANTIBODIES OF LEXINED OXIDATED LDL RECEIVER 1 AND METHODS OF USE
WO2014207224A1 (en) 2013-06-27 2014-12-31 Novozymes A/S Subtilase variants and polynucleotides encoding same
WO2014207227A1 (en) 2013-06-27 2014-12-31 Novozymes A/S Subtilase variants and polynucleotides encoding same
CN105358670A (en) 2013-07-04 2016-02-24 诺维信公司 Polypeptides with xanthan lyase activity having anti-redeposition effect and polynucleotides encoding same
WO2015006105A1 (en) 2013-07-09 2015-01-15 Board Of Trustees Of Michigan State University Transgenic plants produced with a k-domain, and methods and expression cassettes related thereto
WO2015004102A1 (en) 2013-07-09 2015-01-15 Novozymes A/S Polypeptides with lipase activity and polynucleotides encoding same
US11459579B2 (en) 2013-07-09 2022-10-04 Board Of Trustees Of Michigan State University Transgenic plants produced with a K-domain, and methods and expression cassettes related thereto
WO2015007639A1 (en) 2013-07-17 2015-01-22 Novozymes A/S Pullulanase chimeras and polynucleotides encoding same
CN105408492A (en) 2013-07-25 2016-03-16 巴斯夫酶有限责任公司 Phytase
US9926550B2 (en) 2013-07-29 2018-03-27 Novozymes A/S Protease variants and polynucleotides encoding same
EP3339436B1 (en) 2013-07-29 2021-03-31 Henkel AG & Co. KGaA Detergent composition comprising protease variants
RU2670946C9 (en) 2013-07-29 2018-11-26 Новозимс А/С Protease variants and polynucleotides encoding them
BR112016001778A2 (en) 2013-07-31 2017-09-05 Novozymes As TRANSGENIC YEAST CELL, COMPOSITION, AND, METHODS OF PRODUCTION OF 3-HP AND OF PRODUCTION OF ACRYLIC ACID OR A SALT THEREOF
ES2761587T3 (en) 2013-08-07 2020-05-20 Friedrich Miescher Institute For Biomedical Res New screening method for the treatment of Friedreich's ataxia
CA2920031C (en) 2013-08-08 2023-03-14 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides having broad spectrum activity and uses thereof
UY35696A (en) 2013-08-09 2015-03-27 Athenix Corp ? RECOMBINANT DNA MOLECULE THAT INCLUDES AXMI440 TOXIN GENE, VECTOR, GUEST CELL, PLANTS, COMPOSITIONS AND RELATED METHODS ?.
WO2015038262A2 (en) 2013-08-09 2015-03-19 Athenix Corp. Axmi281 toxin gene and methods for its use
EP3033420B1 (en) 2013-08-15 2018-02-21 Novozymes A/S Polypeptides having beta-1,3-galactanase activity and polynucleotides encoding same
WO2015023846A2 (en) 2013-08-16 2015-02-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US10395758B2 (en) 2013-08-30 2019-08-27 10X Genomics, Inc. Sequencing methods
CA2923629A1 (en) 2013-09-11 2015-03-19 Pioneer Hi-Bred International, Inc. Plant regulatory elements and methods of use thereof
EA031651B1 (en) 2013-09-13 2019-02-28 Пайонир Хай-Бред Интернэшнл, Инк. Insecticidal proteins and methods for their use
SG11201707615YA (en) 2013-09-19 2017-10-30 Univ Leland Stanford Junior Methods and compositions for producing hepatocyte-like cells
WO2015048016A2 (en) 2013-09-24 2015-04-02 E. I. Du Pont De Nemours And Company Fasciated inflorescence (fin) sequences and methods of use
ES2693150T3 (en) 2013-09-27 2018-12-07 Codexis, Inc. Automatic filtration of enzyme variants
WO2015050959A1 (en) 2013-10-01 2015-04-09 Yale University Anti-kit antibodies and methods of use thereof
WO2015049370A1 (en) 2013-10-03 2015-04-09 Novozymes A/S Detergent composition and use of detergent composition
WO2015059133A1 (en) 2013-10-22 2015-04-30 Novozymes A/S Cellobiose dehydrogenase variants and polynucleotides encoding same
EP3068793B1 (en) 2013-11-13 2021-02-17 Codexis, Inc. Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
CN111616159B (en) 2013-11-25 2022-08-26 巴斯夫农业解决方案种子美国有限责任公司 Control of hemipteran insects using AXMI-011
EP2876156A1 (en) 2013-11-26 2015-05-27 Commissariat A L'energie Atomique Et Aux Energies Alternatives New enzymes and method for preparing hydroxylated L-lysine or L-ornithine and analogs thereof
CN110219055A (en) * 2013-11-27 2019-09-10 Gen9股份有限公司 Nucleic acid library and its manufacturing method
CN105764330B (en) 2013-11-27 2019-04-30 纳幕尔杜邦公司 Locus associated with the response to abiotic stress
CN105793418A (en) 2013-11-29 2016-07-20 诺维信公司 Peroxygenase variants
US9309314B2 (en) 2013-12-03 2016-04-12 Agency For Science, Technology And Research (A*Star) Polypeptides, nucleic acids and uses thereof
BR122021024287B1 (en) 2013-12-09 2022-10-11 Athenix Corp CONSTRUCT, VECTOR, BACTERIAL HOST CELL, POLYPEPTIDE, COMPOSITION, METHOD OF CONTROL AND EXTERMINATION OF LEPIDOPTER PEST, METHOD OF PLANT PROTECTION AND METHOD OF PRODUCTION OF POLYPEPTIDE WITH PESTICIDE ACTIVITY
KR102268532B1 (en) 2013-12-11 2021-06-24 노보자임스 에이/에스 Cutinase variants and polynucleotides encoding same
US9824068B2 (en) 2013-12-16 2017-11-21 10X Genomics, Inc. Methods and apparatus for sorting data
EP2886656A1 (en) 2013-12-18 2015-06-24 Commissariat A L'energie Atomique Et Aux Energies Alternatives New enzyme and method for preparing 4-hydroxyl benzyl alcohol and derivatives thereof
WO2015094527A1 (en) 2013-12-19 2015-06-25 Danisco Us Inc. Use of hydrophobins to increase gas transferin aerobic fermentation processes
US10030239B2 (en) 2013-12-20 2018-07-24 Novozymes A/S Polypeptides having protease activity and polynucleotides encoding same
EP4122945A1 (en) 2013-12-23 2023-01-25 University of Rochester Methods and compositions for ribosomal synthesis of macrocyclic peptides
NZ630311A (en) 2013-12-27 2016-03-31 Celgene Corp Romidepsin formulations and uses thereof
EP2896698A1 (en) 2014-01-17 2015-07-22 BASF Plant Science Company GmbH Plants having one or more enhanced yield-related traits and a method for making the same
DK3097192T3 (en) 2014-01-22 2018-11-19 Novozymes As PULLULANASE VARIATIONS AND POLYNUCLEOTIDES CODING THEM
EP3097112B1 (en) 2014-01-22 2020-05-13 Novozymes A/S Polypeptides with lipase activity and polynucleotides encoding same
UA119863C2 (en) 2014-01-24 2019-08-27 Нгм Біофармасьютікалс, Інк. Binding proteins and methods of use thereof
JP6602772B2 (en) 2014-01-28 2019-11-06 ダイス モレキュルズ エスヴィー, エルエルシー Recognition compound-attached monoliths, arrays thereof, and uses thereof
US20170166920A1 (en) 2014-01-30 2017-06-15 Two Blades Foundation Plants with enhanced resistance to phytophthora
US10480007B2 (en) 2014-02-07 2019-11-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants
ES2806473T3 (en) 2014-02-07 2021-02-17 Pioneer Hi Bred Int Insecticidal proteins and methods for their use
EP2910640B1 (en) 2014-02-25 2018-12-05 Biopract GmbH A method for improving substrate degradation in agricultural biogas plants
GB201403775D0 (en) 2014-03-04 2014-04-16 Kymab Ltd Antibodies, uses & methods
US20170021033A1 (en) 2014-03-12 2017-01-26 Novartis Ag Specific sites for modifying antibodies to make immunoconjugates
US10155935B2 (en) 2014-03-12 2018-12-18 Novozymes A/S Polypeptides with lipase activity and polynucleotides encoding same
WO2015143144A1 (en) 2014-03-19 2015-09-24 Novozymes A/S Method for enhancing activity of an x143 polypeptide
WO2015140275A1 (en) 2014-03-19 2015-09-24 Novozymes A/S Polypeptides having phospholipase c activity and polynucleotides encoding same
WO2015150372A1 (en) 2014-04-01 2015-10-08 Dupont Nutrition Biosciences Aps Method for increasing crude palm oil yields
WO2015150457A1 (en) 2014-04-01 2015-10-08 Novozymes A/S Polypeptides having alpha amylase activity
US9546214B2 (en) 2014-04-04 2017-01-17 Bionomics, Inc. Humanized antibodies that bind LGR5
CN106170545A (en) 2014-04-10 2016-11-30 诺维信公司 Alpha-amylase variants and the polynucleotide that it is encoded
EP3129143B1 (en) 2014-04-10 2022-11-23 10X Genomics, Inc. Method for partitioning microcapsules
RU2737535C2 (en) 2014-04-11 2020-12-01 Новозимс А/С Detergent composition
WO2015158237A1 (en) 2014-04-15 2015-10-22 Novozymes A/S Polypeptides with lipase activity and polynucleotides encoding same
WO2015161019A1 (en) 2014-04-16 2015-10-22 Codexis, Inc. Engineered tyrosine ammonia lyase
BR112016023769A2 (en) 2014-04-17 2017-10-17 Boehringer Ingelheim Rcv Gmbh recombinant host cell for expression of proteins of interest
EP3132028B1 (en) 2014-04-17 2023-10-25 Boehringer Ingelheim RCV GmbH & Co KG Recombinant host cell engineered to overexpress helper proteins
AR100159A1 (en) 2014-04-22 2016-09-14 Du Pont GENES OF PLASID CARBON ANHYDRAINE FOR OIL INCREASE IN SEEDS WITH INCREASED DGAT EXPRESSION
WO2015171603A1 (en) 2014-05-06 2015-11-12 Two Blades Foundation Methods for producing plants with enhanced resistance to oomycete pathogens
DK3139745T3 (en) 2014-05-09 2020-09-28 Univ Louisiana State VACCINES FOR GENITAL HERPES-SIMPLEX INFECTIONS
AR102318A1 (en) 2014-05-15 2017-02-22 Novozymes As COMPOSITIONS THAT INCLUDE POLIPEPTIDES THAT HAVE ACTIVITY PHOSPHOLIPASE C AND ITS USES
US10023852B2 (en) 2014-05-27 2018-07-17 Novozymes A/S Lipase variants and polynucleotides encoding same
EP3149028B1 (en) 2014-05-30 2021-09-15 Novozymes A/S Variants of gh family 11 xylanase and polynucleotides encoding same
EP2952584A1 (en) 2014-06-04 2015-12-09 Boehringer Ingelheim RCV GmbH & Co KG Improved protein production
CN106414729A (en) 2014-06-12 2017-02-15 诺维信公司 Alpha-amylase variants and polynucleotides encoding same
WO2015189816A1 (en) 2014-06-13 2015-12-17 Friedrich Miescher Institute For Biomedical Research New treatment against influenza virus
EP2957571B1 (en) 2014-06-17 2018-08-15 Centre National De La Recherche Scientifique (Cnrs) Monoclonal anti-pvhl antibodies and uses thereof
WO2015196070A1 (en) 2014-06-20 2015-12-23 Genentech, Inc. Chagasin-based scaffold compositions, methods, and uses
WO2015198243A2 (en) 2014-06-25 2015-12-30 Novartis Ag Compositions and methods for long acting proteins
ES2712402T3 (en) 2014-06-25 2019-05-13 Novozymes As Variants of xylanase and polynucleotides that encode them
WO2015198240A2 (en) 2014-06-25 2015-12-30 Novartis Ag Compositions and methods for long acting proteins
CN110211637B (en) 2014-06-26 2023-10-27 10X基因组学有限公司 Method and system for assembling nucleic acid sequences
CN106795553B (en) 2014-06-26 2021-06-04 10X基因组学有限公司 Methods of analyzing nucleic acids from individual cells or cell populations
WO2016001319A1 (en) 2014-07-03 2016-01-07 Novozymes A/S Improved stabilization of non-protease enzyme
EP3164486B1 (en) 2014-07-04 2020-05-13 Novozymes A/S Subtilase variants and polynucleotides encoding same
EP3327122B1 (en) 2014-07-04 2021-02-17 Novozymes A/S Subtilase variants and polynucleotides encoding same
EP3167052B1 (en) 2014-07-09 2020-01-01 Codexis, Inc. P450-bm3 variants with improved activity
WO2016020791A1 (en) 2014-08-05 2016-02-11 Novartis Ag Ckit antibody drug conjugates
TW201613977A (en) 2014-08-07 2016-04-16 Novartis Ag Angiopoetin-like 4 (ANGPTL4) antibodies and methods of use
CU24453B1 (en) 2014-08-07 2019-11-04 Novartis Ag ANTI-PROTEIN ANTIBODIES SIMILAR TO ANGIOPOYETIN 4
US20170218384A1 (en) 2014-08-08 2017-08-03 Pioneer Hi-Bred International, Inc. Ubiquitin promoters and introns and methods of use
EP3180360A1 (en) 2014-08-12 2017-06-21 Novartis AG Anti-cdh6 antibody drug conjugates
WO2016028999A1 (en) 2014-08-20 2016-02-25 Novozymes A/S Xyloglucan endotransglycosylase variants and polynucleotides encoding same
JP6594955B2 (en) 2014-08-27 2019-10-23 ニユー・イングランド・バイオレイブス・インコーポレイテツド Synthon formation
US9963687B2 (en) 2014-08-27 2018-05-08 New England Biolabs, Inc. Fusion polymerase and method for using the same
EP3189137A1 (en) 2014-09-05 2017-07-12 Novozymes A/S Carbohydrate binding module variants and polynucleotides encoding same
EP3191600A1 (en) 2014-09-11 2017-07-19 Promega Corporation Luciferase sequences utilizing infrared-emitting substrates to produce enhanced luminescence
RU2017112324A (en) 2014-09-12 2018-10-15 Пайонир Хай-Бред Интернэшнл, Инк. CREATION OF WEBSITES OF SITE-SPECIFIC INTEGRATION FOR COMPLEX SIGNS LOCUSES IN CORN AND SOY, AND ALSO WAYS OF APPLICATION
US20170298360A1 (en) 2014-09-24 2017-10-19 Friedrich Miescher Institute For Biomedical Research Lats and breast cancer
MA41044A (en) 2014-10-08 2017-08-15 Novartis Ag COMPOSITIONS AND METHODS OF USE FOR INCREASED IMMUNE RESPONSE AND CANCER TREATMENT
US20170292153A1 (en) * 2014-10-14 2017-10-12 Bgi Shenzhen Co., Limited Method for breaking nucleic acid and adding adaptor by means of transposase, and reagent
CA2963550A1 (en) 2014-10-16 2016-04-21 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides having broad spectrum activity and uses thereof
US20170226164A1 (en) 2014-10-16 2017-08-10 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides having improved activity spectrum and uses thereof
RU2733898C2 (en) 2014-10-16 2020-10-09 Пайонир Хай-Бред Интернэшнл, Инк. Insecticidal proteins and methods of using them
GB201418713D0 (en) 2014-10-21 2014-12-03 Kymab Ltd Bindings Proteins
WO2016062875A2 (en) 2014-10-23 2016-04-28 Novozymes A/S Glucoamylase variants and polynucleotides encoding same
BR112017007949A2 (en) 2014-10-24 2018-01-23 Danisco Us Inc method for alcohol production by the use of a tripeptidyl peptidase
BR112017008406A2 (en) 2014-10-24 2018-02-27 Dupont Nutrition Biosciences Aps use of proline tolerant tripeptidyl peptidases in food additive compositions
EP3212807B1 (en) 2014-10-29 2020-09-02 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequencing
US9975122B2 (en) 2014-11-05 2018-05-22 10X Genomics, Inc. Instrument systems for integrated sample processing
WO2016073610A1 (en) 2014-11-07 2016-05-12 Novozymes A/S Xylanase based bleach boosting
MA40913A (en) 2014-11-14 2017-09-20 Novartis Ag ANTIBODY-DRUG CONJUGATES
WO2016079305A1 (en) 2014-11-20 2016-05-26 Novozymes A/S Alicyclobacillus variants and polynucleotides encoding same
JP6719463B2 (en) 2014-11-25 2020-07-15 コデクシス, インコーポレイテッド Engineered imine reductase and method for reductive amination of ketone and amine compounds
WO2016087327A1 (en) 2014-12-01 2016-06-09 Novozymes A/S Polypeptides having pullulanase activity comprising the x25, x45 and cbm41 domains
EP3227438A1 (en) 2014-12-02 2017-10-11 Novozymes A/S Laccase variants and polynucleotides encoding same
CN107075493B (en) 2014-12-04 2020-09-01 诺维信公司 Subtilase variants and polynucleotides encoding same
MX2017007103A (en) 2014-12-05 2017-08-24 Novozymes As Lipase variants and polynucleotides encoding same.
ES2934940T3 (en) 2014-12-11 2023-02-28 Pf Medicament Anti-C10orf54 antibodies and uses thereof
MA41142A (en) 2014-12-12 2017-10-17 Amgen Inc ANTI-SCLEROSTINE ANTIBODIES AND THE USE OF THEM TO TREAT BONE CONDITIONS AS PART OF THE TREATMENT PROTOCOL
RU2741833C2 (en) 2014-12-12 2021-01-29 Зингента Партисипейшнс Аг Compositions and methods for controlling plant pests
US20170369902A1 (en) 2014-12-16 2017-12-28 Pioneer Hi-Bred International, Inc. Restoration of male fertility in wheat
EP3233894A1 (en) 2014-12-16 2017-10-25 Novozymes A/S Polypeptides having n-acetyl glucosamine oxidase activity
UY36449A (en) 2014-12-19 2016-07-29 Novartis Ag COMPOSITIONS AND METHODS FOR ANTIBODIES DIRECTED TO BMP6
CN114717217A (en) 2014-12-19 2022-07-08 诺维信公司 Compositions comprising a polypeptide having xylanase activity and a polypeptide having arabinofuranosidase activity
EP3741848A3 (en) 2014-12-19 2021-02-17 Novozymes A/S Protease variants and polynucleotides encoding same
WO2016100910A1 (en) 2014-12-19 2016-06-23 Novozymes A/S Recombinant host cells for the production of 3-hydroxypropionic acid
US11518987B2 (en) 2014-12-19 2022-12-06 Novozymes A/S Protease variants and polynucleotides encoding same
SI3237621T1 (en) 2014-12-22 2023-11-30 Codexis, Inc. Human alpha-galactosidase variants
US10221436B2 (en) 2015-01-12 2019-03-05 10X Genomics, Inc. Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same
WO2016115273A1 (en) 2015-01-13 2016-07-21 10X Genomics, Inc. Systems and methods for visualizing structural variation and phasing information
CA2971462A1 (en) 2015-02-04 2016-08-11 Pioneer Hi-Bred International, Inc. Novel bt toxin receptors and methods of use
US10854315B2 (en) 2015-02-09 2020-12-01 10X Genomics, Inc. Systems and methods for determining structural variation and phasing using variant call data
CA2976045A1 (en) 2015-02-09 2016-08-18 Bioconsortia, Inc. Agriculturally beneficial microbes, microbial compositions, and consortia
WO2016130412A1 (en) 2015-02-10 2016-08-18 Codexis, Inc. Ketoreductase polypeptides for the synthesis of chiral compounds
JP6957797B2 (en) 2015-02-12 2021-11-02 ディーエスエム アイピー アセッツ ビー.ブイ.Dsm Ip Assets B.V. Methods for improving feed digestibility in bovinae
DK3262165T3 (en) 2015-02-24 2020-08-24 Novozymes As CELLOBIOHYDROLASE VARIANTS AND POLYNUCLEOTIDES ENCODING THEM
EP3262188B1 (en) 2015-02-24 2021-05-05 10X Genomics, Inc. Methods for targeted nucleic acid sequence coverage
WO2016137973A1 (en) 2015-02-24 2016-09-01 10X Genomics Inc Partition processing methods and systems
WO2016138315A1 (en) 2015-02-25 2016-09-01 Danisco Us Inc Alpha-glucosidase, compositions & methods
EP3262161B1 (en) 2015-02-27 2021-06-30 Novozymes A/S Mutant host cells for the production of 3-hydroxypropionic acid
HUE061070T2 (en) 2015-03-03 2023-05-28 Kymab Ltd Antibodies, uses & methods
CN108699578A (en) 2015-03-11 2018-10-23 杰能科国际有限公司 The enzymatic activity of cracking performance polysaccharide monooxygenase
CA2977026A1 (en) 2015-03-11 2016-09-15 E.I. Du Pont De Nemours And Company Insecticidal combinations of pip-72 and methods of use
CA2977675A1 (en) 2015-03-12 2016-09-15 Medimmune, Llc Method of purifying albumin-fusion proteins
EP3070103A1 (en) 2015-03-19 2016-09-21 Institut Hospitalier Universitaire De Strasbourg Anti-Claudin 1 monoclonal antibodies for the prevention and treatment of hepatocellular carcinoma
PL3271447T3 (en) 2015-03-19 2019-09-30 Novozymes A/S A brewing method
CN107429258A (en) 2015-03-19 2017-12-01 先锋国际良种公司 The method and composition that character for acceleration is gradually oozed
US20180073017A1 (en) 2015-04-07 2018-03-15 Novozymes A/S Methods for selecting enzymes having enhanced activity
CN107864658A (en) 2015-04-07 2018-03-30 诺维信公司 Method for selecting the enzyme with lipase active
US20180112156A1 (en) 2015-04-10 2018-04-26 Novozymes A/S Laundry method, use of polypeptide and detergent composition
WO2016162558A1 (en) 2015-04-10 2016-10-13 Novozymes A/S Detergent composition
MA44954A (en) 2015-04-17 2019-03-20 Agbiome Inc PESTICIDE GENES AND THEIR METHODS OF USE
CN108064234A (en) 2015-04-22 2018-05-22 农业生物群落股份有限公司 Killing gene and application method
CN104805508B (en) * 2015-04-29 2017-07-14 江南大学 A kind of method based on synthesizing single-stranded DNA library evolution metabolic pathway
US20180142251A1 (en) 2015-05-06 2018-05-24 Pioneer Hi-Bred International, Inc. Methods and compositions for the production of unreduced, non-recombined gametes and clonal offspring
US9944916B2 (en) 2015-05-07 2018-04-17 Codexis, Inc. Penicillin-G acylases
AU2016259703B2 (en) 2015-05-08 2021-12-23 Novozymes A/S Alpha-amylase variants and polynucleotides encoding same
AR105416A1 (en) 2015-05-08 2017-10-04 Novozymes As A-AMYLASE AND POLINUCLEOTIDE VARIANTS CODING THEM
EP3294883A1 (en) 2015-05-08 2018-03-21 Novozymes A/S Alpha-amylase variants having improved performance and stability
CA2985458A1 (en) 2015-05-09 2016-11-17 Two Blades Foundation Late blight resistance gene from solanum americanum and methods of use
CN116333064A (en) 2015-05-19 2023-06-27 先锋国际良种公司 Insecticidal proteins and methods of use thereof
WO2016187101A2 (en) 2015-05-21 2016-11-24 Full Spectrum Genetics, Inc. Method of improving characteristics of proteins
CN116676293A (en) 2015-05-27 2023-09-01 国投生物科技投资有限公司 Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2016196202A1 (en) 2015-05-29 2016-12-08 Novozymes A/S Polypeptides having protease activity and polynucleotides encoding same
MA46313A (en) 2015-06-03 2021-04-28 Agbiome Inc PESTICIDE GENES AND THEIR METHODS OF USE
WO2016193420A1 (en) 2015-06-04 2016-12-08 Novozymes A/S Use of m4 metalloprotease in wort production
JP2018522540A (en) 2015-06-05 2018-08-16 ノバルティス アーゲー Antibodies targeting bone morphogenetic protein 9 (BMP9) and methods therefor
EP3310908B1 (en) 2015-06-16 2020-08-05 Novozymes A/S Polypeptides with lipase activity and polynucleotides encoding same
EP3310921B8 (en) 2015-06-17 2023-03-15 Corteva Agriscience LLC Plant regulatory elements and methods of use thereof
WO2016203432A1 (en) 2015-06-17 2016-12-22 Novartis Ag Antibody drug conjugates
WO2016202839A2 (en) 2015-06-18 2016-12-22 Novozymes A/S Subtilase variants and polynucleotides encoding same
CA2981342A1 (en) 2015-06-18 2016-12-22 Novozymes A/S Polypeptides having trehalase activity and the use thereof in process of producing fermentation products
EP3106508B1 (en) 2015-06-18 2019-11-20 Henkel AG & Co. KGaA Detergent composition comprising subtilase variants
CN107849097A (en) 2015-06-22 2018-03-27 农业生物群落股份有限公司 Killing gene and application method
WO2016207351A2 (en) 2015-06-24 2016-12-29 Genencor International B.V. Polypeptides having demethylating activity
MX2017016924A (en) 2015-06-25 2018-08-15 Ascus Biosciences Inc Methods, apparatuses, and systems for analyzing microorganism strains from complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon.
US9938558B2 (en) 2015-06-25 2018-04-10 Ascus Biosciences, Inc. Methods, apparatuses, and systems for analyzing microorganism strains from complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
US10851399B2 (en) 2015-06-25 2020-12-01 Native Microbials, Inc. Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
US10632157B2 (en) 2016-04-15 2020-04-28 Ascus Biosciences, Inc. Microbial compositions and methods of use for improving fowl production
WO2016210395A1 (en) 2015-06-26 2016-12-29 Dupont Nutrition Biosciences Aps Aminopeptidases for protein hydrlyzates
CA2987164C (en) 2015-06-26 2023-09-19 Novozymes A/S Method for producing a coffee extract
WO2016207373A1 (en) 2015-06-26 2016-12-29 Novozymes A/S Polypeptides having peroxygenase activity
EP3313990A4 (en) 2015-06-26 2019-01-23 Novozymes A/S Biofinishing system
JOP20200312A1 (en) 2015-06-26 2017-06-16 Novartis Ag Factor xi antibodies and methods of use
CA3175255A1 (en) 2015-07-01 2017-01-05 Novozymes A/S Methods of reducing odor
US10945449B2 (en) 2015-07-02 2021-03-16 Novozymes A/S Animal feed compositions and uses thereof
WO2017005816A1 (en) 2015-07-06 2017-01-12 Novozymes A/S Lipase variants and polynucleotides encoding same
JP6691558B2 (en) 2015-07-07 2020-04-28 コデクシス, インコーポレイテッド Novel P450-BM3 variants with improved activity
US20180216089A1 (en) 2015-07-24 2018-08-02 Novozymes, Inc. Polypeptides Having Beta-Xylosidase Activity And Polynucleotides Encoding Same
CN108138153A (en) 2015-07-24 2018-06-08 诺维信股份有限公司 Polypeptide with nofuranosidase activity and encode their polynucleotides
AU2016297929B2 (en) 2015-07-25 2021-07-01 Bioconsortia, Inc. Agriculturally beneficial microbes, microbial compositions, and consortia
CA2994516A1 (en) 2015-08-03 2017-02-09 Novartis Ag Methods of treating fgf21-associated disorders
RU2762832C2 (en) 2015-08-06 2021-12-23 Пайонир Хай-Бред Интернэшнл, Инк. Insecticide proteins of plant origin and their application methods
JP7007261B2 (en) 2015-08-20 2022-01-24 アルブミディクス リミティド Albumin variants and conjugates
WO2017035270A1 (en) 2015-08-24 2017-03-02 Novozymes A/S Beta-alanine aminotransferases for the production of 3-hydroxypropionic acid
EP3347466B1 (en) 2015-09-08 2024-01-03 Cold Spring Harbor Laboratory Genetic copy number determination using high throughput multiplex sequencing of smashed nucleotides
PT3347377T (en) 2015-09-09 2021-04-30 Novartis Ag Thymic stromal lymphopoietin (tslp)-binding antibodies and methods of using the antibodies
TWI799366B (en) 2015-09-15 2023-04-21 美商建南德克公司 Cystine knot scaffold platform
EP3353195B1 (en) 2015-09-22 2021-11-10 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2017050722A1 (en) 2015-09-22 2017-03-30 Genia Technologies, Inc. Ompg variants
US10428145B2 (en) 2015-09-29 2019-10-01 Celgene Corporation PD-1 binding proteins and methods of use thereof
EP3763818A1 (en) 2015-10-06 2021-01-13 Pierce Biotechnology, Inc. Devices and methods for producing proteins
WO2017060475A2 (en) 2015-10-07 2017-04-13 Novozymes A/S Polypeptides
WO2017062790A1 (en) 2015-10-09 2017-04-13 Two Blades Foundation Cold shock protein receptors and methods of use
EP4324919A2 (en) 2015-10-14 2024-02-21 Novozymes A/S Polypeptide variants
EP3362558A1 (en) 2015-10-14 2018-08-22 Novozymes A/S Polypeptides having protease activity and polynucleotides encoding same
US10675589B2 (en) 2015-10-14 2020-06-09 Novozymes A/S Cleaning of water filtration membranes
DK3362574T3 (en) 2015-10-14 2021-10-11 Novozymes As Glucoamylase variants and polynucleotides encoding them
WO2017070219A1 (en) 2015-10-20 2017-04-27 Novozymes A/S Lytic polysaccharide monooxygenase (lpmo) variants and polynucleotides encoding same
US20180348224A1 (en) 2015-10-28 2018-12-06 Friedrich Miescher Institute For Biomedical Resear Ch Tenascin-w and biliary tract cancers
MX2018004683A (en) 2015-10-28 2018-07-06 Novozymes As Detergent composition comprising protease and amylase variants.
ITUB20155272A1 (en) 2015-11-02 2017-05-02 Scuola Normale Superiore Intracellular antibody
EP3370509A1 (en) 2015-11-03 2018-09-12 Two Blades Foundation Wheat stripe rust resistance genes and methods of use
US20180258438A1 (en) 2015-11-06 2018-09-13 Pioneer Hi-Bred International, Inc. Generation of complex trait loci in soybean and methods of use
US11001821B2 (en) 2015-11-24 2021-05-11 Novozymes A/S Polypeptides having protease activity and polynucleotides encoding same
BR112018011174A2 (en) 2015-11-30 2018-11-21 Du Pont recombinant polynucleotide, dna construct, heterologous cell, transgenic plant or plant cell, transgenic plant seed, method for expressing a polynucleotide in a plant and method for enhancing transcription of a polynucleotide in a host cell
EP3384019B1 (en) 2015-12-01 2020-06-24 Novozymes A/S Methods for producing lipases
EP3384048B1 (en) 2015-12-04 2021-03-24 10X Genomics, Inc. Methods and compositions for nucleic acid analysis
PL3387125T3 (en) 2015-12-07 2023-01-09 Henkel Ag & Co. Kgaa Dishwashing compositions comprising polypeptides having beta-glucanase activity and uses thereof
US9988624B2 (en) 2015-12-07 2018-06-05 Zymergen Inc. Microbial strain improvement by a HTP genomic engineering platform
US11208649B2 (en) 2015-12-07 2021-12-28 Zymergen Inc. HTP genomic engineering platform
JP6821598B2 (en) 2015-12-07 2021-01-27 ザイマージェン インコーポレイテッド Promoter derived from Corynebacterium glutamicum
EP3858996B1 (en) 2015-12-07 2022-08-03 Zymergen Inc. Microbial strain improvement by a htp genomic engineering platform
US10035995B2 (en) 2015-12-07 2018-07-31 Eastman Chemical Company CALB variants
WO2017105987A1 (en) 2015-12-18 2017-06-22 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP3389711A1 (en) 2015-12-18 2018-10-24 Novartis AG Antibodies targeting cd32b and methods of use thereof
BR112018012893A2 (en) 2015-12-22 2018-12-04 AgBiome, Inc. pesticide genes and methods of use
BR112018012851A2 (en) 2015-12-22 2018-12-26 Albumedix Ltd improved protein expression strains
US11104911B2 (en) 2015-12-22 2021-08-31 Pioneer Hi-Bred International, Inc. Embryo-preferred Zea mays promoters and methods of use
CN114921442A (en) 2015-12-30 2022-08-19 诺维信公司 Enzyme variants and polynucleotides encoding same
AU2017206051B2 (en) 2016-01-07 2023-04-20 Native Microbials, Inc. Methods for improving milk production by administration of microbial consortia
ES2847155T3 (en) 2016-01-21 2021-08-02 Novartis Ag Multispecific molecules targeting CLL-1
CA3007148A1 (en) 2016-01-29 2017-08-03 Novozymes A/S Beta-glucanase variants and polynucleotides encoding same
US11081208B2 (en) 2016-02-11 2021-08-03 10X Genomics, Inc. Systems, methods, and media for de novo assembly of whole genome sequence data
GB201602535D0 (en) 2016-02-12 2016-03-30 Univ Edinburgh Improved flu vaccine yield
WO2017140807A1 (en) 2016-02-16 2017-08-24 Monaghan Mushrooms Group Fungal polypeptides having lysozyme activity
EP3419992B1 (en) 2016-02-22 2020-11-18 Danisco US Inc. Fungal high-level protein production system
US10988749B2 (en) 2016-02-23 2021-04-27 Da Volterra Beta-lactamase variants
EP3420078B1 (en) 2016-02-23 2022-05-25 Da Volterra Beta-lactamase variants
WO2017147060A1 (en) 2016-02-25 2017-08-31 Dupont Nutrition Biosciences Aps Method for producing a protein hydrolysate employing an aspergillus fumigatus tripeptidyl peptidase
ES2880331T3 (en) 2016-02-29 2021-11-24 Genia Tech Inc Polymerase variants
US10738293B2 (en) 2016-03-02 2020-08-11 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
GB201604124D0 (en) 2016-03-10 2016-04-27 Ucb Biopharma Sprl Pharmaceutical formulation
CA3018081A1 (en) 2016-03-22 2017-09-28 Bionomics Limited Administration of an anti-lgr5 monoclonal antibody
CN109072209A (en) 2016-03-24 2018-12-21 诺维信公司 Cellobiohydrolase variant and the polynucleotides for encoding it
US9988641B2 (en) 2016-04-05 2018-06-05 Corn Products Development, Inc. Compositions and methods for producing starch with novel functionality
WO2017177153A1 (en) 2016-04-07 2017-10-12 Novozymes A/S Methods for selecting enzymes having protease activity
WO2017180715A2 (en) 2016-04-14 2017-10-19 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides having improved activity spectrum and uses thereof
CA3020833A1 (en) 2016-04-15 2017-10-19 Ascus Biosciences, Inc. Methods for improving agricultural production of fowl by administration of microbial consortia or purified strains thereof
BR112018071502A2 (en) 2016-04-19 2019-04-30 Pioneer Hi-Bred International, Inc. DNA construction, transgenic or offspring plant, composition and method for controlling a population of insect pests
JP7138567B2 (en) 2016-04-27 2022-09-16 ノバルティス アーゲー Antibodies against growth differentiation factor 15 and their uses
RU2740913C2 (en) 2016-04-29 2021-01-21 Дефенсин Терапьютикс Апс Treatment of liver, bile duct and pancreatic disorders
CN109312271A (en) 2016-04-29 2019-02-05 诺维信公司 Detergent composition and application thereof
CN109415421B (en) 2016-05-03 2023-02-28 诺维信公司 Alpha-amylase variants and polynucleotides encoding same
CN109068660B (en) 2016-05-04 2023-05-02 先锋国际良种公司 Insecticidal proteins and methods of use thereof
KR102382489B1 (en) 2016-05-05 2022-04-01 코덱시스, 인코포레이티드 Penicillin G acylase
CN109312319B (en) 2016-05-09 2023-05-16 诺维信公司 Variant polypeptides with improved properties and uses thereof
WO2017197338A1 (en) 2016-05-13 2017-11-16 10X Genomics, Inc. Microfluidic systems and methods of use
EP3246401A1 (en) 2016-05-20 2017-11-22 Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives New fatty acid decarboxylase and its uses
CN109196098A (en) 2016-05-24 2019-01-11 诺维信公司 Composition comprising the polypeptide with galactanase activity and the polypeptide with betagalactosidase activity
BR112018073890A2 (en) 2016-05-24 2019-02-26 Novozymes As composition, granule, animal feed additive, animal feed, liquid formulation, use of composition, granule, animal feed additive or liquid formulation, isolated polypeptide, methods for releasing galactose from plant-based material to improve one or more performance parameters of an animal and to produce the polypeptide, polynucleotide, nucleic acid construct or expression vector, and recombinant host cell.
CN109153981A (en) 2016-05-24 2019-01-04 诺维信公司 Polypeptide with alpha-galactosidase activity and the polynucleotides for encoding it
EP3462904A1 (en) 2016-05-24 2019-04-10 Novozymes A/S Polypeptides having alpha-galactosidase activity and polynucleotides encoding same
TW201802121A (en) 2016-05-25 2018-01-16 諾華公司 Reversal binding agents for anti-factor XI/XIa antibodies and uses thereof
WO2017205535A1 (en) 2016-05-27 2017-11-30 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
US20190218479A1 (en) 2016-05-31 2019-07-18 Novozymes A/S Stabilized Liquid Peroxide Compositions
WO2017207762A1 (en) 2016-06-03 2017-12-07 Novozymes A/S Subtilase variants and polynucleotides encoding same
WO2017211803A1 (en) 2016-06-07 2017-12-14 Novozymes A/S Co-expression of heterologous polypeptides to increase yield
IL263448B2 (en) 2016-06-09 2023-10-01 Codexis Inc Biocatalysts and methods for hydroxylation of chemical compounds
CN110381988A (en) 2016-06-15 2019-10-25 诺华股份有限公司 Use the method for the inhibitor for treating disease of Bone Morphogenetic Protein 6 (BMP6)
WO2017218325A1 (en) 2016-06-15 2017-12-21 Codexis, Inc. Engineered beta-glucosidases and glucosylation methods
EP3472186A1 (en) 2016-06-17 2019-04-24 National Hellenic Research Foundation Systems for recombinant protein production
US20190194676A1 (en) 2016-06-24 2019-06-27 Pioneer Hi-Bred International, Inc. Plant regulatory elements and methods of use thereof
EP3478827A1 (en) 2016-06-30 2019-05-08 Novozymes A/S Lipase variants and compositions comprising surfactant and lipase variant
WO2018005793A1 (en) 2016-06-30 2018-01-04 Zymergen Inc. Methods for generating a glucose permease library and uses thereof
JP2019519242A (en) 2016-06-30 2019-07-11 ザイマージェン インコーポレイテッド Method for generating a bacterial hemoglobin library and its use
WO2018002379A2 (en) 2016-07-01 2018-01-04 Novozymes A/S Enzymatic preparation of indigo dyes and in situ dyeing process
WO2018005411A1 (en) 2016-07-01 2018-01-04 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2018002261A1 (en) 2016-07-01 2018-01-04 Novozymes A/S Detergent compositions
CN109715794A (en) 2016-07-05 2019-05-03 诺维信公司 Pectin lyase enzyme variants and the polynucleotides for encoding them
MX2019000139A (en) 2016-07-08 2019-06-10 Novozymes As Polypeptides having xylanase activity and polynucleotides encoding same.
WO2018007573A1 (en) 2016-07-08 2018-01-11 Novozymes A/S Detergent compositions with galactanase
CN109415710A (en) 2016-07-08 2019-03-01 诺维信公司 Zytase variant and the polynucleotides that it is encoded
CN109642222A (en) 2016-07-13 2019-04-16 诺维信公司 Food bacillus DNA enzymatic variant
US11326152B2 (en) 2016-07-18 2022-05-10 Novozymes A/S Lipase variants, polynucleotides encoding same and the use thereof
US10927364B2 (en) 2016-07-20 2021-02-23 Novozymes A/S Heat-stable metagenomic carbonic anhydrases and their use
EP3487996A1 (en) 2016-07-21 2019-05-29 Novozymes A/S Serine protease variants and polynucleotides encoding same
MX2019000743A (en) 2016-07-21 2019-05-20 Novozymes As Serine protease variants and polynucleotides encoding same.
WO2018015444A1 (en) 2016-07-22 2018-01-25 Novozymes A/S Crispr-cas9 genome editing with multiple guide rnas in filamentous fungi
WO2018026868A1 (en) 2016-08-01 2018-02-08 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2018037064A1 (en) 2016-08-24 2018-03-01 Henkel Ag & Co. Kgaa Detergent compositions comprising xanthan lyase variants i
CN109844110B (en) 2016-08-24 2023-06-06 诺维信公司 Xanthan gum lyase variants and polynucleotides encoding same
KR102507692B1 (en) 2016-08-24 2023-03-09 헨켈 아게 운트 코. 카게아아 Detergent composition comprising GH9 endoglucanase variant I
EP3504330A1 (en) 2016-08-24 2019-07-03 Novozymes A/S Gh9 endoglucanase variants and polynucleotides encoding same
EP3515926B1 (en) 2016-08-26 2023-10-25 Codexis, Inc. Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
EP3510159B1 (en) 2016-09-06 2023-03-01 AgBiome, Inc. Pesticidal genes and methods of use
JP2019534858A (en) 2016-09-09 2019-12-05 ジェネンテック, インコーポレイテッド Selective peptide inhibitor of FRIZZLED
US11578316B2 (en) 2016-09-16 2023-02-14 Dupont Nutrition Biosciences Aps Acetolactate decarboxylase variants having improved specific activity
US10766958B2 (en) 2016-09-19 2020-09-08 Celgene Corporation Methods of treating vitiligo using PD-1 binding antibodies
CA3036701A1 (en) 2016-09-19 2018-03-22 Celgene Corporation Methods of treating immune disorders using pd-1 binding proteins
AU2017332638A1 (en) 2016-09-20 2019-04-11 22Nd Century Limited, Llc Trichome specific promoters for the manipulation of cannabinoids and other compounds in glandular trichomes
EP3519547A1 (en) 2016-09-29 2019-08-07 Novozymes A/S Spore containing granule
CA3038972A1 (en) 2016-09-30 2018-04-05 Dow Agrosciences Llc Binary insecticidal cry toxins
WO2018077938A1 (en) 2016-10-25 2018-05-03 Novozymes A/S Detergent compositions
EP3535285B1 (en) 2016-11-01 2022-04-06 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2018085370A1 (en) 2016-11-02 2018-05-11 Novozymes A/S Processes for reducing production of primeverose during enzymatic saccharification of lignocellulosic material
WO2018083248A1 (en) 2016-11-03 2018-05-11 Kymab Limited Antibodies, combinations comprising antibodies, biomarkers, uses & methods
KR102489902B1 (en) 2016-11-11 2023-01-19 바이오 래드 래버러토리스 인코오포레이티드 Methods of processing nucleic acid samples
US20210284991A1 (en) 2016-11-21 2021-09-16 Novozymes A/S Yeast Cell Extract Assisted Construction of DNA Molecules
EP3545086A1 (en) 2016-11-23 2019-10-02 Novozymes A/S Polypeptides having protease activity and polynucleotides encoding same
WO2018098214A1 (en) 2016-11-23 2018-05-31 Bayer Cropscience Lp Axmi669 and axmi991 toxin genes and methods for their use
WO2018102348A1 (en) 2016-11-29 2018-06-07 Novozymes A/S Alpha-amylase variants and polynucleotides encoding same
MX2019006122A (en) 2016-11-30 2019-08-14 Novozymes As Method of baking.
CN110168095A (en) 2016-12-06 2019-08-23 诺维信公司 For using engineered yeast bacterial strain from the ameliorative way of the cellulose matrix production ethyl alcohol containing xylose
BR112019011844A2 (en) 2016-12-13 2022-05-10 Defensin Therapeutics Aps Use of at least one defensin
US11174295B2 (en) 2016-12-14 2021-11-16 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US11041166B2 (en) 2016-12-16 2021-06-22 Two Blades Foundation Late blight resistance genes and methods of use
EP3541353B1 (en) 2016-12-20 2022-09-07 Colgate-Palmolive Company Oral care compositions and methods for increasing the stability of the same
CN110114465A (en) 2016-12-20 2019-08-09 诺维信公司 Restructuring yeast strains for pentose fermentation
EP3538063B1 (en) 2016-12-20 2022-11-30 Colgate-Palmolive Company Oral care composition and methods for whitening teeth
RU2743118C2 (en) 2016-12-20 2021-02-15 Колгейт-Палмолив Компани Compositions for oral care and methods of teeth whitening
WO2018119336A1 (en) 2016-12-22 2018-06-28 Athenix Corp. Use of cry14 for the control of nematode pests
CA3046226A1 (en) 2016-12-22 2018-06-28 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US10011872B1 (en) 2016-12-22 2018-07-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10550429B2 (en) 2016-12-22 2020-02-04 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10815525B2 (en) 2016-12-22 2020-10-27 10X Genomics, Inc. Methods and systems for processing polynucleotides
AU2017383232A1 (en) 2016-12-23 2019-06-27 Novartis Ag Factor XI antibodies and methods of use
CA3048157A1 (en) 2016-12-23 2018-06-28 Novartis Ag Methods of treatment with anti-factor xi/xia antibodies
US11891647B2 (en) 2016-12-28 2024-02-06 Native Microbials, Inc. Methods, apparatuses, and systems for analyzing complete microorganism strains in complex heterogeneous communities, determining functional relationships and interactions thereof, and identifying and synthesizing bioreactive modificators based thereon
TW201825515A (en) 2017-01-04 2018-07-16 美商伊繆諾金公司 Met antibodies and immunoconjugates and uses thereof
EP3565893A4 (en) 2017-01-05 2020-12-09 Codexis, Inc. Penicillin-g acylases
BR112019014720A2 (en) 2017-01-18 2020-04-07 BASF Agricultural Solutions Seed US LLC methods to confer disease resistance on a plant and to increase yield on a plant
BR112019014727A2 (en) 2017-01-18 2020-04-07 BASF Agricultural Solutions Seed US LLC nucleic acid molecule, vector, cell, plant, seed, polypeptide, composition, methods for controlling a pest population, to kill a pest, to produce a polypeptide, to protect a plant and to increase yield on a plant, use of nucleic acid and basic product
WO2018140214A1 (en) 2017-01-24 2018-08-02 Pioneer Hi-Bred International, Inc. Nematicidal protein from pseudomonas
CN110506117A (en) 2017-01-30 2019-11-26 农业生物群落股份有限公司 Killing gene and application method
EP4029939B1 (en) 2017-01-30 2023-06-28 10X Genomics, Inc. Methods and systems for droplet-based single cell barcoding
EP4276179A2 (en) 2017-02-01 2023-11-15 Novozymes A/S Alpha-amylase variants
BR112019015689A2 (en) 2017-02-01 2020-04-14 Procter & Gamble cleaning compositions comprising amylase variants
JOP20190187A1 (en) 2017-02-03 2019-08-01 Novartis Ag Anti-ccr7 antibody drug conjugates
MX2019009233A (en) 2017-02-03 2019-09-19 Codexis Inc Engineered glycosyltransferases and steviol glycoside glucosylation methods.
BR112019016344A2 (en) 2017-02-08 2020-04-07 Novartis Ag mimetic antibodies fgf21 and uses thereof
CA3052794A1 (en) 2017-02-08 2018-08-16 Pioneer Hi-Bred International, Inc. Insecticidal combinations of plant derived insecticidal proteins and methods for their use
AU2018217495B2 (en) 2017-02-13 2021-03-04 Codexis, Inc. Engineered phenylalanine ammonia lyase polypeptides
BR112019017271A2 (en) 2017-02-20 2020-04-14 Novozymes As lipolytic enzyme for use in baking
EP3363900A1 (en) * 2017-02-21 2018-08-22 ETH Zurich Evolution-guided multiplexed dna assembly of dna parts, pathways and genomes
WO2018164737A1 (en) 2017-03-07 2018-09-13 Danisco Us Inc. Thermostable glucoamylase and methods of use, thereof
US11479798B2 (en) 2017-03-29 2022-10-25 Boehringer Ingelheim Rcv Gmbh & Co Kg Recombinant host cell with altered membrane lipid composition
WO2018177938A1 (en) 2017-03-31 2018-10-04 Novozymes A/S Polypeptides having dnase activity
EP3601551A1 (en) 2017-03-31 2020-02-05 Novozymes A/S Polypeptides having rnase activity
WO2018177936A1 (en) 2017-03-31 2018-10-04 Novozymes A/S Polypeptides having dnase activity
WO2018185618A1 (en) 2017-04-03 2018-10-11 Novartis Ag Anti-cdh6 antibody drug conjugates and anti-gitr antibody combinations and methods of treatment
US20200109354A1 (en) 2017-04-04 2020-04-09 Novozymes A/S Polypeptides
CN110651029B (en) 2017-04-04 2022-02-15 诺维信公司 Glycosyl hydrolase
US20200109352A1 (en) 2017-04-04 2020-04-09 Novozymes A/S Polypeptide compositions and uses thereof
WO2018191162A1 (en) 2017-04-11 2018-10-18 AgBiome, Inc. Pesticidal genes and methods of use
BR112019021223A2 (en) 2017-04-11 2020-04-28 Novozymes As glucoamylase variant, methods for increasing the crude starch hydrolysis activity of a glucoamylase and for producing a glucoamylase variant, composition, use of a glucoamylase variant, processes for producing a fermentation product and for producing a syrup product, polynucleotide encoding the glucoamylase variant, nucleic acid construct, expression vector, and host cell.
TWI783993B (en) 2017-04-24 2022-11-21 美商提薩羅有限公司 Methods of manufacturing of niraparib
WO2018197520A1 (en) 2017-04-24 2018-11-01 Dupont Nutrition Biosciences Aps Methods and compositions of anti-crispr proteins for use in plants
JP7045725B2 (en) 2017-04-27 2022-04-01 コデクシス, インコーポレイテッド Ketoreductase polypeptides and polynucleotides
CN110913702A (en) 2017-04-28 2020-03-24 埃斯库斯生物科技股份公司 Method for supporting a cereal fortifier and/or energy fortifier diet in ruminants using an artificial pool of microorganisms
WO2018202846A1 (en) 2017-05-05 2018-11-08 Novozymes A/S Compositions comprising lipase and sulfite
US20210130743A1 (en) 2017-05-08 2021-05-06 Novozymes A/S Mannanase Variants and Polynucleotides Encoding Same
WO2018206535A1 (en) 2017-05-08 2018-11-15 Novozymes A/S Carbohydrate-binding domain and polynucleotides encoding the same
CA3058092A1 (en) 2017-05-08 2018-11-15 Novozymes A/S Mannanase variants and polynucleotides encoding same
SG11201909957TA (en) 2017-05-08 2019-11-28 Codexis Inc Engineered ligase variants
WO2018208882A1 (en) 2017-05-11 2018-11-15 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP3625715A4 (en) 2017-05-19 2021-03-17 10X Genomics, Inc. Systems and methods for analyzing datasets
EP3625351A1 (en) 2017-05-19 2020-03-25 Zymergen Inc. Genomic engineering of biosynthetic pathways leading to increased nadph
WO2018215937A1 (en) 2017-05-24 2018-11-29 Novartis Ag Interleukin-7 antibody cytokine engrafted proteins and methods of use in the treatment of cancer
KR20200010468A (en) 2017-05-24 2020-01-30 노파르티스 아게 Antibody-Cytokine Implanted Proteins and Methods of Use in Cancer Treatment
JP2020520671A (en) 2017-05-24 2020-07-16 ノバルティス アーゲー Antibody-cytokine grafted proteins and methods of use
JOP20190271A1 (en) 2017-05-24 2019-11-21 Novartis Ag Antibody-cytokine engrafted proteins and methods of use for immune related disorders
SG11201901822QA (en) 2017-05-26 2019-03-28 10X Genomics Inc Single cell analysis of transposase accessible chromatin
US10844372B2 (en) 2017-05-26 2020-11-24 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
BR112019024827A2 (en) 2017-05-26 2020-06-16 Pioneer Hi-Bred International, Inc. DNA CONSTRUCTION, TRANSGENIC PLANT OR PROGENY OF THE SAME, COMPOSITION AND METHOD TO CONTROL A POPULATION OF INSECT PEST
WO2018220116A1 (en) 2017-05-31 2018-12-06 Novozymes A/S Xylose fermenting yeast strains and processes thereof for ethanol production
US20200157581A1 (en) 2017-06-02 2020-05-21 Novozymes A/S Improved Yeast For Ethanol Production
JP2020524490A (en) 2017-06-06 2020-08-20 ザイマージェン インコーポレイテッド HTP genome manipulation platform to improve Escherichia coli
JP7350659B2 (en) 2017-06-06 2023-09-26 ザイマージェン インコーポレイテッド High-throughput (HTP) genome manipulation platform for the improvement of Saccharopolyspora spinosa
US20200102554A1 (en) 2017-06-06 2020-04-02 Zymergen Inc. High throughput transposon mutagenesis
WO2018226900A2 (en) 2017-06-06 2018-12-13 Zymergen Inc. A htp genomic engineering platform for improving fungal strains
WO2018224544A1 (en) 2017-06-08 2018-12-13 Novozymes A/S Compositions comprising polypeptides having cellulase activity and amylase activity, and uses thereof in cleaning and detergent compositions
CA3064574A1 (en) 2017-06-14 2018-12-20 Codexis, Inc. Engineered transaminase polypeptides for industrial biocatalysis
WO2018229715A1 (en) 2017-06-16 2018-12-20 Novartis Ag Compositions comprising anti-cd32b antibodies and methods of use thereof
CN111032871A (en) 2017-06-20 2020-04-17 阿尔布梅迪克斯医疗有限公司 Improved protein expressing strains
WO2018234465A1 (en) 2017-06-22 2018-12-27 Novozymes A/S Xylanase variants and polynucleotides encoding same
JP7244089B2 (en) 2017-06-27 2023-03-22 コデクシス, インコーポレイテッド penicillin G acylase
CN110997701A (en) 2017-06-28 2020-04-10 诺维信公司 Polypeptides having trehalase activity and polynucleotides encoding same
US20200181542A1 (en) 2017-06-30 2020-06-11 Novozymes A/S Enzyme Slurry Composition
WO2019005540A1 (en) 2017-06-30 2019-01-03 Codexis, Inc. T7 rna polymerase variants
EP3645712A4 (en) 2017-06-30 2021-07-07 Codexis, Inc. T7 rna polymerase variants
WO2019016772A2 (en) 2017-07-21 2019-01-24 Novartis Ag Compositions and methods to treat cancer
KR20200035060A (en) 2017-07-24 2020-04-01 노보자임스 에이/에스 GH5 and GH30 in wet milling
WO2019023587A1 (en) 2017-07-28 2019-01-31 Two Blades Foundation Potyvirus resistance genes and methods of use
EP3661950A2 (en) 2017-08-03 2020-06-10 Agbiome, Inc. Pesticidal genes and methods of use
BR112020002115A2 (en) 2017-08-08 2020-08-11 Novozymes A/S trehalase variant polypeptide, polynucleotide, nucleic acid construct, expression vector, host cell, composition, whole broth formulation or cell culture composition, method of producing a trehalase variant, and, production process of a product of fermentation
US11634400B2 (en) 2017-08-19 2023-04-25 University Of Rochester Micheliolide derivatives, methods for their preparation and their use as anticancer and antiinflammatory agents
WO2019038058A1 (en) 2017-08-24 2019-02-28 Novozymes A/S Gh9 endoglucanase variants and polynucleotides encoding same
EP3673060A1 (en) 2017-08-24 2020-07-01 Henkel AG & Co. KGaA Detergent composition comprising xanthan lyase variants ii
US11624059B2 (en) 2017-08-24 2023-04-11 Henkel Ag & Co. Kgaa Detergent compositions comprising GH9 endoglucanase variants II
CA3071078A1 (en) 2017-08-24 2019-02-28 Novozymes A/S Xanthan lyase variants and polynucleotides encoding same
US11834484B2 (en) 2017-08-29 2023-12-05 Novozymes A/S Bakers's yeast expressing anti-staling/freshness amylases
US11377650B2 (en) 2017-08-31 2022-07-05 Novozymes A/S Polypeptides having D-psicose 3 epimerase activity and polynucleotides encoding same
MX2020002177A (en) 2017-09-01 2020-07-14 Novozymes As Animal feed additives comprising a polypeptide having protease activity and uses thereof.
AU2018322865B2 (en) 2017-09-01 2023-11-09 Novozymes A/S Animal feed additives comprising polypeptide having protease activity and uses thereof
WO2019046703A1 (en) 2017-09-01 2019-03-07 Novozymes A/S Methods for improving genome editing in fungi
WO2019052907A2 (en) 2017-09-07 2019-03-21 Total Raffinage Chimie Method for detecting hydrogen peroxide conversion activity of an enzyme
WO2019047199A1 (en) 2017-09-11 2019-03-14 Danisco Us Inc. Glucoamylase and methods of use, thereof
WO2019060383A1 (en) 2017-09-25 2019-03-28 Pioneer Hi-Bred, International, Inc. Tissue-preferred promoters and methods of use
EP3461892A3 (en) 2017-09-27 2019-06-12 The Procter & Gamble Company Detergent compositions comprising lipases
WO2019063499A1 (en) 2017-09-27 2019-04-04 Novozymes A/S Lipase variants and microcapsule compositions comprising such lipase variants
WO2019068715A1 (en) 2017-10-02 2019-04-11 Novozymes A/S Polypeptides having mannanase activity and polynucleotides encoding same
EP3692147A1 (en) 2017-10-02 2020-08-12 Novozymes A/S Polypeptides having mannanase activity and polynucleotides encoding same
US20200248159A1 (en) 2017-10-04 2020-08-06 Novozymes A/S Polypeptides having protease activity and polynucleotides encoding same
WO2019074598A1 (en) 2017-10-13 2019-04-18 Pioneer Hi-Bred International, Inc. Virus-induced gene silencing technology for insect control in maize
MX2020003992A (en) 2017-10-18 2020-08-13 Native Microbials Inc Improving fowl production by administration of a synthetic bioensemble of microbes or purified strains thereof.
EP3701021A1 (en) 2017-10-23 2020-09-02 Novozymes A/S Improving expression of a protease by co-expression with propeptide
BR112020007974A2 (en) 2017-10-25 2020-10-27 Da Volterra beta-lactamase variants
WO2019081983A1 (en) 2017-10-25 2019-05-02 Novartis Ag Antibodies targeting cd32b and methods of use thereof
WO2019084349A1 (en) 2017-10-27 2019-05-02 The Procter & Gamble Company Detergent compositions comprising polypeptide variants
MX2020004149A (en) 2017-10-27 2020-08-03 Novozymes As Dnase variants.
DE102017125560A1 (en) 2017-11-01 2019-05-02 Henkel Ag & Co. Kgaa CLEANSING COMPOSITIONS CONTAINING DISPERSINE III
DE102017125558A1 (en) 2017-11-01 2019-05-02 Henkel Ag & Co. Kgaa CLEANING COMPOSITIONS CONTAINING DISPERSINE I
DE102017125559A1 (en) 2017-11-01 2019-05-02 Henkel Ag & Co. Kgaa CLEANSING COMPOSITIONS CONTAINING DISPERSINE II
EP3706780A4 (en) 2017-11-07 2021-12-22 Codexis, Inc. Transglutaminase variants
CN111556759A (en) 2017-11-10 2020-08-18 防御素治疗学公司 Maturation of mucosal defenses and intestinal/pulmonary function in premature infants
EP3707252A1 (en) 2017-11-10 2020-09-16 Novozymes A/S Temperature-sensitive cas9 protein
BR112020009525A2 (en) 2017-11-14 2020-11-03 Danisco Us Inc. alpha-amylase, composition and method
EP3954782A1 (en) 2017-11-15 2022-02-16 10X Genomics, Inc. Functionalized gel beads
US10829815B2 (en) 2017-11-17 2020-11-10 10X Genomics, Inc. Methods and systems for associating physical and genetic properties of biological particles
WO2019096903A1 (en) 2017-11-20 2019-05-23 Novozymes A/S New galactanases (ec 3.2.1.89) for use in soy processing
BR112020010180A2 (en) 2017-11-24 2021-01-12 Defensin Therapeutics Aps B-DEFENSIN AND / OR A-HUMAN DEFENSIN FOR USE IN PREVENTING OR TREATING GRAFT DISEASE AGAINST ACUTE HOST
WO2019108619A1 (en) 2017-11-28 2019-06-06 Two Blades Foundation Methods and compositions for enhancing the disease resistance of plants
WO2019110462A1 (en) 2017-12-04 2019-06-13 Novozymes A/S Lipase variants and polynucleotides encoding same
BR112020011278A2 (en) 2017-12-08 2020-11-17 Novozymes A/S alpha-amylase variant, composition, polynucleotide, nucleic acid construct, expression vector, host cell, methods for producing an alpha-amylase variant and for increasing the stability of a parent alpha-amylase, use of the variant, and, process for producing a syrup from material containing starch
US11384347B2 (en) 2017-12-08 2022-07-12 Novozymes A/S Alpha-amylase variants and polynucleotides encoding same
CA3085261A1 (en) 2017-12-13 2019-06-20 Glaxosmithkline Intellectual Property Development Limited Carboxyesterase biocatalysts
KR20200098564A (en) 2017-12-13 2020-08-20 코덱시스, 인코포레이티드 Carboxysterase Polypeptides for Amide Coupling
EP4122947A1 (en) 2017-12-19 2023-01-25 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides and uses thereof
US11634698B2 (en) 2017-12-20 2023-04-25 Basf Se Nucleases and methods for making and using them
EP3728333A1 (en) 2017-12-22 2020-10-28 Novozymes A/S Wheat milling process and gh8 xylanases
BR112020012763A2 (en) 2017-12-22 2020-12-15 AgBiome, Inc. PESTICIDAL GENES AND METHODS OF USE
US11173136B2 (en) 2018-01-10 2021-11-16 Brightseed, Inc. Method for modulating metabolism
US11732271B2 (en) 2018-01-12 2023-08-22 The Sainsbury Laboratory Stem rust resistance genes and methods of use
MX2020007914A (en) 2018-01-29 2020-10-28 Novozymes As Microorganisms with improved nitrogen utilization for ethanol production.
CN111868239A (en) 2018-02-08 2020-10-30 诺维信公司 Lipase, lipase variants and compositions thereof
US20210071157A1 (en) 2018-02-08 2021-03-11 Novozymes A/S Lipase Variants and Compositions Thereof
US11905518B2 (en) 2018-02-12 2024-02-20 Curators Of The University Of Missouri Small auxin upregulated (SAUR) gene for the improvement of root system architecture, waterlogging tolerance, drought resistance and yield in plants and methods of uses
CN111684056A (en) 2018-02-28 2020-09-18 宝洁公司 Cleaning method
EP3762504A1 (en) 2018-03-09 2021-01-13 Danisco US Inc. Glucoamylases and methods of use thereof
EP3765185B1 (en) 2018-03-13 2023-07-19 Novozymes A/S Microencapsulation using amino sugar oligomers
JP2021518128A (en) 2018-03-20 2021-08-02 ザイマージェン インコーポレイテッド HTP platform for genetic engineering of Chinese hamster ovary cells
TW202003551A (en) 2018-03-28 2020-01-16 美商必治妥美雅史谷比公司 Interleukin-2/interleukin-2 receptor alpha fusion proteins and methods of use
WO2019185726A1 (en) 2018-03-29 2019-10-03 Novozymes A/S Mannanase variants and polynucleotides encoding same
SG11202008206TA (en) 2018-03-30 2020-09-29 Amgen Inc C-terminal antibody variants
WO2019195842A1 (en) 2018-04-06 2019-10-10 Braskem S.A. Novel nadh-dependent enzyme mutants to convert acetone into isopropanol
WO2019195166A1 (en) 2018-04-06 2019-10-10 10X Genomics, Inc. Systems and methods for quality control in single cell processing
EP3775191A1 (en) 2018-04-09 2021-02-17 Novozymes A/S Polypeptides having alpha-amylase activity and polynucleotides encoding same
WO2019201785A1 (en) 2018-04-19 2019-10-24 Novozymes A/S Stabilized cellulase variants
CN112272701A (en) 2018-04-19 2021-01-26 诺维信公司 Stabilized cellulase variants
CA3097621A1 (en) 2018-04-20 2019-10-24 AgBiome, Inc. Pesticidal proteins and methods of use
WO2019222226A2 (en) 2018-05-17 2019-11-21 Bp Corporation North America Inc. Production of 2-keto-3-deoxy-d-gluconic acid in filamentous fungi
CA3096516A1 (en) 2018-05-22 2019-11-28 Pioneer Hi-Bred International, Inc. Plant regulatory elements and methods of use thereof
AR126019A1 (en) 2018-05-30 2023-09-06 Novartis Ag ANTIBODIES AGAINST ENTPD2, COMBINATION THERAPIES AND METHODS OF USE OF ANTIBODIES AND COMBINATION THERAPIES
WO2019229228A1 (en) 2018-05-31 2019-12-05 Novozymes A/S Method for treating dissolving pulp using lytic polysaccharide monooxygenase
TW202016136A (en) 2018-06-01 2020-05-01 瑞士商諾華公司 Binding molecules against bcma and uses thereof
CA3102840A1 (en) 2018-06-05 2019-12-12 Lifeedit, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
JP2021526799A (en) 2018-06-06 2021-10-11 ザイマージェン インコーポレイテッド Manipulation of genes involved in signal transduction to control fungal morphology during fermentation and production
EP3807409A4 (en) 2018-06-12 2022-08-03 Codexis, Inc. Engineered tyrosine ammonia lyase
JP2021528985A (en) 2018-06-27 2021-10-28 ベーリンガー インゲルハイム エルツェーファウ ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディトゲゼルシャフト Means and methods for increasing protein expression by the use of transcription factors
BR112020026640A2 (en) 2018-06-28 2021-04-06 Pioneer Hi-Bred International, Inc. METHODS FOR SELECTING TRANSFORMED PLANTS
WO2020002575A1 (en) 2018-06-28 2020-01-02 Novozymes A/S Polypeptides having pectin lyase activity and polynucleotides encoding same
BR112021000059A2 (en) 2018-07-04 2021-04-06 Danisco Us Inc. GLUCOAMYLASES AND METHODS FOR USE OF THE SAME
MX2021000326A (en) 2018-07-09 2021-03-25 Codexis Inc Engineered deoxyribose-phosphate aldolases.
EP3820502A4 (en) 2018-07-09 2022-07-20 Codexis, Inc. Engineered purine nucleoside phosphorylase variant enzymes
MX2021000323A (en) 2018-07-09 2021-03-25 Codexis Inc Engineered pantothenate kinase variant enzymes.
AU2019302422B2 (en) 2018-07-09 2022-08-04 Codexis, Inc. Engineered phosphopentomutase variant enzymes
WO2020014049A1 (en) 2018-07-09 2020-01-16 Codexis, Inc. Engineered galactose oxidase variant enzymes
WO2020014306A1 (en) 2018-07-10 2020-01-16 Immunogen, Inc. Met antibodies and immunoconjugates and uses thereof
JP2021531749A (en) 2018-07-12 2021-11-25 コデクシス, インコーポレイテッド Manipulated Phenylalanine Ammonia Riase Polypeptide
JP2021531007A (en) 2018-07-20 2021-11-18 ピエール、ファーブル、メディカマン Receptor for VISTA
US11866751B2 (en) 2018-07-25 2024-01-09 Novozymes A/S Yeast expressing a heterologous alpha-amylase for ethanol production
AU2019314181A1 (en) 2018-07-30 2021-02-04 Tate & Lyle Solutions Usa Llc Engineered glycosyltransferases and steviol glycoside glucosylation methods
EP3829608B1 (en) 2018-08-01 2024-01-10 Board of Supervisors of Louisiana State University and Agricultural and Mechanical College Compositions comprising herpes simplex virus-1 for use in methods of treating and preventing cancer
MX2021002290A (en) 2018-08-29 2021-04-28 Pioneer Hi Bred Int Insecticidal proteins and methods for their use.
US11473076B2 (en) 2018-08-31 2022-10-18 Novozymes A/S Animal feed additives and compositions comprising an S8 serine protease
CA3110664A1 (en) 2018-09-07 2020-03-12 Basf Plant Science Company Gmbh Improved method for the production of high levels of pufa in plants
AU2019334671A1 (en) 2018-09-07 2021-03-25 Basf Plant Science Company Gmbh Improved method for the production of high levels of PUFA in plants
CA3110651A1 (en) 2018-09-07 2020-03-12 Basf Plant Science Company Gmbh Improved method for the production of high levels of pufa in plants
CN113056476A (en) 2018-10-03 2021-06-29 诺维信公司 Polypeptides having alpha-mannan degrading activity and polynucleotides encoding same
BR112021006692A2 (en) 2018-10-08 2021-08-10 Novozymes A/S yeast expressing enzymes for ethanol production
UY38407A (en) 2018-10-15 2020-05-29 Novartis Ag TREM2 STABILIZING ANTIBODIES
US11060075B2 (en) 2018-10-29 2021-07-13 Codexis, Inc. Engineered DNA polymerase variants
EP3874051A1 (en) 2018-10-31 2021-09-08 Novozymes A/S Genome editing by guided endonuclease and single-stranded oligonucleotide
JP2022512817A (en) 2018-10-31 2022-02-07 パイオニア ハイ-ブレッド インターナショナル, インコーポレイテッド Compositions and Methods for Ochrobactrum-mediated Plant Transformation
WO2020089811A1 (en) 2018-10-31 2020-05-07 Novartis Ag Dc-sign antibody drug conjugates
JP2022513408A (en) 2018-10-31 2022-02-07 ザイマージェン インコーポレイテッド Multiplexing deterministic assembly of DNA libraries
CN113302293A (en) 2018-12-12 2021-08-24 诺维信公司 Polypeptides having xylanase activity and polynucleotides encoding same
CA3122524A1 (en) 2018-12-14 2020-06-18 Codexis, Inc. Engineered tyrosine ammonia lyase
WO2020132252A2 (en) 2018-12-20 2020-06-25 Codexis, Inc. Human alpha-galactosidase variants
EP3898962A2 (en) 2018-12-21 2021-10-27 Novozymes A/S Polypeptides having peptidoglycan degrading activity and polynucleotides encoding same
CU20210047A7 (en) 2018-12-21 2022-01-13 Novartis Ag ANTI-PMEL 17 ANTIBODIES AND CONJUGATES THEREOF
MX2021007839A (en) 2018-12-27 2021-08-11 Colgate Palmolive Co Oral care compositions.
US20220145296A1 (en) 2018-12-27 2022-05-12 LifeEDIT Therapeutics, Inc. Polypeptides useful for gene editing and methods of use
EP3917571A4 (en) 2019-01-31 2022-10-12 Agency For Science, Technology And Research Cnx/erp57 inhibitor for use in the treatment or prevention of cancer
EP3918060A1 (en) 2019-01-31 2021-12-08 Novozymes A/S Polypeptides having xylanase activity and use thereof for improving the nutritional quality of animal feed
US11053515B2 (en) 2019-03-08 2021-07-06 Zymergen Inc. Pooled genome editing in microbes
KR20210136997A (en) 2019-03-08 2021-11-17 지머젠 인코포레이티드 Iterative genome editing in microorganisms
CA3123457A1 (en) 2019-03-11 2020-09-17 Pioneer Hi-Bred International, Inc. Methods for clonal plant production
JP2022524490A (en) 2019-03-21 2022-05-06 ノボザイムス アクティーゼルスカブ Alpha-amylase mutants and polynucleotides encoding them
EP3947425A1 (en) 2019-03-27 2022-02-09 Pioneer Hi-Bred International, Inc. Plant explant transformation
AU2020248645A1 (en) 2019-03-27 2021-10-28 Tigatx, Inc. Engineered IgA antibodies and methods of use
CA3127173A1 (en) 2019-03-28 2020-10-01 Pioneer Hi-Bred International, Inc. Modified agrobacterium strains and use thereof for plant transformation
US20220169706A1 (en) 2019-03-28 2022-06-02 Danisco Us Inc Engineered antibodies
CN113785039A (en) 2019-04-03 2021-12-10 诺维信公司 Polypeptides having beta-glucanase activity, polynucleotides encoding same and use thereof in cleaning and detergent compositions
US20220364138A1 (en) 2019-04-10 2022-11-17 Novozymes A/S Polypeptide variants
MX2021012289A (en) 2019-04-12 2021-11-12 Novozymes As Stabilized glycoside hydrolase variants.
US20220275354A1 (en) 2019-05-15 2022-09-01 Novozymes A/S TEMPERATURE-SENSITIVE RNA- Guided Endonuclease
CN113874398A (en) 2019-05-21 2021-12-31 诺华股份有限公司 CD19 binding molecules and uses thereof
WO2020236797A1 (en) 2019-05-21 2020-11-26 Novartis Ag Variant cd58 domains and uses thereof
CN114173810A (en) 2019-05-21 2022-03-11 诺华股份有限公司 Trispecific binding molecules directed against BCMA and uses thereof
US20220251528A1 (en) 2019-06-24 2022-08-11 Novozymes A/S Alpha-Amylase Variants
CN114040972A (en) 2019-06-24 2022-02-11 宝洁公司 Cleaning compositions comprising amylase variants
CN114450405A (en) 2019-06-27 2022-05-06 双刃基金会 Engineered ATRLP23 pattern recognition receptors and methods of use
WO2021001400A1 (en) 2019-07-02 2021-01-07 Novozymes A/S Lipase variants and compositions thereof
CN114391038A (en) 2019-07-25 2022-04-22 诺维信公司 Filamentous fungal expression systems
WO2021018751A1 (en) 2019-07-26 2021-02-04 Novozymes A/S Enzymatic treatment of paper pulp
BR112021026761A2 (en) 2019-07-26 2022-02-15 Novozymes As Yeast cell, composition, and methods of producing a derivative of a yeast strain, producing ethanol, and producing a fermentation product from a starch-containing or cellulose-containing material
MX2022001225A (en) 2019-07-29 2022-03-17 Brightseed Inc Method for improving digestive health.
EP4010469A1 (en) 2019-08-06 2022-06-15 Novozymes A/S Fusion proteins for improved enzyme expression
TW202120688A (en) 2019-08-12 2021-06-01 美商生命編輯公司 Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2021032881A1 (en) 2019-08-22 2021-02-25 Basf Se Amylase variants
MX2022002834A (en) 2019-09-16 2022-04-06 Novozymes As Polypeptides having beta-glucanase activity and polynucleotides encoding same.
TW202124446A (en) 2019-09-18 2021-07-01 瑞士商諾華公司 Combination therapies with entpd2 antibodies
CN114502590A (en) 2019-09-18 2022-05-13 诺华股份有限公司 ENTPD2 antibodies, combination therapies, and methods of using these antibodies and combination therapies
AU2020358714A1 (en) 2019-10-02 2022-05-12 Abbott Diabetes Care Inc. Detection of analytes by protein switches
EP4038170A1 (en) 2019-10-03 2022-08-10 Novozymes A/S Polypeptides comprising at least two carbohydrate binding domains
JP2023501483A (en) 2019-11-11 2023-01-18 ブライトシード・インコーポレイテッド Extracts, consumable products and methods for enriching bioactive metabolites in extracts
EP3821946A1 (en) 2019-11-12 2021-05-19 Université de Strasbourg Anti-claudin-1 monoclonal antibodies for the prevention and treatment of fibrotic diseases
BR112022011271A2 (en) 2019-12-10 2022-09-06 Novozymes As RECOMBINANT HOST CELL, COMPOSITION, METHODS FOR PRODUCING A CELL DERIVATIVE AND FERMENTATION PRODUCT, AND, USE OF A RECOMBINANT HOST CELL
EP4077622A1 (en) 2019-12-19 2022-10-26 The Procter & Gamble Company Cleaning compositions comprising polypeptides having alpha amylase activity
WO2021122687A1 (en) 2019-12-19 2021-06-24 Basf Se Increasing space-time-yield, carbon-conversion-efficiency and carbon substrate flexibility in the production of fine chemicals
MX2022007361A (en) 2019-12-19 2022-07-19 Novozymes As Alpha-amylase variants.
WO2021122867A2 (en) 2019-12-19 2021-06-24 Novozymes A/S Xylanase variants and polynucleotides encoding same
WO2021123307A2 (en) 2019-12-20 2021-06-24 Novozymes A/S Polypeptides having proteolytic activity and use thereof
EP4077658A1 (en) 2019-12-20 2022-10-26 Basf Se Decreasing toxicity of terpenes and increasing the production potential in micro-organisms
EP4085133A1 (en) 2019-12-30 2022-11-09 Lifeedit Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2021148278A1 (en) 2020-01-24 2021-07-29 Novozymes A/S Mutants of a filamentous fungal cell having increased productivity in the production of a polypeptide
EP4097226A1 (en) 2020-01-31 2022-12-07 Novozymes A/S Mannanase variants and polynucleotides encoding same
JP2023511739A (en) 2020-01-31 2023-03-22 ノボザイムス アクティーゼルスカブ Mannanase variants and polynucleotides encoding them
EP4103709A2 (en) 2020-02-10 2022-12-21 Novozymes A/S Polypeptides having alpha-amylase activity and polynucleotides encoding same
US20230235367A1 (en) 2020-02-10 2023-07-27 Novozymes A/S Process for producing ethanol from raw starch using alpha-amylase variants
US20230142991A1 (en) 2020-02-10 2023-05-11 Novozymes A/S Alpha-amylase variants and polynucleotides encoding same
EP4103580A4 (en) 2020-02-13 2024-03-06 Zymergen Inc Metagenomic library and natural product discovery platform
US20230090771A1 (en) * 2020-02-21 2023-03-23 Regents Of The University Of Minnesota Novel methods for creating alpha-n-methylated polypeptides
WO2021170559A1 (en) 2020-02-26 2021-09-02 Novozymes A/S Polypeptide variants
WO2021202473A2 (en) 2020-03-30 2021-10-07 Danisco Us Inc Engineered antibodies
WO2021202463A1 (en) 2020-03-30 2021-10-07 Danisco Us Inc Anti-rsv antibodies
JP2023520312A (en) 2020-04-08 2023-05-17 ノボザイムス アクティーゼルスカブ Carbohydrate binding module variant
US11788071B2 (en) 2020-04-10 2023-10-17 Codexis, Inc. Engineered transaminase polypeptides
CA3175633A1 (en) 2020-04-17 2021-10-21 Danisco Us Inc. Glucoamylase and methods of use thereof
US20230167384A1 (en) 2020-04-21 2023-06-01 Novozymes A/S Cleaning compositions comprising polypeptides having fructan degrading activity
TW202208626A (en) 2020-04-24 2022-03-01 美商生命編輯公司 Rna-guided nucleases and active fragments and variants thereof and methods of use
US20230181756A1 (en) 2020-04-30 2023-06-15 Novartis Ag Ccr7 antibody drug conjugates for treating cancer
CN116096758A (en) 2020-05-01 2023-05-09 诺华股份有限公司 Engineered immunoglobulins
US20230167193A1 (en) 2020-05-01 2023-06-01 Novartis Ag Immunoglobulin variants
CA3173882A1 (en) 2020-05-11 2021-11-18 Alexandra Briner CRAWLEY Rna-guided nucleic acid binding proteins and active fragments and variants thereof and methods of use
US20230235310A1 (en) 2020-06-04 2023-07-27 Isobionics B.V. Synthetic santalene synthases
WO2022003142A1 (en) 2020-07-03 2022-01-06 Engenes Biotech Gmbh PYRROLYSYL-tRNA SYNTHETASE VARIANTS AND USES THEREOF
CN115867564A (en) 2020-07-14 2023-03-28 先锋国际良种公司 Insecticidal proteins and methods of use thereof
CN116157144A (en) 2020-07-15 2023-05-23 生命编辑制药股份有限公司 Uracil stabilizing proteins and active fragments and variants thereof and methods of use
US11479779B2 (en) 2020-07-31 2022-10-25 Zymergen Inc. Systems and methods for high-throughput automated strain generation for non-sporulating fungi
EP4192232A2 (en) 2020-08-10 2023-06-14 E. I. du Pont de Nemours and Company Compositions and methods for enhancing resistance to northern leaf blight in maize
WO2022035649A1 (en) 2020-08-10 2022-02-17 Pioneer Hi-Bred International, Inc. Plant regulatory elements and methods of use thereof
KR20230050402A (en) 2020-08-13 2023-04-14 노보자임스 에이/에스 Phytase variants and polynucleotides encoding them
EP4200424A1 (en) 2020-08-18 2023-06-28 Novozymes A/S Dispersins expressed with amylase signal peptides
EP4204551A2 (en) 2020-08-25 2023-07-05 Novozymes A/S Variants of a family 44 xyloglucanase
US11767519B2 (en) 2020-08-28 2023-09-26 Codexis, Inc. Engineered amylase variants
JP2023538773A (en) 2020-08-28 2023-09-11 ノボザイムス アクティーゼルスカブ Protease variants with improved solubility
IL301139A (en) 2020-09-11 2023-05-01 Lifeedit Therapeutics Inc Dna modifying enzymes and active fragments and variants thereof and methods of use
US20230365639A1 (en) 2020-09-14 2023-11-16 Aesculus Bio Aps Defensin fragment derived lipopeptides for treatment of drug-resistant microorganisms
AU2021355365A1 (en) 2020-09-30 2023-04-06 Corteva Agriscience Llc Rapid transformation of monocot leaf explants
EP4225905A2 (en) 2020-10-07 2023-08-16 Novozymes A/S Alpha-amylase variants
CN116529375A (en) 2020-10-13 2023-08-01 诺维信公司 Glycosyltransferase variants for enhanced protein production
US20230399588A1 (en) 2020-10-28 2023-12-14 Novozymes A/S Use of lipoxygenase
US20240035005A1 (en) 2020-10-29 2024-02-01 Novozymes A/S Lipase variants and compositions comprising such lipase variants
EP4237430A1 (en) 2020-11-02 2023-09-06 Novozymes A/S Leader peptides and polynucleotides encoding the same
WO2022090564A1 (en) 2020-11-02 2022-05-05 Novozymes A/S Glucoamylase variants and polynucleotides encoding same
AU2021374083A1 (en) 2020-11-06 2023-06-01 Novartis Ag Anti-cd19 agent and b cell targeting agent combination therapy for treating b cell malignancies
US20240025993A1 (en) 2020-11-06 2024-01-25 Novartis Ag Cd19 binding molecules and uses thereof
WO2022106400A1 (en) 2020-11-18 2022-05-27 Novozymes A/S Combination of immunochemically different proteases
WO2022106404A1 (en) 2020-11-18 2022-05-27 Novozymes A/S Combination of proteases
EP4251755A2 (en) 2020-11-24 2023-10-04 AgBiome, Inc. Pesticidal genes and methods of use
CA3200858A1 (en) 2020-11-24 2022-06-02 Novartis Ag Anti-cd48 antibodies, antibody drug conjugates, and uses thereof
CA3201757A1 (en) 2020-12-11 2022-06-16 Mathias Schuetz Recombinant acyl activating enzyme (aae) genes for enhanced biosynthesis of cannabinoids and cannabinoid precursors
CN116710471A (en) 2020-12-15 2023-09-05 诺维信公司 Mutant host cells with reduced cell motility
US11898174B2 (en) 2020-12-18 2024-02-13 Codexis, Inc. Engineered uridine phosphorylase variant enzymes
EP4015626A1 (en) 2020-12-18 2022-06-22 Isobionics B.V. Enzymes and methods for fermentative production of monoterpene esters
JP2024502832A (en) 2020-12-31 2024-01-23 アラマー バイオサイエンシーズ, インコーポレイテッド Binding agent molecules with high affinity and/or specificity and methods for their production and use
CN112725331B (en) * 2021-01-25 2021-07-20 深圳市狂风生命科技有限公司 Construction method of high-throughput mutant library
WO2022162043A1 (en) 2021-01-28 2022-08-04 Novozymes A/S Lipase with low malodor generation
WO2022171780A2 (en) 2021-02-12 2022-08-18 Novozymes A/S Alpha-amylase variants
WO2022175440A1 (en) 2021-02-18 2022-08-25 Novozymes A/S Inactive heme polypeptides
JP2024508766A (en) 2021-02-22 2024-02-28 ベーアーエスエフ・エスエー amylase variant
EP4047088A1 (en) 2021-02-22 2022-08-24 Basf Se Amylase variants
WO2022189521A1 (en) 2021-03-12 2022-09-15 Novozymes A/S Polypeptide variants
EP4060036A1 (en) 2021-03-15 2022-09-21 Novozymes A/S Polypeptide variants
US20240060061A1 (en) 2021-03-15 2024-02-22 Novozymes A/S Dnase variants
WO2022197512A1 (en) 2021-03-15 2022-09-22 The Procter & Gamble Company Cleaning compositions containing polypeptide variants
AU2022242754A1 (en) 2021-03-22 2023-11-02 LifeEDIT Therapeutics, Inc. Dna modifyng enzymes and active fragments and variants thereof and methods of use
WO2022199418A1 (en) 2021-03-26 2022-09-29 Novozymes A/S Detergent composition with reduced polymer content
CA3214973A1 (en) 2021-04-02 2022-10-06 Codexis, Inc. Engineered guanylate kinase variant enzymes
IL305919A (en) 2021-04-02 2023-11-01 Codexis Inc Engineered adenylate kinase variant enzymes
US20220325285A1 (en) 2021-04-02 2022-10-13 Codexis, Inc. ENGINEERED CYCLIC GMP-AMP SYNTHASE (cGAS) VARIANT ENZYMES
IL305928A (en) 2021-04-02 2023-11-01 Codexis Inc Engineered acetate kinase variant enzymes
CN117529235A (en) 2021-04-12 2024-02-06 诺维信公司 Method for producing meat analogue products
CA3216880A1 (en) 2021-04-16 2022-10-20 Novartis Ag Antibody drug conjugates and methods for making thereof
WO2022236060A1 (en) 2021-05-06 2022-11-10 AgBiome, Inc. Pesticidal genes and methods of use
EP4336997A1 (en) 2021-05-11 2024-03-20 Two Blades Foundation Methods for preparing a library of plant disease resistance genes for functional testing for disease resistance
WO2022251056A1 (en) 2021-05-27 2022-12-01 Novozymes A/S Transcriptional regulators and polynucleotides encoding the same
CA3222371A1 (en) 2021-06-07 2022-12-15 Novozymes A/S Engineered microorganism for improved ethanol fermentation
AU2022290278A1 (en) 2021-06-11 2024-01-04 LifeEDIT Therapeutics, Inc. Rna polymerase iii promoters and methods of use
CA3220135A1 (en) 2021-06-16 2022-12-22 Novozymes A/S Method for controlling slime in a pulp or paper making process
WO2023285348A1 (en) 2021-07-13 2023-01-19 Novozymes A/S Recombinant cutinase expression
WO2023288294A1 (en) 2021-07-16 2023-01-19 Novozymes A/S Compositions and methods for improving the rainfastness of proteins on plant surfaces
CA3227215A1 (en) 2021-07-30 2023-02-02 Trish Choudhary Recombinant prenyltransferase polypeptides engineered for enhanced biosynthesis of cannabinoids
WO2023012111A2 (en) 2021-08-02 2023-02-09 Basf Se Novel production of aroma compounds with ionylideneethane synthases
WO2023023621A1 (en) 2021-08-19 2023-02-23 Willow Biosciences, Inc. Recombinant olivetolic acid cyclase polypeptides engineered for enhanced biosynthesis of cannabinoids
WO2023056269A1 (en) 2021-09-30 2023-04-06 Two Blades Foundation Plant disease resistance genes against stem rust and methods of use
WO2023061928A1 (en) 2021-10-12 2023-04-20 Novozymes A/S Endoglucanase with improved stability
WO2023064955A1 (en) 2021-10-15 2023-04-20 Cytomx Therapeutics, Inc. Activatable anti-cd3, anti-egfr, heteromultimeric bispecific polypeptide complex
WO2023064905A1 (en) 2021-10-15 2023-04-20 Danisco Us Inc. Glucoamylase variants and methods for use thereof
US20230183382A1 (en) 2021-10-15 2023-06-15 Cytomx Therapeutics, Inc. Activatable polypeptide complex
US20230174995A1 (en) 2021-10-15 2023-06-08 Cytomx Therapeutics, Inc. Activatable polypeptide complex
WO2023069921A1 (en) 2021-10-19 2023-04-27 Epimeron Usa, Inc. Recombinant thca synthase polypeptides engineered for enhanced biosynthesis of cannabinoids
WO2023107943A1 (en) 2021-12-07 2023-06-15 AgBiome, Inc. Pesticidal genes and methods of use
WO2023104846A1 (en) 2021-12-10 2023-06-15 Novozymes A/S Improved protein production in recombinant bacteria
WO2023118436A1 (en) 2021-12-22 2023-06-29 Novozymes A/S Method of producing a milk-based product
WO2023118565A1 (en) 2021-12-23 2023-06-29 Novozymes A/S Reduction of residual dna in microbial fermentation products
US20230227545A1 (en) 2022-01-07 2023-07-20 Johnson & Johnson Enterprise Innovation Inc. Materials and methods of il-1beta binding proteins
WO2023139557A1 (en) 2022-01-24 2023-07-27 LifeEDIT Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2023152220A1 (en) 2022-02-10 2023-08-17 Novozymes A/S Improved expression of recombinant proteins
WO2023161245A1 (en) 2022-02-22 2023-08-31 Novozymes A/S Oral care composition comprising enzymes
WO2023165507A1 (en) 2022-03-02 2023-09-07 Novozymes A/S Use of xyloglucanase for improvement of sustainability of detergents
EP4242303A1 (en) 2022-03-08 2023-09-13 Novozymes A/S Fusion polypeptides with deamidase inhibitor and deamidase domains
WO2023170177A1 (en) 2022-03-08 2023-09-14 Novozymes A/S Fusion polypeptides with deamidase inhibitor and deamidase domains
WO2023173084A1 (en) 2022-03-11 2023-09-14 University Of Rochester Cyclopeptibodies and uses thereof
WO2023209568A1 (en) 2022-04-26 2023-11-02 Novartis Ag Multispecific antibodies targeting il-13 and il-18
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections
WO2023222648A2 (en) 2022-05-17 2023-11-23 Novozymes A/S Process for reducing free fatty acids
WO2023247348A1 (en) 2022-06-21 2023-12-28 Novozymes A/S Mannanase variants and polynucleotides encoding same
WO2023247514A1 (en) 2022-06-22 2023-12-28 Novozymes A/S Recombinant mannanase expression
WO2023247664A2 (en) 2022-06-24 2023-12-28 Novozymes A/S Lipase variants and compositions comprising such lipase variants
WO2024003143A1 (en) 2022-06-30 2024-01-04 Novozymes A/S Mutanases and oral care compositions comprising same
WO2024013727A1 (en) 2022-07-15 2024-01-18 Janssen Biotech, Inc. Material and methods for improved bioengineered pairing of antigen-binding variable regions
WO2024015953A1 (en) 2022-07-15 2024-01-18 Danisco Us Inc. Methods for producing monoclonal antibodies
WO2024012912A1 (en) 2022-07-15 2024-01-18 Novozymes A/S Polypeptides having deamidase inhibitor activity
EP4311430A1 (en) 2022-07-28 2024-01-31 Limagrain Europe Chlorotoluron tolerance gene and methods of use thereof
WO2024026406A2 (en) 2022-07-29 2024-02-01 Vestaron Corporation Next Generation ACTX Peptides
WO2024033133A2 (en) 2022-08-11 2024-02-15 Basf Se Enzyme compositions comprising an amylase
WO2024033134A1 (en) 2022-08-11 2024-02-15 Basf Se Enzyme compositions comprising protease, mannanase, and/or cellulase
WO2024033901A1 (en) 2022-08-12 2024-02-15 LifeEDIT Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2024040020A1 (en) 2022-08-15 2024-02-22 Absci Corporation Quantitative affinity activity specific cell enrichment
WO2024044596A1 (en) 2022-08-23 2024-02-29 AgBiome, Inc. Pesticidal genes and methods of use
WO2024042489A1 (en) 2022-08-25 2024-02-29 LifeEDIT Therapeutics, Inc. Chemical modification of guide rnas with locked nucleic acid for rna guided nuclease-mediated gene editing
EP4273249A2 (en) 2023-07-07 2023-11-08 Novozymes A/S Improved expression of recombinant proteins

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6251674B1 (en) * 1997-01-17 2001-06-26 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US6287862B1 (en) * 1997-01-17 2001-09-11 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US6319713B1 (en) * 1994-02-17 2001-11-20 Maxygen, Inc. Methods and compositions for polypeptide engineering
US6358742B1 (en) * 1996-03-25 2002-03-19 Maxygen, Inc. Evolving conjugative transfer of DNA by recursive recombination
US6372497B1 (en) * 1994-02-17 2002-04-16 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6395547B1 (en) * 1994-02-17 2002-05-28 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6420175B1 (en) * 1994-02-17 2002-07-16 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6537746B2 (en) * 1997-12-08 2003-03-25 Maxygen, Inc. Method for creating polynucleotide and polypeptide sequences

Family Cites Families (146)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4994379A (en) * 1983-03-09 1991-02-19 Cetus Corporation Modified signal peptides
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5422266A (en) 1984-12-31 1995-06-06 University Of Georgia Research Foundation, Inc. Recombinant DNA vectors capable of expressing apoaequorin
US4965188A (en) * 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5176995A (en) * 1985-03-28 1993-01-05 Hoffmann-La Roche Inc. Detection of viruses by amplification and hybridization
DE229046T1 (en) 1985-03-30 1987-12-17 Marc Genf/Geneve Ch Ballivet METHOD FOR OBTAINING DNA, RNS, PEPTIDES, POLYPEPTIDES OR PROTEINS BY DNA RECOMBINANT METHOD.
US6492107B1 (en) 1986-11-20 2002-12-10 Stuart Kauffman Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
US4959312A (en) 1985-05-31 1990-09-25 The University Of Tennessee Research Corporation Full spectrum mutagenesis
DE3688920D1 (en) * 1985-07-03 1993-09-30 Genencor Int Hybrid polypeptides and process for their preparation.
US5866363A (en) 1985-08-28 1999-02-02 Pieczenik; George Method and means for sorting and identifying biological information
US4800159A (en) * 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
DK311186D0 (en) * 1986-06-30 1986-06-30 Novo Industri As ENZYMES
US5824469A (en) * 1986-07-17 1998-10-20 University Of Washington Method for producing novel DNA sequences with biological activity
US4994368A (en) * 1987-07-23 1991-02-19 Syntex (U.S.A.) Inc. Amplification method for polynucleotide assays
US4994367A (en) 1988-10-07 1991-02-19 East Carolina University Extended shelf life platelet preparations and process for preparing the same
FR2641793B1 (en) * 1988-12-26 1993-10-01 Setratech METHOD OF IN VIVO RECOMBINATION OF DNA SEQUENCES HAVING BASIC MATCHING
DD282028A5 (en) 1989-02-16 1990-08-29 Akad Wissenschaften Ddr PROCESS FOR PREPARING THERMOSTABILES, HYBRIDEN BACILLUS BETA-1,3-1,4-GLUCANASE
US5043272A (en) * 1989-04-27 1991-08-27 Life Technologies, Incorporated Amplification of nucleic acid sequences using oligonucleotides of random sequence as primers
US5106727A (en) * 1989-04-27 1992-04-21 Life Technologies, Inc. Amplification of nucleic acid sequences using oligonucleotides of random sequences as primers
US5556750A (en) * 1989-05-12 1996-09-17 Duke University Methods and kits for fractionating a population of DNA molecules based on the presence or absence of a base-pair mismatch utilizing mismatch repair systems
CA2016842A1 (en) * 1989-05-16 1990-11-16 Richard A. Lerner Method for tapping the immunological repertoire
CA2016841C (en) * 1989-05-16 1999-09-21 William D. Huse A method for producing polymers having a preselected activity
IT1231157B (en) * 1989-07-20 1991-11-19 Crc Ricerca Chim NEW FUNCTIONAL HYBRID GENES OF BACILLUS THURINGIENSIS OBTAINED THROUGH IN VIVO RECOMBINATION.
US5574205A (en) 1989-07-25 1996-11-12 Cell Genesys Homologous recombination for universal donor cells and chimeric mammalian hosts
US5023171A (en) * 1989-08-10 1991-06-11 Mayo Foundation For Medical Education And Research Method for gene splicing by overlap extension using the polymerase chain reaction
WO1991006570A1 (en) * 1989-10-25 1991-05-16 The University Of Melbourne HYBRID Fc RECEPTOR MOLECULES
US5071743A (en) 1989-10-27 1991-12-10 Her Majesty The Queen In Right Of Canada, As Represented By The National Research Council Of Canada Process for conducting site-directed mutagenesis
DE3936258C1 (en) 1989-10-31 1991-04-25 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften Ev, 3400 Goettingen, De
WO1991007506A1 (en) * 1989-11-08 1991-05-30 The United States Of America, Represented By The Secretary, United States Department Of Commerce A method of synthesizing double-stranded dna molecules
ATE126535T1 (en) * 1990-04-05 1995-09-15 Roberto Crea ''WALK-THROUGH'' MUTAGENesis.
EP0528881A4 (en) 1990-04-24 1993-05-26 Stratagene Methods for phenotype creation from multiple gene populations
US5877402A (en) 1990-05-01 1999-03-02 Rutgers, The State University Of New Jersey DNA constructs and methods for stably transforming plastids of multicellular plants and expressing recombinant proteins therein
US5851813A (en) * 1990-07-12 1998-12-22 President And Fellows Of Harvard College Primate lentivirus antigenic compositions
US5571208A (en) 1990-07-13 1996-11-05 Caspers; Carl A. Reinforced prosthetic polyurethane hypobaric sleeve
CA2087724C (en) 1990-07-24 2003-09-16 John J. Sninsky Reduction of non-specific amplification during in vitro nucleic acid amplification using modified nucleic acid bases
US5169764A (en) 1990-08-08 1992-12-08 Regeneron Pharmaceuticals, Inc. Multitrophic and multifunctional chimeric neurotrophic factors, and nucleic acids and plasmids encoding the chimeras
JP3306063B2 (en) * 1990-08-24 2002-07-24 イグジス, インコーポレイテッド Method for synthesizing oligonucleotides having random codons
US5264563A (en) * 1990-08-24 1993-11-23 Ixsys Inc. Process for synthesizing oligonucleotides with random codons
IL99553A0 (en) * 1990-09-28 1992-08-18 Ixsys Inc Compositions containing oligonucleotides linked to expression elements,a kit for the preparation of vectors useful for the expression of a diverse population of random peptides and methods utilizing the same
US5871974A (en) * 1990-09-28 1999-02-16 Ixsys Inc. Surface expression libraries of heteromeric receptors
US5770434A (en) * 1990-09-28 1998-06-23 Ixsys Incorporated Soluble peptides having constrained, secondary conformation in solution and method of making same
US5698426A (en) * 1990-09-28 1997-12-16 Ixsys, Incorporated Surface expression libraries of heteromeric receptors
US5858725A (en) * 1990-10-10 1999-01-12 Glaxo Wellcome Inc. Preparation of chimaeric antibodies using the recombinant PCR strategy
US5187083A (en) * 1990-11-13 1993-02-16 Specialty Laboratories, Inc. Rapid purification of DNA
US5234824A (en) * 1990-11-13 1993-08-10 Specialty Laboratories, Inc. Rapid purification of DNA
US5489523A (en) * 1990-12-03 1996-02-06 Stratagene Exonuclease-deficient thermostable Pyrococcus furiosus DNA polymerase I
AU9166691A (en) * 1990-12-20 1992-07-22 Ixsys, Inc. Optimization of binding proteins
US5795747A (en) * 1991-04-16 1998-08-18 Evotec Biosystems Gmbh Process for manufacturing new biopolymers
DE4112440C1 (en) * 1991-04-16 1992-10-22 Diagen Institut Fuer Molekularbiologische Diagnostik Gmbh, 4000 Duesseldorf, De
JPH06510116A (en) * 1991-04-17 1994-11-10 ベミス・マニファクチュアリング・カンパニー Method and apparatus for data point analysis
US5512463A (en) * 1991-04-26 1996-04-30 Eli Lilly And Company Enzymatic inverse polymerase chain reaction library mutagenesis
US5514568A (en) * 1991-04-26 1996-05-07 Eli Lilly And Company Enzymatic inverse polymerase chain reaction
DK0519338T3 (en) 1991-06-20 1996-10-28 Hoffmann La Roche Improved Methods for Nucleic Acid Amplification
WO1993000103A1 (en) * 1991-06-21 1993-01-07 The Wistar Institute Of Anatomy And Biology Chimeric envelope proteins for viral targeting
AU2317192A (en) 1991-07-01 1993-02-11 Berlex Laboratories, Inc. Novel mutagenesis methods and compositions
US6071889A (en) 1991-07-08 2000-06-06 Neurospheres Holdings Ltd. In vivo genetic modification of growth factor-responsive neural precursor cells
US5223408A (en) * 1991-07-11 1993-06-29 Genentech, Inc. Method for making variant secreted proteins with altered properties
GB9115364D0 (en) * 1991-07-16 1991-08-28 Wellcome Found Antibody
US5279952A (en) * 1991-08-09 1994-01-18 Board Of Regents, The University Of Texas System PCR-based strategy of constructing chimeric DNA molecules
DE69229477T2 (en) * 1991-09-23 1999-12-09 Cambridge Antibody Tech Methods for the production of humanized antibodies
GB9125979D0 (en) * 1991-12-06 1992-02-05 Wellcome Found Antibody
FR2685004A1 (en) * 1991-12-11 1993-06-18 Pasteur Institut METHOD FOR GENERATING A STRUCTURAL AND FUNCTIONAL DIVERSITY IN A PEPTIDE SEQUENCE.
JP3431146B2 (en) * 1992-01-30 2003-07-28 ジェンザイム・リミテッド Chiral synthesis with modified enzymes
US5773267A (en) 1992-02-07 1998-06-30 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University D29 shuttle phasmids and uses thereof
GB9202796D0 (en) * 1992-02-11 1992-03-25 Wellcome Found Antiviral antibody
US5843643A (en) 1992-02-21 1998-12-01 Ratner; Paul L. Site-specific transfection of eukaryotic cells using polypeptide-linked recombinant nucleic acid
WO1993018141A1 (en) * 1992-03-02 1993-09-16 Biogen, Inc. Thrombin receptor antagonists
EP1477558A1 (en) 1992-03-16 2004-11-17 WOHLSTADTER, Jacob Nathaniel Selection methods
JP3507073B2 (en) 1992-03-24 2004-03-15 ケンブリッジ アンティボディー テクノロジー リミティド Methods for producing members of a specific binding pair
US5316935A (en) 1992-04-06 1994-05-31 California Institute Of Technology Subtilisin variants suitable for hydrolysis and synthesis in organic media
GEP20012448B (en) 1992-06-03 2001-05-25 Genentech Inc Tissue Plasminogen Activators (Improved Variants)
WO1993025237A1 (en) * 1992-06-15 1993-12-23 City Of Hope Chimeric anti-cea antibody
CA2139876A1 (en) * 1992-07-10 1994-01-20 John Rambosek Recombinant dna encoding a desulfurization biocatalyst
US6107062A (en) * 1992-07-30 2000-08-22 Inpax, Inc. Antisense viruses and antisense-ribozyme viruses
FR2694754B1 (en) 1992-08-12 1994-09-16 Bio Merieux Mycobacteria DNA fragments, amplification primers, hybridization probes, reagents and method for detecting detection of mycobacteria.
US5837821A (en) * 1992-11-04 1998-11-17 City Of Hope Antibody construct
DE69334261D1 (en) * 1992-11-10 2009-03-19 Applied Molecular Evolution SOLUBLE PEPTIDES THAT HAVE SOLVED AN EXCITING SECONDARY STRUCTURE AND METHOD FOR THE MANUFACTURE THEREOF
US5360728A (en) * 1992-12-01 1994-11-01 Woods Hole Oceanographic Institution (W.H.O.I.) Modified apoaequorin having increased bioluminescent activity
CA2150262C (en) * 1992-12-04 2008-07-08 Kaspar-Philipp Holliger Multivalent and multispecific binding proteins, their manufacture and use
US5571708A (en) 1993-04-19 1996-11-05 Bristol-Myers Squibb Company Thrombin-activatable plasminogen activator
IT1264712B1 (en) * 1993-07-13 1996-10-04 Eniricerche Spa BIOLOGICAL SYNTHESIS METHOD OF PEPTIDES
US5498531A (en) 1993-09-10 1996-03-12 President And Fellows Of Harvard College Intron-mediated recombinant techniques and reagents
US5474920A (en) 1993-11-23 1995-12-12 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University Modified thermo-resistant DNA polymerases
US5556772A (en) * 1993-12-08 1996-09-17 Stratagene Polymerase compositions and uses thereof
DE4343591A1 (en) * 1993-12-21 1995-06-22 Evotec Biosystems Gmbh Process for the evolutionary design and synthesis of functional polymers based on shape elements and shape codes
US6995017B1 (en) * 1994-02-17 2006-02-07 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6165793A (en) * 1996-03-25 2000-12-26 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5837458A (en) 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US5834252A (en) 1995-04-18 1998-11-10 Glaxo Group Limited End-complementary polymerase reaction
US5928905A (en) * 1995-04-18 1999-07-27 Glaxo Group Limited End-complementary polymerase reaction
US5521077A (en) * 1994-04-28 1996-05-28 The Leland Stanford Junior University Method of generating multiple protein variants and populations of protein variants prepared thereby
EP0714980A1 (en) 1994-12-02 1996-06-05 Institut Pasteur Hypermutagenesis
WO1996017056A1 (en) 1994-12-02 1996-06-06 Institut Pasteur Hypermutagenesis
US5514588A (en) 1994-12-13 1996-05-07 Exxon Research And Engineering Company Surfactant-nutrients for bioremediation of hydrocarbon contaminated soils and water
US5629179A (en) * 1995-03-17 1997-05-13 Novagen, Inc. Method and kit for making CDNA library
KR19990008000A (en) * 1995-04-24 1999-01-25 로버트 에스. 화이트 헤드 How to create a new metabolic pathway and screen it
US6004788A (en) 1995-07-18 1999-12-21 Diversa Corporation Enzyme kits and libraries
US6030779A (en) 1995-07-18 2000-02-29 Diversa Corporation Screening for novel bioactivities
US5958672A (en) * 1995-07-18 1999-09-28 Diversa Corporation Protein activity screening of clones having DNA from uncultivated microorganisms
US6168919B1 (en) 1996-07-17 2001-01-02 Diversa Corporation Screening methods for enzymes and enzyme kits
US6057103A (en) 1995-07-18 2000-05-02 Diversa Corporation Screening for novel bioactivities
ATE216427T1 (en) * 1995-08-11 2002-05-15 Novozymes As METHOD FOR PRODUCING POLYPEPTIDE DERIVATIVES
US5962258A (en) 1995-08-23 1999-10-05 Diversa Corporation Carboxymethyl cellulase fromthermotoga maritima
FR2738840B1 (en) 1995-09-19 1997-10-31 Rhone Poulenc Rorer Sa GENETICALLY MODIFIED YEAST STRAINS
US6051409A (en) 1995-09-25 2000-04-18 Novartis Finance Corporation Method for achieving integration of exogenous DNA delivered by non-biological means to plant cells
US5756316A (en) * 1995-11-02 1998-05-26 Genencor International, Inc. Molecular cloning by multimerization of plasmids
US6358709B1 (en) 1995-12-07 2002-03-19 Diversa Corporation End selection in directed evolution
US6352842B1 (en) 1995-12-07 2002-03-05 Diversa Corporation Exonucease-mediated gene assembly in directed evolution
US5965408A (en) * 1996-07-09 1999-10-12 Diversa Corporation Method of DNA reassembly by interrupting synthesis
US6238884B1 (en) 1995-12-07 2001-05-29 Diversa Corporation End selection in directed evolution
US6361974B1 (en) 1995-12-07 2002-03-26 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
US5830696A (en) * 1996-12-05 1998-11-03 Diversa Corporation Directed evolution of thermophilic enzymes
US5814473A (en) 1996-02-09 1998-09-29 Diversa Corporation Transaminases and aminotransferases
US20030215798A1 (en) 1997-06-16 2003-11-20 Diversa Corporation High throughput fluorescence-based screening for novel enzymes
US5962283A (en) 1995-12-07 1999-10-05 Diversa Corporation Transminases and amnotransferases
US6171820B1 (en) 1995-12-07 2001-01-09 Diversa Corporation Saturation mutagenesis in directed evolution
US5939250A (en) * 1995-12-07 1999-08-17 Diversa Corporation Production of enzymes having desired activities by mutagenesis
JP2000502568A (en) * 1996-01-10 2000-03-07 ノボ ノルディスク アクティーゼルスカブ Method for in vivo production of a mutation library in cells
US5942430A (en) 1996-02-16 1999-08-24 Diversa Corporation Esterases
US5958751A (en) 1996-03-08 1999-09-28 Diversa Corporation α-galactosidase
US5783431A (en) * 1996-04-24 1998-07-21 Chromaxome Corporation Methods for generating and screening novel metabolic pathways
US5789228A (en) 1996-05-22 1998-08-04 Diversa Corporation Endoglucanases
US5877001A (en) 1996-06-17 1999-03-02 Diverso Corporation Amidase
US5763239A (en) 1996-06-18 1998-06-09 Diversa Corporation Production and use of normalized DNA libraries
US5939300A (en) 1996-07-03 1999-08-17 Diversa Corporation Catalases
US6093873A (en) 1996-08-19 2000-07-25 Institut National De La Sante Et De La Recherche Medicale Genetically engineered mice containing alterations in the gene encoding RXR
US5834458A (en) 1996-10-09 1998-11-10 Eli Lilly And Company Heterocyclic compounds and their use
EP0948615B1 (en) * 1996-12-20 2004-12-15 Novozymes A/S In vivo recombination
JP2001515356A (en) * 1997-03-18 2001-09-18 ノボ ノルディスク アクティーゼルスカブ Method for preparing DNA library in test tube
DK1015575T3 (en) * 1997-03-18 2010-08-23 Novozymes As Shuffling of heterologous DNA sequences
DE69840382D1 (en) * 1997-03-18 2009-02-05 Novozymes As METHOD FOR THE PRODUCTION OF A LIBRARY THROUGH DNA SHUFFLING
US5948653A (en) 1997-03-21 1999-09-07 Pati; Sushma Sequence alterations using homologous recombination
US6153410A (en) * 1997-03-25 2000-11-28 California Institute Of Technology Recombination of polynucleotide sequences using random or defined primers
US6051049A (en) 1997-05-29 2000-04-18 Exothermic Distribution Corporation Utilization of strontium aluminate in steelmaking
US5948666A (en) 1997-08-06 1999-09-07 Diversa Corporation Isolation and identification of polymerases
US5876997A (en) 1997-08-13 1999-03-02 Diversa Corporation Phytase
US6087341A (en) 1998-02-12 2000-07-11 The Board Of Trustees Of The Leland Standford Junior University Introduction of nucleic acid into skin cells by topical application
US6365408B1 (en) 1998-06-19 2002-04-02 Maxygen, Inc. Methods of evolving a polynucleotides by mutagenesis and recombination
MXPA01000224A (en) 1998-07-28 2002-10-17 California Inst Of Techn Expression of functional eukaryotic proteins.
WO2000009727A2 (en) 1998-08-12 2000-02-24 Maxygen, Inc. Dna shuffling to produce herbicide selective crops
JP2002537758A (en) 1998-09-29 2002-11-12 マキシジェン, インコーポレイテッド Shuffling of codon-modified genes
US6376246B1 (en) * 1999-02-05 2002-04-23 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
US6397053B1 (en) * 1999-12-30 2002-05-28 Koninklijke Philips Electronics N.V. (Kpenv) Reduction of power consumption with increased standby time in wireless communications device
US20050084907A1 (en) * 2002-03-01 2005-04-21 Maxygen, Inc. Methods, systems, and software for identifying functional biomolecules
US20090312196A1 (en) * 2008-06-13 2009-12-17 Codexis, Inc. Method of synthesizing polynucleotide variants

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6319713B1 (en) * 1994-02-17 2001-11-20 Maxygen, Inc. Methods and compositions for polypeptide engineering
US6372497B1 (en) * 1994-02-17 2002-04-16 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6395547B1 (en) * 1994-02-17 2002-05-28 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6413774B1 (en) * 1994-02-17 2002-07-02 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6420175B1 (en) * 1994-02-17 2002-07-16 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6444468B1 (en) * 1994-02-17 2002-09-03 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US7288375B2 (en) * 1994-02-17 2007-10-30 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6335160B1 (en) * 1995-02-17 2002-01-01 Maxygen, Inc. Methods and compositions for polypeptide engineering
US6358742B1 (en) * 1996-03-25 2002-03-19 Maxygen, Inc. Evolving conjugative transfer of DNA by recursive recombination
US6251674B1 (en) * 1997-01-17 2001-06-26 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US6287862B1 (en) * 1997-01-17 2001-09-11 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US6537746B2 (en) * 1997-12-08 2003-03-25 Maxygen, Inc. Method for creating polynucleotide and polypeptide sequences

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8168380B2 (en) 1997-02-12 2012-05-01 Life Technologies Corporation Methods and products for analyzing polymers
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US8314216B2 (en) 2000-12-01 2012-11-20 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US9845500B2 (en) 2000-12-01 2017-12-19 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US8648179B2 (en) 2000-12-01 2014-02-11 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US8202831B2 (en) 2008-06-06 2012-06-19 The Procter & Gamble Company Detergent composition comprising a variant of a family 44 xyloglucanase
US9567629B2 (en) 2009-03-27 2017-02-14 Life Technologies Corporation Labeled enzyme compositions, methods and systems
US9932573B2 (en) 2009-03-27 2018-04-03 Life Technologies Corporation Labeled enzyme compositions, methods and systems
US9365838B2 (en) 2009-03-27 2016-06-14 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US9365839B2 (en) 2009-03-27 2016-06-14 Life Technologies Corporation Polymerase compositions and methods
US8741618B2 (en) 2009-03-27 2014-06-03 Life Technologies Corporation Labeled enzyme compositions, methods and systems
US9695471B2 (en) 2009-03-27 2017-07-04 Life Technologies Corporation Methods and apparatus for single molecule sequencing using energy transfer detection
US8603792B2 (en) 2009-03-27 2013-12-10 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US8999674B2 (en) 2009-03-27 2015-04-07 Life Technologies Corporation Methods and apparatus for single molecule sequencing using energy transfer detection
US10093974B2 (en) 2009-03-27 2018-10-09 Life Technologies Corporation Methods and apparatus for single molecule sequencing using energy transfer detection
US10093973B2 (en) 2009-03-27 2018-10-09 Life Technologies Corporation Polymerase compositions and methods
US10093972B2 (en) 2009-03-27 2018-10-09 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US11008612B2 (en) 2009-03-27 2021-05-18 Life Technologies Corporation Methods and apparatus for single molecule sequencing using energy transfer detection
US11015220B2 (en) 2009-03-27 2021-05-25 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US11453909B2 (en) 2009-03-27 2022-09-27 Life Technologies Corporation Polymerase compositions and methods
US11542549B2 (en) 2009-03-27 2023-01-03 Life Technologies Corporation Labeled enzyme compositions, methods and systems

Also Published As

Publication number Publication date
EP0752008B1 (en) 2002-04-24
US20100222237A1 (en) 2010-09-02
JP2007244399A (en) 2007-09-27
CA2182393A1 (en) 1995-08-24
DE1094108T1 (en) 2002-10-17
ES2165652T3 (en) 2002-03-16
US7981614B2 (en) 2011-07-19
JP2004254709A (en) 2004-09-16
DE69526497D1 (en) 2002-05-29
JPH10500561A (en) 1998-01-20
EP0934999A1 (en) 1999-08-11
RU2157851C2 (en) 2000-10-20
JP2001057893A (en) 2001-03-06
US5811238A (en) 1998-09-22
ES2176324T3 (en) 2002-12-01
US20040241853A1 (en) 2004-12-02
AU703264B2 (en) 1999-03-25
AU2010200270A1 (en) 2010-02-18
US6444468B1 (en) 2002-09-03
JP3485560B2 (en) 2004-01-13
AU703264C (en) 2003-06-12
ATE211761T1 (en) 2002-01-15
JP4067516B2 (en) 2008-03-26
US6132970A (en) 2000-10-17
DK0934999T3 (en) 2002-02-18
JP2003033180A (en) 2003-02-04
EP1094108A3 (en) 2001-05-09
DE752008T1 (en) 2001-04-05
US5605793A (en) 1997-02-25
US6576467B1 (en) 2003-06-10
EP1205547A3 (en) 2002-07-24
EP0752008B2 (en) 2013-04-10
EP0752008A1 (en) 1997-01-08
US7288375B2 (en) 2007-10-30
DE69525084D1 (en) 2002-02-28
DE934999T1 (en) 2001-04-05
ATE216722T1 (en) 2002-05-15
CA2182393C (en) 2005-05-17
US6420175B1 (en) 2002-07-16
US6602986B1 (en) 2003-08-05
DE69526497T2 (en) 2002-11-21
EP0934999B1 (en) 2002-01-09
US6287861B1 (en) 2001-09-11
AU2006202542A1 (en) 2006-07-13
EP1205547A2 (en) 2002-05-15
CN1145641A (en) 1997-03-19
DE69525084T2 (en) 2002-07-11
US6277638B1 (en) 2001-08-21
EP1094108A2 (en) 2001-04-25
EP0752008A4 (en) 1999-03-17
JP3393848B2 (en) 2003-04-07
US6297053B1 (en) 2001-10-02
WO1995022625A1 (en) 1995-08-24
JP2003174881A (en) 2003-06-24
US5830721A (en) 1998-11-03
AU2971495A (en) 1995-09-04
DK0752008T3 (en) 2002-06-17

Similar Documents

Publication Publication Date Title
US7981614B2 (en) Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6365408B1 (en) Methods of evolving a polynucleotides by mutagenesis and recombination
EP0876509B2 (en) Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6995017B1 (en) Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6395547B1 (en) Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US7868138B2 (en) Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
AU747034B2 (en) DNA mutagenesis by random fragmentation and reassembly
CA2497384C (en) Dna mutagenesis by random fragmentation and reassembly
AU2747302A (en) DNA mutagenesis by random fragmentation and reassembly
AU2747402A (en) DNA mutagenesis by random fragmentation and reassembly
AU2080900A (en) Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: CODEXIS, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CODEXIS MAYFLOWER HOLDINGS, LLC;REEL/FRAME:066528/0932

Effective date: 20240206