WO1998026080A1 - Method for expression of heterologous proteins in yeast - Google Patents
Method for expression of heterologous proteins in yeast Download PDFInfo
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- WO1998026080A1 WO1998026080A1 PCT/US1997/022647 US9722647W WO9826080A1 WO 1998026080 A1 WO1998026080 A1 WO 1998026080A1 US 9722647 W US9722647 W US 9722647W WO 9826080 A1 WO9826080 A1 WO 9826080A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/49—Platelet-derived growth factor [PDGF]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Definitions
- the present invention relates to the production of recombinant proteins using yeast host cells as the expression system. More particularly, it relates to compositions and methods for expression of heterologous proteins and their secretion as the biologically active mature proteins.
- Yeast host expression systems have been used to express and secrete proteins foreign to yeast. Numerous approaches have been developed in terms of the degree of expression and the yield of biologically active mature proteins.
- Such approaches have involved modifications to the various molecular components that are involved in expression and secretion of proteins in yeast. These components include the translation and termination regulatory regions for gene expression; signal peptide and secretion leader peptide sequences, which direct the precursor form of the heterologous protein through the yeast secretory pathway; and processing sites, which cleave leader peptide sequences from the polypeptide sequence of the protein of interest.
- Expression of the protein of interest can be enhanced with use of yeast- recognized regulatory regions. Increased yield of the heterologous protein of interest is commonly achieved with the use of yeast-derived signal and secretion leader peptide sequences.
- the use of native signal-leader peptide sequences is believed to improve direction of the protein of interest through the secretory pathway of the yeast host.
- Previous work has demonstrated that full-length yeast ⁇ -factor signal-leader sequences can be used to drive expression and processing of heterologous proteins in yeast host cells. Substantial improvements in efficiency of expression can be accomplished with the use of truncated ⁇ -factor leader sequences, particularly for heterologous proteins that are poorly expressed by the full-length sequence or whose expression is nonresponsive to the full-length sequence.
- compositions and methods for expression of heterologous proteins, more particularly heterologous mammalian proteins, and their secretion in a biologically active mature form using a yeast host cell as the expression system are provided.
- compositions of the invention are nucleotide sequences encoding a signal sequence for a yeast secreted protein, a native propeptide leader sequence for a mature protein of interest, and a peptide sequence for the mature protein of interest.
- Each of these elements is associated with a processing site recognized in vivo by a yeast proteolytic enzyme. Any or all of these processing sites may be a preferred processing site that has been modified or synthetically derived for more efficient cleavage in vivo. In turn, all of these elements are operably linked to a yeast promoter and optionally other regulatory sequences.
- the nucleotide coding sequences of these compositions may additionally comprise a leader peptide sequence for a yeast secreted protein.
- this element which is also associated with a processing site recognized in vivo by a yeast proteolytic enzyme, is positioned 3' to the yeast signal sequence and 5' to the sequence for the mature protein of interest.
- cleavage by a yeast proteolytic enzyme removes the yeast leader sequence from the hybrid precursor molecule comprising the sequence for the mature protein of interest.
- compositions are useful in methods for expression of heterologous mammalian proteins and their secretion in the biologically active mature form.
- vectors comprising these nucleotide coding sequences can be used to transform a yeast host cell, which can then be cultured and screened for secretion of the biologically active mature protein of interest.
- the method of the present invention is particularly useful in production of mammalian proteins whose assumption of a native confirmation is facilitated by the presence of a native propeptide sequence in the precursor polypeptide.
- Figure 1 is a map of plasmid pAB24.
- Figure 2 is a map of the rhPDGF-B expression cassette in pAGL7PB and pYAGL7PB.
- Figure 3 is a map of rhPDGF-B expression plasmid pYAGL7PB.
- Figure 4 is a map of the rhPDGF-B expression cassette in pL7PPB and pYL7PPB.
- Figure 5 shows the final steps in the construction of the rhPDGF-B expression cassette in pL7PPB.
- Figure 6 is a map of rhPDGF-B expression plasmid pYL7PPB.
- compositions and methods for expression of heterologous proteins of interest are particularly heterologous mammalian proteins, and their secretion in a biologically active mature form using a yeast host cell as the expression system.
- biologically active mature form is intended a protein whose conformational form is similar to the native conformation such that its biological activity is substantially the same as the biological activity of the native protein.
- Compositions of the present invention are nucleotide sequences encoding hybrid precursor polypeptides that each comprise the polypeptide sequence for a mature heterologous protein of interest.
- Expression vectors comprising these nucleotide sequences, all under the operational control of a yeast promoter region and a yeast terminator region, are also provided.
- Methods of the invention comprise stably transforming a yeast host cell with said vectors, where expression of the nucleotide sequence encoding the hybrid precursor polypeptide leads to secretion of the mature heterologous protein of interest in a biologically active form.
- heterologous protein of interest is intended a protein that is not expressed by the yeast host cell in nature.
- the heterologous protein will be a mammalian protein, including substantially homologous and functionally equivalent variants thereof.
- variant is intended a polypeptide derived from the native polypeptide by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native polypeptide; or substitution of one or more amino acids at one or more sites in the native polypeptide.
- variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
- amino acid sequence variants of the polypeptide can be prepared by mutations in the cloned DNA sequence encoding the native polypeptide of interest.
- Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York); U.S. Patent No.
- proteins of the invention include the naturally occurring forms as well as variants thereof. These variants will be substantially homologous and functionally equivalent to the native protein.
- a variant of a native protein is "substantially homologous" to the native protein when at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of its amino acid sequence is identical to the amino acid sequence of the native protein.
- a variant may differ by as few as 1, 2, 3, or 4 amino acids.
- Bioactivity can be measured using assays specifically designed for measuring activity of the native protein, including assays described in the present invention. Additionally, antibodies raised against the biologically active native protein can be tested for their ability to bind to the functionally equivalent variant, where effective binding is indicative of a protein having a conformation similar to that of the native protein.
- nucleotide sequences encoding the mature heterologous proteins of interest can be sequences cloned from non-yeast organisms, or they may be synthetically derived sequences, usually prepared using yeast-preferred codons.
- heterologous proteins suitable for the invention include, but are not limited to transforming growth factor (TGF-alpha and TGF-beta), somatostatin (as in SRIF 1), parathryoid hormone, and more particularly platelet-derived growth factor (PDGF) and insulin growth factor (IGF), all of which have a native prosequence as part of the precursor protein.
- TGF-alpha and TGF-beta transforming growth factor-alpha and TGF-beta
- somatostatin as in SRIF 1
- parathryoid hormone and more particularly platelet-derived growth factor (PDGF) and insulin growth factor (IGF)
- PDGF platelet-derived growth factor
- IGF insulin growth factor
- compositions of the present invention are nucleotide sequences encoding hybrid precursor polypeptides that each comprise the polypeptide sequence for a mature heterologous protein of interest or any substantially homologous and functionally equivalent variants thereof. More particularly, nucleotide sequences of the present invention encode in the 5 ' to 3 ' direction a hybrid precursor polypeptide comprising the following primary elements:
- SP comprises a signal peptide sequence for a yeast secreted protein
- PS comprises a processing site cleaved in vivo by a yeast proteolytic enzyme
- LP comprises a leader peptide sequence for a yeast secreted protein
- each of the other elements present in the hybrid precursor polypeptide can be a known naturally occurring polypeptide sequence or can be synthetically derived, including any variants thereof that do not adversely affect the function of the element as described herein.
- adversely affect is intended inclusion of the variant form of the element results in decreased yield of the secreted mature heterologous protein of interest relative to the hybrid precursor polypeptide comprising the native form of the element.
- the hybrid precursor polypeptide may comprise additional elements positioned 5 ' or 3' to any of the primary elements listed above, including the yeast leader peptide sequence and its associated yeast-recognized processing site when present.
- SP is a presequence that is an N- terminal sequence for the precursor polypeptide of the mature form of a yeast secreted protein.
- the signal peptide sequence functions to direct the hybrid precursor polypeptide comprising the mature heterologous protein of interest into the endoplasmic reticulum (ER). Movement into the lumen of the ER represents the initial step into the secretory pathway of the yeast host cell.
- the signal peptide of the invention can be heterologous to the yeast host cell, more preferably the signal peptide will be native to the host cell.
- the signal peptide sequence of the invention may be a known naturally occurring signal sequence or any variant thereof as described above that does not adversely affect the function of the signal peptide.
- Examples of signal peptides appropriate for the present invention include, but are not limited to, the signal peptide sequences for ⁇ -factor (see, for example, U.S. Patent No. 5,602,034; Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81 :4642-4646); invertase (WO 84/01153); PHO5 (DK 3614/83); YAP3 (yeast aspartic protease 3; PCT Publication No. 95/02059); and BAR1 (PCT Publication No.
- the signal peptide sequence may be determined from genomic or cDNA libraries using hybridization probe techniques available in the art (see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York), or even synthetically derived (see, for example, WO 92/11378).
- the signal peptide is cleaved off the precursor polypeptide at a processing site.
- the processing site can comprise any peptide sequence that is recognized in vivo by a yeast proteolytic enzyme.
- This processing site may be the naturally occurring processing site for the signal peptide. More preferably, the naturally occurring processing site will be modified, or the processing site will be synthetically derived, so as to be a preferred processing site.
- preferred processing site is intended a processing site that is cleaved in vivo by a yeast proteolytic enzyme more efficiently than is the naturally occurring site.
- processing sites include, but are not limited to, dibasic peptides, particularly any combination of the two basic residues Lys and Arg, that is Lys-Lys, Lys- Arg, Arg-Lys, or Arg- Arg, most preferably Lys-Arg. These sites are cleaved by the endopeptidase encoded by the KEX2 gene of Saccharomyces cerevisiae (see Fuller et al. Microbiology 1986:273-278) or the equivalent protease of other yeast species (see Julius et al. (1983) Cell 32:839-852).
- KEX2 endopeptidase would cleave a site within the peptide sequence for the mature heterologous protein of interest
- other preferred processing sites could be utilized such that the peptide sequence of interest remains intact (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York).
- a functional signal peptide sequence is essential to bring about extracellular secretion of a heterologous protein from a yeast cell.
- the hybrid precursor polypeptide may comprise a secretion leader peptide sequence of a yeast secreted protein to further facilitate this secretion process.
- the leader peptide sequence is generally positioned immediately 3 ' to the signal peptide sequence processing site.
- secretion leader peptide sequence LP
- secretion leader peptide sequence LP
- LP secretion leader peptide sequence
- the leader peptide sequence may be native or heterologous to the yeast host cell but more preferably is native to the host cell.
- the leader peptide sequence of the present invention may be a naturally occurring sequence for the same yeast secreted protein that served as the source of the signal peptide sequence, a naturally occurring sequence for a different yeast secreted protein, or a synthetic sequence (see, for example, WO 92/11378), or any variants thereof that do not adversely affect the function of the leader peptide.
- the leader peptide sequence when present is preferably derived from the same yeast secreted protein that served as the source of the signal peptide sequence, more preferably an ⁇ -factor protein.
- ⁇ -factor protein a number of genes encoding precursor ⁇ -factor proteins have been cloned and their combined signal- leader peptide sequences identified. See, for example, Singh et al. (1983) Nucleic acid sequences.
- an oligopeptide mating pheromone approximately 13 residues in length is produced from a larger precursor polypeptide of between about 100 and 200 residues in length, more typically about 120-160 residues.
- This precursor polypeptide comprises the signal sequence, which is about 19-23 (more typically 20-22 residues), the leader sequence, which is about 60 residues, and typically 2-6 tandem repeats of the mature pheromone sequence.
- signal peptide sequence and full-length ⁇ -factor leader peptide sequence can be used, more preferably for this invention a truncated ⁇ -factor leader peptide sequence will be used with the signal peptide when both elements are present in the hybrid precursor molecule.
- truncated ⁇ -factor leader peptide sequence is intended a portion of the full-length ⁇ -factor leader peptide sequence that is about 20 to about 60 amino acid residues, preferably about 25 to about 50 residues, more preferably about 30 to about 40 residues in length.
- Methods for using truncated ⁇ -factor leader sequences to direct secretion of heterologous proteins in yeast are known in the art. See particularly U.S. Patent No. 5,602,034.
- the hybrid precursor polypeptide sequence comprises a truncated ⁇ -factor leader peptide
- deletions to the full-length leader will preferably be from the C-terminal end and will be done in such a way as to retain at least one glycosylation site (-Asn-Y-Thr/Ser-, where Y is any amino acid residue) in the truncated peptide sequence.
- This glycosylation site whose modification is within skill in the art, is retained to facilitate secretion (see particularly WO 89/02463).
- the hybrid precursor polypeptide sequence of the present invention comprises a leader peptide sequence, such as the ⁇ -factor leader sequence, there will be a processing site immediately adjacent to the 3 ' end of the leader peptide sequence.
- This processing site enables a proteolytic enzyme native to the yeast host cell to cleave the yeast secretion leader peptide sequence from the 5' end of the native N- terminal propeptide sequence of the mature heterologous protein of interest, when present, or from the 5' end of the peptide sequence for the mature heterologous protein of interest.
- the processing site can comprise any peptide sequence that is recognized in vivo by a yeast proteolytic enzyme such that the mature heterologous protein of interest can be processed correctly.
- the peptide sequence for this processing site may be a naturally occurring peptide sequence for the native processing site of the leader peptide sequence. More preferably, the naturally occurring processing site will be modified, or the processing site will be synthetically derived, so as to be a preferred processing site as described above.
- the nucleotide sequence encoding the hybrid precursor polypeptide comprises a native propeptide sequence (PRO MHP ) for the mature heterologous protein of interest.
- native propeptide sequence or “native prosequence” is intended that portion of an intermediate precursor polypeptide (which is called a "pro-protein") for a mature secreted protein that remains attached to the N- terminal and/or C-terminal end of the mature protein sequence following cleavage of the native signal peptide sequence (or presequence) from the initial precursor polypeptide (or "prepro-protein”). The residues of the propeptide sequence are not contained in the mature secreted protein.
- the present invention provides for the presence of propeptide sequences that naturally occur at the N-terminal and/or C-terminal end of the native pro-protein precursor form of the mature heterologous protein of interest.
- a propeptide sequence may be positioned between the 3' end of the signal peptide sequence processing site, or the 3 ' end of the yeast-recognized processing site adjacent to the leader peptide sequence if present, and the 5' end of the peptide sequence for the mature heterologous protein of interest (an N-terminal propeptide sequence, PRO MHP ) or immediately adjacent to the 3' end of the peptide sequence for the mature heterologous protein of interest (a C-terminal propeptide sequence, CPRO MHP ), depending on its orientation within the native pro-protein.
- the invention also provides for inclusion of both an N-terminal and a C-terminal propeptide sequence flanking the peptide sequence for the mature heterologous protein of interest when both propeptide sequences exist in the native pro-protein.
- both an N-terminal and a C-terminal propeptide sequence exists in the native pro-protein, preference for inclusion of both propeptide sequences in the hybrid precursor polypeptide will be experimentally determined.
- Methods are available in the art for determining the naturally occurring processing sites for the native signal peptide and propeptide sequences of a prepro- protein (see, for example, von Heijne (1983) Eur. J. Biochem. 133:17-21, (1984) J. Mol. Biol. 173: 243-251, (1986) J. Mol. Biol. 184:99-105, and (1986) Nucleic Acids 1 t Res. 14:4683-4690) such that the native N-terminal and/or C-terminal propeptide sequence can be determined for use in the invention.
- a processing site that is recognized in vivo by a yeast proteolytic enzyme.
- This processing site allows for cleavage of the propeptide sequence from the peptide sequence for the mature heterologous protein of interest (MHP). It is recognized that this processing site may be the naturally occurring processing site for the propeptide sequence if the naturally occurring site is recognized in vivo by a proteolytic enzyme of the yeast host cell. More preferably, the naturally occurring processing site will be modified, or the processing site will be synthetically derived, so as to be a preferred processing site.
- yeast signal peptide and secretion leader peptide sequences represent those parts of the hybrid precursor polypeptide of the invention that can direct the sequence for the mature heterologous protein of interest through the secretory pathway of a yeast host cell.
- the nucleotide sequence of the hybrid precursor polypeptide comprises in the 5' to 3' direction:
- AFSP comprises an ⁇ -factor signal peptide sequence and a processing site
- tAFLP comprises a truncated ⁇ -factor secretion leader peptide sequence
- PS L comprises a preferred processing site for the leader peptide sequence
- NPRO PDGF comprises the peptide sequence for a native N-terminal propeptide of a mature platelet-derived growth factor (PDGF);
- PS NPRO comprises a preferred processing site for the N-terminal propeptide sequence
- M PDGF comprises the sequence for said mature PDGF.
- the ⁇ -factor signal peptide and truncated ⁇ -factor secretion leader peptide sequences are derived from the Mat ⁇ gene of S. cerevisiae as outlined in the examples.
- the preferred truncated ⁇ -factor leader peptide sequence will include the
- N-terminal portion of the full-length leader sequence that is, the leader sequence will start with the first amino acid residue of the full-length sequence and run the length of about 20 to about 60 amino acid residues, preferably about 25 to about 50 residues, more preferably about 30 to about 40 residues. In one embodiment, a leader of about 35 residues is used.
- the mature protein of this preferred embodiment is human platelet-derived growth factor (PDGF).
- PDGF the primary mitogen in serum for mesenchymal- derived cells, is stored in platelet alpha-granules. Injury to blood vessels activates the release of PDGF from these granules in the vicinity of the injured vessels. This mitogen acts as a potent chemoattractant for fibroblasts and smooth muscle cells, as well as monocytes and neutrophils. The mitogenic activity of the localized PDGF results in proliferation of these cells at the site of injury, contributing to the process of wound repair.
- PDGF platelet-derived growth factor
- Purified native platelet-derived growth factor a glycoprotein of about 30,000 daltons, is composed of two disulfide-linked polypeptide chains. Two forms of these chains, designated A and B, have been identified.
- the native protein occurs as the homodimer AA or BB or the heterodimer AB, or a mixture thereof.
- a partial amino acid sequence for the PDGF-A chain has been identified (Johnsson et #/.(1984) EMBOJ. 3:921-928) and cDNAs encoding two forms of PDGF A-chain precursors have been described (U.S. Patent No. 5,219,759).
- the A-chain is derived by proteolytic processing of a 211 amino acid precursor polypeptide.
- the cDNA encoding the PDGF-B chain has also been described (Nature (1985) 316:748-750).
- the B-chain is derived by proteolytic processing of a 241 amino acid precursor.
- the mature PDGF protein of the present invention will be the biologically active dimeric form, including the homodimers PDGF-AA and PDGF-BB or the heterodimer PDGF-AB, and any substantially homologous and functionally equivalent variants thereof as defined above.
- the native amino acid sequence for the A-chain or the B-chain may be truncated at either the N-terminal or C-terminal end.
- removal of up to 15 or up to 10 amino acids from the N- terminal or C-terminal end, respectively, of the B-chain does not affect biological activity of the variant.
- amino-acid substitutions may be made.
- an amino acid such as serine may be substituted for any of the cysteine residues at positions 43, 52, 53, and 97 of the native human B-chain and at corresponding positions in the native A-chain to obtain substantially homologous and functionally equivalent variants of the native chain.
- Variants of the A-chain are known based on cloned DNA sequences, such as, for example, variants having an additional 6 or 19 amino acids at the C-terminal end. See, for example, Tong et al. (1987) Nature 328:619-621; Betsholtz et al. (1986) Nature 320:695-699.
- One PDGF B-chain variant may be the corresponding substantially homologous portion of the amino-acid sequence encoded by the v-sis gene of simian sarcoma virus.
- the homologous region of the product of this gene, p28 s ⁇ s begins at amino acid 67 and continues to amino acid 175, and differs from the human B-chain by only 4 amino acid residues (see, for example, European Patent Application No. 0 487 116 Al).
- Functionally equivalent variants can be determined with assays for biological activity as described in the examples.
- the nucleotide sequence encoding the mature PDGF protein of the present invention may be genomic, cDNA, or synthetic DNA.
- the genes encoding the native forms of PDGF have been sequenced, and several variants are well known in the art. Expression of PDGF homodimers and heterodimers is described in, for example, U.S. Patent Nos. 4,766,073; 4,769,328; 4,801,542; 4,845,075; 4,849,407; 5,045,633;
- nucleotide sequences encoding PDGF A-chain and B-chain polypeptides may be made in vitro using methods available in the art. See particularly Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York). Where the mature protein of interest is the heterodimer PDGF-AB, the nucleotide sequences encoding the hybrid precursor polypeptides comprising the A- and B-chain polypeptides may be assembled as part of one expression cassette or assembled into separate expression cassettes for cotransformation of a yeast host cell.
- the C-terminal end of the truncated ⁇ -factor secretion leader peptide sequence and of the native N-terminal propeptide sequence will terminate in a preferred processing site, preferably a dibasic processing site that is specific for the KEX2 endopeptidase of S. cerevisiae.
- the dipeptides can be any combination of the basic residues Lys and Arg, more preferably a Lys-Arg dipeptide.
- the native prepro-PDGF-B additionally comprises a 51 amino acid C-terminal propeptide.
- the nucleotide sequence encoding the hybrid precursor polypeptide comprises in the 5 ' to 3 ' direction the following modified sequence:
- CPRO PDGF comprises a C-terminal propeptide sequence for said PDGF mature heterologous protein of interest; and PS CPRO comprises a preferred processing site for the C-terminal propeptide sequence.
- the preferred processing site for the C-terminal propeptide sequence is similar to that of the leader peptide sequence and the N-terminal propeptide sequence, such that the same yeast proteolytic enzyme brings about cleavage of the ⁇ -factor leader peptide sequence and the sequences for both of the native propeptides.
- nucleotide sequence of the hybrid precursor polypeptide comprises in the 5' to 3 ' direction:
- AFSP comprises an ⁇ -factor signal peptide sequence and a processing site; AFLP comprises an ⁇ -factor secretion leader peptide sequence
- PS L comprises a preferred processing site for the leader peptide sequence
- MtGF comprises the peptide sequence for a mature insulin-like growth factor
- PS CPRO comprises a preferred processing site for the C-terminal propeptide sequence
- CPRO IG F comprises the peptide sequence for a native C-terminal propeptide of said mature IGF.
- the ⁇ -factor signal peptide and ⁇ -factor secretion leader peptide sequences are derived from the Mat ⁇ gene of S. cerevisiae as outlined for the preferred embodiment for PDGF.
- the mature protein of this preferred embodiment is insulin-like growth factor (IGF), more particularly IGF-I.
- IGF-I insulin-like growth factor
- IGF-I belongs to a family of polypeptides known as somatomedins. IGF-I stimulates growth and division of a variety of cell types, particularly during development. See, for example, European Patent Application Nos. 560,723 A and 436,469 B. Thus, processes such as skeletal growth and cell replication are affected by IGF-I levels.
- IGF-I is structurally and functionally similar to, but antigenically distinct from, insulin.
- IGF-I is a single-chain polypeptide with three intrachain disulfide bridges and four domains known as the A, B, C, and D domains, respectively.
- the A and B domains are connected by the C domain, and are homologous to the corresponding domains of proinsulin.
- the D domain, a C-terminal prosequence is present in IGF-I but is absent from proinsulin.
- IGF-I has 70 amino acid residues and a molecular mass of approximately 7.5 kDa. See Rinderknecht (1978) J Biol. Chem..253:2769 and FEBS Lett. 89:283. For a review of IGF, see Humbel (1990) Eur. J. Biochem. 190:445-462.
- the mature IGF protein of the present invention will be the biologically active form and any substantially homologous and functionally equivalent variants thereof as defined above.
- Functionally equivalent variants can be determined with assays for biological activity, including the assay, as described in the examples.
- Representative assays include known radioreceptor assays using placental membranes (see, for example, U.S. Patent No. 5,324,639; Hall et al. (1974) J. Clin. Endocrinol. and Metab. 39:973-976; and Marshall et al. (1974)7. Clin. Endocrinol andMetab.
- fragment of IGF-I will generally include at least about 10 contiguous amino acid residues of the full-length molecule, preferably about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably about 20-50 or more contiguous amino acid residues of full-length IGF-I.
- the term "IGF-I analog” also captures peptides having one or more peptide mimics ("peptoids"), such as those described in International Publication No. WO 91/04282.
- eptoids peptides having one or more peptide mimics
- IGF-I analogs and fragments are known in the art and include those described in, for example, Proc. Natl. Acad. Sci. USA (1986) 83:4904-4907; Biochem. Biophys. Res.
- Representative analogs include one with a deletion of Glu-3 of the mature molecule, analogs with up to five amino acids truncated from the N-terminus, an analog with a truncation of the first three N-terminal amino acids and an analog including the first
- the nucleotide sequence encoding the mature IGF protein of the present invention may be genomic, cDNA, or synthetic DNA.
- the genes encoding the native forms of IGF have been sequenced, and several variants are well known in the art. .
- IGF-I and variants thereof can be produced in any number of wys that are well known in the art.
- the IGF-I polypeptides can be isolated directly from blood, such as from serum or plasma, by known methods. See, for example, U.S. Patent No. 4,769,361; Svoboda et al. (1980) Biochemistry 19:790-797; Georgia and Boughdady (1982) Prep. Biochem. 12:57 and (1984) Prep. Biochem. 14:123; herein inco ⁇ orated by reference.
- IGF-I can be synthesized chemically, by any of several techniques that are known to those skilled in the art.
- the IGF-I polypeptides of the present invention can also be chemically prepared by the method of simultaneous multiple peptide synthesis. See, for example, Houghten (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135; U.S. Patent No. 4,631,211; herein inco ⁇ orated by reference.
- the C-terminal end of the truncated ⁇ -factor secretion leader peptide sequence and the N-terminal end of the native C-terminal propeptide sequence will terminate in a preferred processing site, preferably a dibasic processing site that is specific for the KEX2 endopeptidase of 5. cerevisiae.
- the dipeptides can be any combination of the basic residues Lys and Arg, more preferably a Lys-Arg dipeptide.
- the nucleotide sequences of the present invention are useful for producing biologically active mature heterologous proteins of interest in a yeast host cell when operably linked to a yeast promoter.
- nucleotide sequences encoding the hybrid precursor polypeptides of the invention are provided in expression cassettes for introduction into a yeast host cell. These expression cassettes will comprise a transcriptional initiation region linked to the nucleotide sequence encoding the hybrid precursor polypeptide. Such an expression cassette is provided with a plurality of restriction sites for insertion of the nucleotide sequence to be under the transcriptional regulation of the regulatory regions.
- the expression cassette may additionally contain selectable marker genes.
- Such an expression cassette comprises in the 5 ' to 3 ' direction and operably linked a yeast-recognized transcription and translation initiation region, a nucleotide coding sequence for the hybrid precursor polypeptide comprising the sequence for the mature protein of interest, and a yeast-recognized transcription and translation termination region.
- operably linked is intended expression of the coding sequence for the hybrid precursor polypeptide is under the regulatory control of the yeast-recognized transcription and translation initiation and termination regions.
- yeast-recognized transcription and translation initiation and termination regions is intended regulatory regions that flank a coding sequence, in this case the nucleotide sequence encoding the hybrid polypeptide sequence, and control transcription and translation of the coding sequence in a yeast. These regulatory regions must be functional in the yeast host.
- the transcription initiation region the yeast promoter, provides a binding site for RNA polymerase to initiate downstream (3 ') translation of the coding sequence.
- the promoter may be a constitutive or inducible promoter, and may be native or analogous or foreign or heterologous to the specific yeast host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By foreign is intended that the transcription initiation region is not found in the native yeast of interest into which the transcription initiation region is introduced.
- Suitable native yeast promoters include, but are not limited to the wild-type ⁇ - factor promoter, as well as other yeast promoters.
- the promoter is selected from the list including promoters for the glycolytic enzymes phosphoglucoisomerase, phosphofructokinase, phosphotrioseisomerase, phosphoglucomutase, enolase, pyruvate kinase (PyK), glyceraldehyde-3 -phosphate dehydrogenase (GAP or
- GAPDH GAPDH
- alcohol dehydrogenase ADH
- EPO Publication No. 284,044 See, for example, EPO Publication Nos. 120,551 and 164,556.
- Synthetic hybrid promoters consisting of the upstream activator sequence of one yeast promoter, which allows for inducible expression, and the transcription activation region of another yeast promoter also serve as functional promoters in a yeast host.
- hybrid promoters include ADH/GAP, where the inducible region of the ADH promoter is combined with the activation region of the GAP promoter (U.S. Patent Nos. 4,876,197 and 4,880,734).
- Other hybrid promoters using upstream activator sequences of either the ADH2, GAL4, GAL 10, or PHO5 genes combined with the transcriptional activation region of a glycolytic enzyme such as
- yeast promoter is the inducible ADH/GAP hybrid promoter.
- Yeast-recognized promoters also include naturally occurring non-yeast promoters that bind yeast RNA polymerase and initiate translation of the coding sequence. Such promoters are available in the art . See, for example, Cohen et al.
- the termination regulatory region of the expression cassette may be native with the transcription initiation region, or may be derived from another source, providing that it is recognized by the yeast host.
- the termination regions may be those of the native ⁇ -factor transcription termination sequence, or another yeast- recognized termination sequence, such as those for the glycolytic enzymes mentioned above. More preferably the transcription terminator is the ⁇ t- ( ⁇ -factor) transcription terminator described in U.S. Patent No. 4,870,008.
- the nucleotide sequences encoding the hybrid precursor polypeptides of the invention are provided in expression cassettes for expression in a yeast host.
- the cassette will include 5' and 3' regulatory sequences operably linked to the nucleotide sequence encoding the hybrid precursor polypeptide of interest.
- the cassette may also contain at least one additional nucleotide sequence of interest to be cotransformed into the yeast host. Alternatively, the additional nucleotide sequences can be provided on another expression cassette.
- the nucleotide sequence encoding the hybrid precursor polypeptide and any additional nucleotide sequences of interest may be optimized for increased expression in the transformed yeast. That is, these nucleotide sequences can be synthesized using yeast-preferred codons for improved expression. Methods are available in the art for synthesizing yeast-preferred nucleotide sequences of interest (see, for example, U.S. Patent Nos.
- nucleotide coding sequences are known to enhance expression of nucleotide coding sequences in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression.
- the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell.
- the nucleotide coding sequence is modified to avoid predicted hai ⁇ in secondary mRNA structures.
- the various nucleotide sequence fragments may be manipulated, so as to provide for the sequences in the proper orientation and, as appropriate, in the proper reading frame.
- adapters or linkers may be employed to join the nucleotide fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous nucleotides, removal of restriction sites, or the like.
- in vitro mutagenesis primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. See particularly Sambrook etal.
- the expression cassettes of the present invention can be ligated into a replicon (e.g., plasmid, cosmid, virus, mini-chromosome), thus forming an expression vector that is capable of autonomous DNA replication in vivo.
- a replicon e.g., plasmid, cosmid, virus, mini-chromosome
- the replicon will be a plasmid.
- Such a plasmid expression vector will be maintained in one or more replication systems, preferably two replications systems, that allow for stable maintenance within a yeast host cell for expression pmposes, and within a prokaryotic host for cloning pu ⁇ oses.
- yeast-bacteria shuttle vectors include Yep24 (Botstein et al. (1979) Gene 8:17-24; pCl/1 (Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81:4642-4646), and Y ⁇ l7 (Stnichomb
- a plasmid expression vector may be a high or low copy number plasmid, the copy number generally ranging from about 1 to about 200.
- high copy number yeast vectors there will generally be at least 10, preferably at least 20, and usually not exceeding about 150 copies in a single host.
- either a high or low copy number vector may be desirable, depending upon the effect of the vector and the foreign protein on the host. See, for example, Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81 :4642-4646.
- DNA constructs of the present invention can also be integrated into the yeast genome by an integrating vector. Examples of such vectors are known in the art. See, for example,
- the host chosen for expression of the heterologous proteins of the invention will preferably be a yeast.
- yeast is intended ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeast belonging to the Fungi Imperfecti (Blastomycetes).
- the ascosporogenous yeasts are divided into two families, Spermophthoraceae and Saccharomycetaceae.
- the later is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces, and Saccharomyces).
- Schizosaccharomycoideae e.g., genus Schizosaccharomyces
- Nadsonioideae e.g., genus Schizosaccharomyces
- Lipomycoideae e.g., Lipomycoideae
- Saccharomycoideae e.g., genera Pichia, Kluyveromyces, and Saccharomyces.
- the basidiosporogenous yeasts include the genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, and
- Filobasidiella Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sporobolomyces, Bullera) and Cryptococcaceae (e.g., genus Candida).
- Sporobolomycetaceae e.g., genera Sporobolomyces, Bullera
- Cryptococcaceae e.g., genus Candida
- species within the genera Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces, and Candida Of particular interest are the Saccharomyces species S. cerevisiae, S. carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis, and S. oviformis.
- yeast shall be defined as described in Skinner et al., eds. 1980) Biology and Activities of Yeast (Soc. App. Bacteriol. Symp. Series No. 9).
- those of ordinary skill in the art are presumably familiar with the biology of yeast and the manipulation of yeast genetics. See, for example, Bacila et al., eds. (1978) Biochemistry and Genetics of Yeast; Rose and Harrison, eds. (1987) The Yeasts (2 nd ed.); Strathern et al., eds. (1981) The Molecular Biology of the Yeast Saccharomyces; herein inco ⁇ orated by reference.
- suitable yeast and other microorganism hosts for the practice of the present invention is within the skill of the art.
- suitable hosts may include those shown to have, inter alia, good secretion capacity, low proteolytic activity, and overall vigor.
- Yeast and other microorganisms are generally available from a variety of sources, including the Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, University of California, Berkeley, California; and the American Type Culture Collection, Rockville, Maryland.
- yeast hosts Methods of introducing exogenous DNA into yeast hosts are well known in the art.
- spheroplast transformation is taught by Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1919- 1933 and Stinchcomb et al, EPO Publication No. 45,573; herein inco ⁇ orated by reference.
- Transformants are grown in an appropriate nutrient medium, and, where appropriate, maintained under selective pressure to insure retention of endogenous DNA. Where expression is inducible, growth can be permitted of the yeast host to yield a high density of cells, and then expression is induced.
- the secreted, mature heterologous protein can be harvested by any conventional means, and purified by chromatography, electrophoresis, dialysis, solvent-solvent extraction, and the like.
- Additional examples describe an expression vector comprising the nucleotide sequence encoding mature human IGF-I in accordance with the disclosed invention and demonstrate the use of this expression vector to produce biologically active mature IGF-I in a yeast host.
- the vector selected for expressing rhPDGF-BB, pAB24 is a yeast-bacteria shuttle vector.
- the plasmid is a chimera of sequences from pBR322, derived from several naturally occurring bacterial plasmids, and sequences of the endogenous S. cerevisiae 2- ⁇ plasmid (Broach (1981) in Molecular Biology of the Yeast Saccharomyces (Cold Spring Harbor Press, New York), 1 :445-470). It also encodes genes enabling selection in both E. coli and S. cerevisiae hosts.
- the pBR322 part of pAB24 includes the ampicillin resistance (Ap r )-conferring gene encoding ⁇ - lactamase, as well as a gene conferring tetracycline resistance (Tc r ). These genes allow transformation of competent E. coli and selection of plasmid-containing bacteria. A unique BamHI cloning site, present in the gene encoding tetracycline resistance, is the site utilized for insertion of an expression cassette.
- the pBR322 portion of the vector also includes a Col ⁇ l-like replication origin enabling replication in E. coli.
- Two S. cerevisiae genes derived from Y ⁇ p24 Botstein et al. (1979) Gene
- URA3 and leu2d enable selection in yeast host strains lacking either or both of these genes.
- the latter gene, leu2d lacks a portion of the 5 '-untranslated promoter region and requires high plasmid copy number for growth in leucine-deficient medium. This is necessary to achieve sufficient LEU2 protein expression for complementation of yeast strains lacking LEU2 (Erhart and Hollenberg (1983) J.
- Plasmid pYAGL7PB includes an expression cassette with the following features. Transcription is mediated by the inducible, hybrid yeast promoter ADH/GAP.
- This promoter includes ADR2 transcription factor responsive sequences from the S. cerevisiae ADH2 gene (Beier and Young (1982) Nature 300:724-728) and promoter sequences from the S. cerevisiae gene TDH3, encoding the glycolytic enzyme glyceraldehyde-3 -phosphate dehydrogenase (GAP).
- GAP glycolytic enzyme glyceraldehyde-3 -phosphate dehydrogenase
- the ADR2 transcription factor responsive sequences confer inducible gene transcription upon downstream sequences. Induction is achieved by glucose depletion in the growth medium.
- Termination of transcription is mediated by the terminator derived from the S. cerevisiae mating factor type alpha (Mata) gene (Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81:4642-4646).
- the cassette further includes an open reading frame encoding a truncated Mata sequence fused to a sequence encoding the human PDGF-B gene.
- the truncated ⁇ -factor leader mediates secretion of in-frame protein fusions. It is a derivative of S. cerevisiae ⁇ -factor leader, the product of the Mata gene (Kurjan and Herskowitz (1982) Cell 30:933-943).
- a dibasic amino acid processing site is present at the truncated ⁇ -factor leader/PDGF-B junction to facilitate production of correctly processed rhPDGF-BB polypeptide by yeast.
- Figure 2 shows a map of the pYAGL7PB expression cassette highlighting these features and the restriction enzyme sites relevant to the construction of this expression cassette.
- the nucleotide sequence and predicted amino acid sequence of the open reading frame encoding the truncated ⁇ -factor leader-PDGF-B primary translation product are given in SEQ ID NO: 1 and SEQ ID NO: 2, respectively
- Plasmid pPAG/ ⁇ F is a pBR322 derivative with an expression cassette delineated by BamHI sites.
- the expression cassette includes the ADH/GAP hybrid promoter, as well as the open reading frame encoding the yeast ⁇ -factor leader (BamHl-Xbal), an Xbal-Sall gene fragment, and the Mata ( ⁇ -factor) transcription terminator (Sall-BamHI). Substitution of an Xbal-Sall gene fragment (in-frame) capable of heterologous protein expression into this plasmid allows the expression and secretion of the heterologous protein.
- yeast glyceraldehyde-3- phosphate (GAP) gene promoter The isolation of the yeast glyceraldehyde-3- phosphate (GAP) gene promoter, the origin of the ADH2 component of the promoter, and the construction of a hybrid ADH/GAP promoter are described in U.S. Patent Nos. 4,876,197 and 4,880,734. The isolation of the yeast ⁇ -factor gene including the transcription terminator is described in U.S. Patent No. 4,870,008. Upon dideoxy sequencing, the synthetic gene sequence was found to have a single base pair mutation, which was repaired by standard procedures. Plasmid pPAGBB-1 is the plasmid derived from pPAG/ ⁇ F that contains the correct synthetic PDGF-B (Xbal-Sall) gene.
- the truncated ⁇ -factor leader mediates secretion of in-frame hybrid polypeptides. It is a derivative of S. cerevisiae ⁇ -factor leader, the secretion leader for mating factor type alpha, the product of the Mata gene (Kurjan and Herskowitz (1982) Cell 30:933-943), and consists of the first 35 amino acids of the native leader.
- the construction and use of a truncated ⁇ -factor leader gene to mediate secretion is described in EPO Publication No. 0324 274 B 1.
- Synthetic oligonucleotides encoding a comparable, partial (amino acids 8-35) truncated ⁇ -factor leader (L7) and part of the dibasic processing site were made from oligonucleotides given in SEQ. ID NO: 22 and and when assembled with the complementary strand shown in SEQ ID NO: 23 resulted in a Pstl-Bglll fragment with a 3 ' -ACGTC- and a 5 ' -CTAG- overhang to allow for convenient ligation into the expression cassette.
- pu ⁇ ose of this construction was the substitution of the synthetic, partial truncated ⁇ -factor leader Pstl-Bglll gene fragment described above for most of the full-length ⁇ -factor leader in the PDGF-B expression cassette of pPAGBB-1.
- 1.9 kb Pst I fragment including pBR322 sequences, the ADH/GAP hybrid promoter (marked at the 5' end by a BamHI site) and the 5' partial ⁇ -factor leader gene sequence (encoding the first seven amino acids of the native ⁇ -factor leader) was isolated from pPAGBB-1. It was ligated to kinased, annealed synthetic oligonucleotides 1.49/3°.40. Following digestion with BamHI, a partial expression cassette 5' fragment was obtained including sequences for the ADH/GAP hybrid promoter and the 5' portion of the truncated ⁇ -factor leader.
- a BgHI fragment containing the PDGF-B synthetic gene, the ⁇ - factor terminator (marked at the 3' end by a BamHI site) and pBR322 sequences was isolated from pPAGBB-1. It was ligated to kinased, annealed synthetic oligonucleotides 2.32/4°.50. Following digestion with BamHI, a partial expression cassette 3' fragment was obtained including sequences for the 3' portion of the truncated ⁇ -factor leader, PDGF-B, and the ⁇ -factor leader transcription terminator. The complete PDGF-B expression cassette was obtained following ligation of the 5'and 3' partial expression cassette gene fragments and digestion with BamHI. The
- BamHI expression cassette was cloned into the BamHI site of a pBR322-derived vector (pBR ⁇ Eco-Sal) to give plasmid pAGL7PB.
- pBR ⁇ Eco-Sal pBR ⁇ Eco-Sal
- the PDGF-B expression cassette of pAGL7PB was isolated by BamHI digestion and inserted into the BamHI site of the yeast-bacteria shuttle vector pAB24 described above.
- a yeast expression plasmid, pYAGL7PB was isolated.
- a plasmid map of pYAGL7PB is shown in Figure 3.
- the nucleotide sequence of the complete expression cassette and the predicted amino acid sequence of the open reading frame (ORF) encoding the truncated ⁇ -factor leader-PDGF-B primary translation product are given in SEQ ID NO: 24 and SEQ ID NO: 25, respectively.
- Expression plasmid pYAGL7PB was transformed into S. cerevisiae MB2-1 by standard procedures and prototrophic uracil colonies were selected. Individual colonies from independent transformants were screened for expression following inoculation of single colonies into medium that selects for leucine prototrophs. The medium also is high in glucose to keep expression of sequences under ADR2 regulation (including the PDGF-B gene) repressed. Cultures were subsequently diluted and grown to confluence in low glucose medium lacking uracil. Cell-free culture supernatants were prepared and assayed for PDGF-BB by immunoactivity (ELISA) and by mitogenic activity on 3T3 cells. A high PDGF-BB expressing colony,
- MB2-l(pYAGL7PB #5) was identified.
- Plasmid pYL7PPB (also known as pYAGL7PPB) includes an expression cassette with the following features. Transcription initiation and termination is mediated by the inducible, hybrid yeast promoter ADH/GAP and the Mata transcriptional terminator described above. The gene further includes an open reading frame encoding a truncated yeast ⁇ -factor leader to mediate secretion of rhPDGF-BB.
- the propeptide sequence included in the expression construct is only the native N- terminal propeptide sequence; the native C-terminal propeptide sequence was not included in the construct. Inclusion of the N-terminal propeptide sequence resulted in enhanced expression of rhPDGF-BB, presumably because of improved folding.
- FIG. 4 shows a map of the pYL7PPB expression cassette highlighting these features and the sites relevant to the construction of this expression cassette.
- the nucleotide sequence and predicted amino acid sequence of the open reading frame encoding the truncated ⁇ -factor leader- proPDGF-B primary translation product are shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively.
- Plasmid pSV7d-PDGF A103-B1 was the source of the N-terminal propeptide- PDGF-B cDNA.
- the plasmid was constructed as described below.
- the 3 kb Eco Rl PDGF-B cDNA insert from clone ⁇ hPDGFb-17 was excised and cloned into the unique Eco RI site of the mammalian expression vector pSV7d to give plasmid phPDGF ⁇ -1 (also known as pSV7d-PDGF-Bl).
- Plasmid phPDGF ⁇ -1 was digested with Pstl under conditions favoring cleavage at one of the two plasmid Pstl sites (desired single cleavage at site in ampicillin resistance gene of the pSV7d vector backbone) and ligated with Pstl -digested pSV7d- PDGF-A103(D1).
- This latter plasmid is strictly analogous to the PDGF-B mammalian expression plasmid phPDGF ⁇ -1, except that it includes cDNA encoding the long,
- This plasmid contains a single Pstl site in the ampicillin resistance gene of the pSV7d vector backbone.
- the PDGF-B cDNA was mutagenized: (1) to introduce a Sad site enabling introduction of the truncated ⁇ -factor secretion leader, and (2) to change the hPDGF- B cDNA sequence encoding dibasic amino acids Arg- Arg to encode Lys-Arg. This dibasic combination is more efficiently cleaved than Arg-Arg by the yeast dibasic processing enzyme KEX2 endopeptidase.
- the template for mutagenesis was prepared as follows.
- the ⁇ 3kb EcoRI hPDGF-B cDNA was isolated from pSV7d-PDGF A103-B1 and inserted into the EcoRI site of pBR322 to give plasmid pPPB/6.
- the nucleotide sequence of the 2.7 kb Pstl-EcoRI cDNA fragment was verified.
- the 0.9 kb Pstl-Ncol cDNA fragment was inserted into the Pstl-Ncol sites of Ml 3 and the nucleotide sequence of the insert verified.
- a partial nucleotide sequence and the predicted amino acid sequence of the PDGF-B cDNA are given in SEQ ID NO: 28 and SEQ ID NO: 29, respectively.
- a double mutagenesis of Ml 3 Pstl-Ncol PDGF-B cDNA fragment was performed by standard methods using the following primers.
- Primer 1 (SEQ ID NO: 30) introduces a Sad site;
- Sall portion of the PDGF-B expression cassette in pAGL7PB encoding the C-terminal portion of the truncated ⁇ -factor leader, the Lys-Arg dibasic processing site and PDGF-B ( Figure 2) with an Xhol-Sall gene fragment encoding the C-terminal portion of the truncated ⁇ -factor leader, a Lys-Arg dibasic processing site, the PDGF-B N- terminal propeptide, a Lys-Arg dibasic processing site, and PDGF-B.
- the sequences encoding the N-terminal PDGF-B propeptide and PDGF-B were derived from cDNA as described above. A map of the resulting expression cassette is shown in Figure 4.
- a 447 bp Sacl-Sphl fragment including most of the proPDGF-B gene was isolated from the Ml 3 RF containing the modified preproPDGF-B cDNA.
- Synthetic oligonucleotides including sequences encoding the C-terminal part of truncated ⁇ factor leader, a Lys-Arg dibasic processing site, and the N-terminal portion of the PDGF-B propeptide (SEQ ID NOs: 32-33), were joined to give a fragment with a 3' Sad overhang.
- Synthetic oligonucleotides Sph-Sal I/Sph-Sal II, including sequences encoding the last 14 amino acids of PDGF-B and stop codons were joined to give a Sphl-Sall fragment (SEQ ID NOs: 34-35). These two sets of annealed oligonucleotides were ligated to the 447 bp Sacl-Sphl proPDGF gene fragment. This resulted in a gene fragment including sequences encoding the C-terminal part of truncated ⁇ -factor leader, a Lys-Arg dibasic processing site and proPDGF-B.
- Synthetic oligonucleotides including sequences encoding the middle amino acids of the truncated ⁇ -factor leader were joined resulting in a fragment with a
- the final step in the construction of the PDGF-B expression cassette was the ligation of the gene fragment into the vector/gene fragment to give plasmid pL7PPB (pAGL7PPB), as shown in Figure 5.
- the Pstl-BamHl insert fragment was isolated and nucleotide sequencing confirmed that the desired construction had been obtained.
- a map of the PDGF-B expression cassette in pL7PPB is shown in Figure 4.
- pYL7PPB (pYAGL7PPB) ' D
- the PDGF-B expression cassette of pL7PPB was isolated following BamHI digestion and inserted into the BamHI site of the yeast shuttle vector pAB24, described above, resulting in yeast expression plasmid pYL7PPB.
- a map of pYL7PPB is shown in Figure 6.
- the nucleotide sequence of the complete expression cassette and the predicted amino acid sequence of the open reading frame (ORF) encoding truncated ⁇ -factor leader-Lys-Arg-proPDGF-B are given in SEQ ID NO: 36 and SEQ ID NO: 37, respectively.
- the complete nucleotide sequence of yeast expression plasmid pYL7PPB has been determined.
- Expression plasmid pYL7PPB was transformed into S. cerevisiae MB2-1 by standard procedures and plasmid-harboring, uracil prototrophs were selected as isolated colonies. Individual colonies from independent transformants were screened for expression following inoculation of isolated colonies into growth medium that selects for leucine prototrophs. The medium also is high in glucose to keep expression of sequences under ADR2 regulation (including the PDGF-B gene) repressed. Cultures were subsequently diluted and grown to confluence in low glucose, selective growth medium lacking uracil. Cell-free supernatants were assayed for PDGF-BB by immunoactivity (ELISA) and by mitogenic activity on 3T3 cells. Frozen stocks were prepared of several transformants exhibiting consistently high levels of expression.
- ELISA immunoactivity
- pYJST400 has a single dibasic processing site, which resides at the propeptide/PDGF-B junction. Elimination of this first processing site was done to determine its relative effect on secretion of rhPDGF-BB from yeast as mediated by the ⁇ -factor leader peptide.
- Example 5 Expression of Recombinant Human PDGF-BB Recombinant human PDGF-BB is produced by a strain of the yeast,
- Saccharomyces cerevisiae genetically modified with a multicopy yeast expression plasmid that includes a gene encoding human PDGF-B.
- the preferred S. cerevisiae strain MB2-1 has the genotype: Mata, ura3A, leu2-3, leu2-112, his3-ll, his3-15, pep4 , [cir °J. It is auxotrophic for uracil, leucine, and histidine, requiring these nutritional supplements when grown in minimal medium.
- MB2-1 does not contain an endogenous 2- ⁇ plasmid, which tends to interfere with the stability of the introduced plasmids and encourages recombination between endogenous and introduced plasmids.
- the strain does not express functional protease A, the product of the PEP 4 gene, which interferes with the production of heterologous proteins.
- MB2-1 was designed to impart these favorable characteristics, which include selection for high expression of heterologous proteins.
- Yeast expression plasmids pYAGL7PB, pYL7PPB, and pYJST400 were transformed into yeast strain MB2-1 as described by Hinnen et al (1978) Proc. Natl. Acad. Sci. USA 75:1929-1933 and plated on ura-, 8% glucose, sorbitol plates. Transformants were grown in leu-, 8% glucose liquid medium for 24 hours and then plated onto leu-, 8% glucose sorbitol plates to get individual colonies. Individual colonies were picked and grown in 3 ml of leu-, 8% glucose medium for 24 hours at 30 C, and then inoculated (1:50) into 1 liter of ura-, 1% glucose media and grown for 75 hours at 30 C. Yeast culture medium was assayed for PDGF activity by the human foreskin fibroblast mitogen assay (see Example 5 below).
- 35 PDGF-B a truncated ⁇ -factor leader consisting of the N-terminal amino acids 1-35 fused in-frame with mature human PDGF-B.
- a single processing site ( EX 2 ) separates the leader sequence from the mature PDGF-B sequence.
- DMEM Eagles Medium
- FBS fetal bovine serum
- 1 mM sodium pyruvate 300 ⁇ g/ml L-glutamine, lOOU/ml penicillin, and 100 ⁇ g/ml streptomycin.
- Cells were incubated at 37 C in humidified 7% CO 2 , 93% air atmosphere.
- PBS phosphate buffered saline
- PBS phosphate buffered saline
- HFFs were plated as follows. The cells were rinsed and dissociated with trypsin as above. The trypsinized cells were pelleted and resuspended to a concentration of 1 X 10 5 cells/ml in medium similar to growth medium, except that 5% FBS replaced 20% FBS; 100 ⁇ l of suspension was dispensed into each well of a 96-well microtiter plate; and then the cells were incubated 5-6 days under the above described conditions.
- PDGF in the sample was determined by monitoring 3 H-thymidine inco ⁇ oration into HFF DNA stimulated by PDGF. Samples were added to the wells containing HFF monolayers, and the assay plates incubated as above for 18 hours.
- the HFF cultures were then pulsed with [Methyl- 3 H]thymidine (10 ⁇ C/ml final concentration, 1 ⁇ C/well) at 37 C under the above described incubation conditions for 8 hours. After incubation, the cells were rinsed with PBS and fixed. Fixing was by incubation with 5% trichloracetic acid (TCA) and then 100% methanol for 15 minutes, followed by drying in air. The cells were then solubilized with 0.3N NaOH and then counted in a liquid scintillation counter.
- TCA trichloracetic acid
- Control samples were treated as the samples described above and were prepared as follows.
- PDGF purchased from PDGF, Inc.
- DMEM fetal calf serum
- a standard curve was prepared; the first point was 10 ng/ml, the remaining points were 2-fold serial dilutions. Each dilution was tested in triplicate.
- Example 7 Expression Plasmids pYLUI
- Plasmid pYLUIGF24 includes an expression cassette with the hybrid yeast promoter ADH/GAP and Mata factor leader sequences fused to a sequence encoding the human IGF-I-A gene. This sequence was synthetically derived using yeast preferred codons. A dibasic amino acid processing site is present at the ⁇ -factor leader/I GF-I-A junction.
- the nucleotide sequence and predicted amino acid sequence of the open reading frame encoding ⁇ -factor leader/I GF-I-A primary translation product are given in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.
- Plasmid pYLUIGF34 differs from pYLUIGF24 only in its open reading frame.
- This cassette includes an open reading frame encoding a full length Mata factor leader sequence fused to a sequence encoding the human IGF-I-A gene with its C-terminal prosequence. Dibasic amino acid processing sites are present at the ⁇ - factor leader/I GF-I-A and IGF-I-A/IGF-I-A prosequence junctions.
- the nucleotide sequence and predicted amino acid sequence of the open reading frame encoding ⁇ - factor leader-proIGF-I-A primary translation product are given in SEQ ID NO: 40 and
- Both of these plasmids were generated by inserting the respective expression cassette into the unique BamHI cloning site of the yeast shuttle vector pAB24 as described above.
- Example 8 Expression of Recombinant Human PDGF-BB
- Recombinant human IGF-I-A is produced by a strain of the yeast Saccaromyces cerevisiae, genetically modified with a multicopy yeast expression plasmid that includes a gene encoding human IGF-I-A.
- Yeast expression plasmids pYLUIGF24 and pYLUIGF34 were transformed into a yeast strain by procedures previously mentioned.
- ORGANISM Homo sapiens/Saccharomyces cerevisiae
- CTCTAGATAA AAGATCTTTG GGTTCTTTGA CTATCGCTGA ACCAGCTATG ATCGCTGAAT 60
- AAATCGTTAG AAAGAAGCCA ATCTTCAAGA AGGCTACTGT TACTTTGGAA GACCACTTGG 300
- ORGANISM Homo sapiens
- xi SEQUENCE DESCRIPTION: SEQ ID NO: 10: CAAGTTAGAA AGATCGAAAT CGTTAGAAAG AAGCCAATCT TCAAG 45
- ORGANISM Saccharomyces cerevisiae
- ORGANISM Homo sapiens b ⁇
- ORGANISM Synthetic (derived from Saccharomyces cerevisiae)
- ORGANISM Synthetic (derived from Saccharomyces cerevisiae)
- ORGANISM Homo sapiens/Saccharomyces cerevisiae
- CAACTGGCAC CATCTCTCCC GTAGTCTCAT CTAATTTTTC TTCCGGATGA GGTTCCAGAT 240
- GGAACACCGG GCATCTCCAA CTTATAAGTT GGAGAAATAA GAGAATTTCA GATTGAGAGA 900
- ORGANISM Saccharomyces cerevisiae/Homo sapiens
- ORGANISM Homo sapiens/Saccharomyces cerevisiae
- GAATTCCCAG AAAATGTTGC
- AAAAAAGCTA AGCCGGCGGG CAGAGGAAAA CGCCTGTAGC 60
- CCAGGTCCTC CCCTGCCCCC CAGCGACGGA GCCGGGGCCG GGGGCGGCGG CGCCGGGGGC 300
- GCA GCT GCA CGG* CCT GTG ACC CGA
- AGC CCG GGG GGT TCC CAG GAG CAG 1050 Ala Ala Ala Arg Pro Val Thr Arg Ser Pro Gly Gly Ser Gin Glu Gin 105 110 115
- ORGANISM Synthetic (derived from Homo sapiens sequence)
- ORGANISM Synthetic (derived from Homo sapiens sequence)
- ORGANISM Homo sapiens/Saccharomyces cerevisiae derived sequence
- ORGANISM Homo sapiens derived sequence
- ORGANISM Homo sapiens derived sequence
- ORGANISM Homo sapiens derived sequence
- ORGANISM Homo sapiens/Saccharomyces cerevisiae
- CAACTGGCAC CATCTCTCCC GTAGTCTCAT CTAATTTTTC TTCCGGATGA GGTTCCAGAT 240
- GGAACACCGG GCATCTCCAA CTTATAAGTT GGAGAAATAA GAGAATTTCA GATTGAGAGA 900
- ORGANISM Homo sapiens/Saccharomyces cerevisiae
- ORGANISM Homo sapiens/Saccharomyces cerevisiae
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP97954073A EP0946736B1 (en) | 1996-12-13 | 1997-12-12 | Method for expression of heterologous proteins in yeast |
JP52692698A JP4341859B2 (en) | 1996-12-13 | 1997-12-12 | Methods for the expression of heterologous proteins in yeast |
DE69725881T DE69725881T2 (en) | 1996-12-13 | 1997-12-12 | METHOD FOR EXPRESSING HETEROLOGICAL PROTEINS IN YEAST |
AT97954073T ATE253120T1 (en) | 1996-12-13 | 1997-12-12 | METHOD FOR EXPRESSING HETEROLOGUE PROTEINS IN YEAST |
AU57945/98A AU5794598A (en) | 1996-12-13 | 1997-12-12 | Method for expression of heterologous proteins in yeast |
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US3272096P | 1996-12-13 | 1996-12-13 | |
US60/032,720 | 1996-12-13 |
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WO1998026080A1 true WO1998026080A1 (en) | 1998-06-18 |
Family
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PCT/US1997/022815 WO1998025963A1 (en) | 1996-12-13 | 1997-12-12 | Analysis and separation of platelet-derived growth factor proteins |
PCT/US1997/022647 WO1998026080A1 (en) | 1996-12-13 | 1997-12-12 | Method for expression of heterologous proteins in yeast |
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Application Number | Title | Priority Date | Filing Date |
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PCT/US1997/022815 WO1998025963A1 (en) | 1996-12-13 | 1997-12-12 | Analysis and separation of platelet-derived growth factor proteins |
Country Status (9)
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US (10) | US6017731A (en) |
EP (4) | EP1935901A1 (en) |
JP (4) | JP4275741B2 (en) |
AT (3) | ATE253120T1 (en) |
AU (2) | AU5465498A (en) |
DE (3) | DE69738768D1 (en) |
ES (1) | ES2306462T3 (en) |
PT (1) | PT948538E (en) |
WO (2) | WO1998025963A1 (en) |
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