WO1983004030A1 - The manufacture and expression of genes for urogastrone and polypeptide analogs thereof - Google Patents

The manufacture and expression of genes for urogastrone and polypeptide analogs thereof Download PDF

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Publication number
WO1983004030A1
WO1983004030A1 PCT/US1983/000650 US8300650W WO8304030A1 WO 1983004030 A1 WO1983004030 A1 WO 1983004030A1 US 8300650 W US8300650 W US 8300650W WO 8304030 A1 WO8304030 A1 WO 8304030A1
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ctg
gac
atg
tgc
tac
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PCT/US1983/000650
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French (fr)
Inventor
Allen R. Banks
David L. Hare
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Applied Molecular Genetics, Inc.
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Publication of WO1983004030A1 publication Critical patent/WO1983004030A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C07K14/485—Epidermal growth factor [EGF] (urogastrone)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates generally to the manipulation of genetic materials and, more particularly, to the manufacture of specific DNA sequen ⁇ ces useful in recombinant procedures to secure the production of urogastrone and polypeptide analogs thereof.
  • epidermal growth factor mouse and human epidermal growth factor- urogastrone (EGF-URO) , are examples of a large class of "growth factors" and are widespread in animals and man.
  • EGF-URO like the other growth factors such as insulin, nerve growth factor, the insulin-like growth factors, and the like, is synthesized in mammals as part of a larger "pro-peptide" molecule from which it is cleaved by specific proteases to liberate the active form of the protein [Frey, et al., Pro ⁇ . Nat. Acad. Sci., 76, 6294 (1979)].
  • pro-peptide Pro-peptide
  • EGF-URO in both the mouse and in man, is composed of 53 amino acids. Further processing in the body also gives rise to a 51 amino acid-contain ⁇ ing form which lacks the two amino acid residues at the carboxyl terminus of the peptide.
  • the 53 and 51 amino acid forms of the peptide are called beta- and gamma- EGF-URO, respectively. Both forms have shown high activity as inhibitors of gastric acid secretion and as stimulators of growth of epidermoid tissue. High gastric secretion inhibitory activi ⁇ ties have also been reported for the 46 and 47 amino acid products of selective enzymatic degradation. [See, U.S. Patent Nos. 4,032,633 and 4,035,485.]
  • EGF-URO EGF-URO
  • EGF-URO is presently isolated in small quantities by purification from mouse salivary glands or by a complex purification from human urine [Hollenberg, Vitamins and Hormones, 37, 69 (1979); Gregory et al., U.S. Patent No. 3,883,497].
  • polypeptide substance is too large to be readily synthesized by the well-known Merrifield procedure.
  • Recombinant DNA techniques for the manufac ⁇ ture, cloning and expression of a structural gene for urogastrone and genes for polypeptide analogs which differ therefrom in terms of the identity and/or location of one or more amino acids have not been brought to bear on this problem.
  • the base sequence includes one or more codons selected from among alterna ⁇ tive codons specifying the same amino acid on the basis of preferential expression characteristics of the codon in a projected host microorganism, e.g., ⁇ - coli « Other preferred forms of manufactured genes - 4 -
  • a base codon specifying additional amino acid in the polypeptide synthesized which facilitates direct expression in E. coli organisms e.g., an initial Met residue
  • base codons specifying urogastrone are preceded and/or followed by a sequence of bases comprising a portion of a base sequence which provides for restriction endonuclease cleavage of a DNA sequence (e.g., a Bell or BamHI site) and consequently facilitates formation of expres- sion vectors.
  • Also provided by the present invention are: (1) a manufactured gene capable of directing the synthe ⁇ sis in a selected host microorganism of a urogastrone polypeptide analogs which differ from urogastrone in terms of the identity and/or location of one or more amino acids (e.g., [Asp 25] urogastrone and [Pro52, Pro 53] urogastrone) ?
  • a fusion gene comprising a manufactured gene accordi'ng to the invention fused to a second gene capable of directing synthesis of a second polypeptide (e.g., ⁇ -lactamase and ⁇ -galacto- sidas ⁇ ) in a manner permitting the synthesis of a fused polypeptide including urogastrone polypeptide or. a urogastrone analog.
  • a second polypeptide e.g., ⁇ -lactamase and ⁇ -galacto- sidas ⁇
  • DNA sequences including manufac ⁇ tured genes are inserted into a viral or circular plasmid DNA vector to form a hybrid vector and the hybrid vectors are employed to transform host micro ⁇ organisms such as bacteria (e.g., E. coli) or yeast cells.
  • the transformed microorganisms are thereafter grown under appropriate nutrient conditions and express the polypeptide products of the invention.
  • Novel DNA sequences of the invention are preferably synthesized from nucleotide bases according to the methods disclosed in the aforementioned co- owned, concurrently-filed U.S. Patent Application Serial No. 375,493, by Yitzhak Stabinsky, entitled “Manufacture and Expression of Structural Genes". Briefly summarized, the general method comprises the steps of:
  • each duplex strand including a double stranded region of 12 or more selected complemen ⁇ tary base pairs and further including a top single stranded terminal sequence of from 3 to 7 selected bases at one end of the strand and/or a bottom single stranded terminal sequence of from 3 to 7 selected bases at the other end of the strand, each single stranded terminal sequence of each duplex DNA strand comprising the entire base complement of at most one single stranded terminal sequence of any other duplex DNA strand prepared;
  • each duplex DNA strand prepared in step (1) to one or two different duplex strands prepared in step (1) having a complementary single stranded terminal sequence, thereby to form a single continuous double stranded DNA sequence which has a duplex region of at least 27 selected base pairs including at least three base pairs formed by complemen ⁇ tary association of single stranded terminal sequences of duplex DNA strands prepared in step (1) and which has from 0 to 2 single stranded top or bottom terminal regions of from 3 to 7 bases.
  • duplex DNA strand preparation step (1) of the DNA sequence manufacturing process noted above preferably comprises the steps of:
  • first and second linear deoxyoligonucleotide segments having 15 or more bases in a selected linear sequence, the linear sequence of bases of the second segment comprising the total complement of the sequence of bases of the first segment except that at least one end of the second segment shall either include an additional linear sequence of from 3 to 7 selected bases beyond those fully comple ⁇ menting the first segment, or shall lack a linear sequence of from 3 to 7 bases complementary to a ter ⁇ minal sequence of the first segment, provided, however, that the second segment shall not have an additional sequence of bases or be lacking a sequence of bases at both of its ends; and,
  • sequence of bases in the double stranded DNA subunit sequences formed preferably includes one or more triplet codons selected from among alternative codons specifying the same amino acid on the basis of preferential expression characteristics of the codon in a projected host microorganism, such as yeast cells or bacteria, especially E. coli bacteria.
  • the term "manufactured" as applied to a DNA sequence or gene shall designate a product either totally chemically synthesized by assembly of nucleotide bases or derived from the bio ⁇ logical replication of a product thus chemically synthe ⁇ sized.
  • the term is exclusive of products "synthesized” by cDNA methods or genomic cloning method- ologies which involve starting materials which are initially of biological origin.
  • Table I provides a tabular correlation between the 64 alternate triplet nucleotide base codons of DNA and the 20 amino acids and transcription termina ⁇ tion ("stop") function specified thereby.
  • Oligonucleotide fragments were synthesized using a four-step procedure and several intermediate washes.
  • Polymer bound dimethoxytrityl protected nucleo- side in a sintered glass funnel was first stripped of its 5'-protecting group (dimethoxytrityl) using 3% trichloroacetic acid in dichloromethane for 1-1/2 minutes.
  • the polymer was then washed with methanol, tetrahydrofuran and acetonitrile.
  • the washed polymer was then rinsed with dry acetonitrile, placed under argon and then treated in the condensation step as follows.
  • the final oligonucleotide chain was treated with thiophenol dioxane, triethylamine 1:2:2, for 45 minutes at room temperature. Then, after rinsing with dioxane, methanol and diethylether, the oligonucleo- tide was cleaved from the polymer, with concentrated ammonia at room temperature. After decanting the solution from the polymer, the concentrated ammonia solution was heated at 60°C for 16 hours in a sealed tube. Each oligonucleotide solution was then extrac ⁇ ted four times with 1-butanol. The solution was loaded into a 20% polyacrylamide 7 molar urea electrophoresis gel and, after running, the appropriate product band was isolated.
  • the following example illustrates the prepara ⁇ tion of a DNA sequence which comprises a gene coding for [Met " ] urogastrone and which includes terminal base sequences facilitative of insertion of the sequence into DNA plasmid restriction sites.
  • oligonucleotide sequences purified by polyacrylamide gel electrophoresis were phosphorylated at the 5' ends using ATP and T 4 polynucleotide kinase in a standard reaction using one nanomole of DNA, a two fold excess of ATP and 1 unit of T. kinase in 20 ⁇ l of buffer made with 50 mM hydroxyethylpiperazine ethane sulfonic acid, 10 mM MgCl 2 , 10 mM dithiothreitol, pH 7.6. After reaction, the kinase was destroyed by boiling for 5 minutes. These phosphorylated oligo- nucleotides in the buffer were then used directly for ligation. These sequences are shown in Table 1.
  • duplexes The oligonucleotides in 20 ⁇ l standard buffer were combined to form short duplexes. Each duplex was formed by combining two complementary sequences in equimolar amounts, boiling the mixture, then slow cooling over a 1/2 hour period to room temperature. In this way, the duplexes in Table II were formed.
  • duplexes were combined sequentially, annealing each set of duplexes at 37°C for 5 minutes until the final structural gene was in a single tube ready for ligation. .
  • the oligonucleotide mixture was then made 150 ⁇ molar in ATP and treated with 84 units of T.DNA ligase for 16 hours at 4°C.
  • the fully ligated structural gene was then purified by polyacrylamide gel electrophoresis. The final structural gene with appropriate restriction sites is shown in Table III.
  • Mutant genes coding for polypeptide sequences different from the natural sequence were also prepared. This was done by changing selected segments and repeat ⁇ ing the ligation step to obtain the new genes. By altering segments 9 and 16, the alanine at residue 25 was changed to aspartic acid. The codon modification was from GCT to GAT. This changes a neutral amino acid residue to an acidic residue and may produce a peptide with novel characteristics. Another mutant gene was prepared by changing codons in segments 1
  • the 175 base pair HEGF-URO synthetic gene was inserted into the E. coli cloning vector pBR325 using the restriction endonuclease sites Bell and BamHl. Because the restriction sites have the same cohesive termini, the gene was insertable in both orientations. However, because both restriction sites are destroyed by insertion of the gene in the incorrect orientation, only those clones which contained the gene in the correct orientation were excisable with Bell and BamHl. Those clones with the gene in the . correct orientation were ' characterized by polyacrylamide gel electrophoresis to verify the estimated molecular weight for the urogastrone structural gene.
  • the 175 base pair fragment was excised from the chimeric pBR325 plasmid (pHEGFl) and inserted into single-strand bacteriophage M13mp8 relicative form DNA at its BamHl site. Clones with the inserted
  • DNA in a defined orientation were isolated and character ⁇ ized by polyacrylamide gel eletrophoresis. Single- strand phage for one orientation were isolated and the DNA sequence for the urogastrone structural gene has been determined using the Sanger Dideoxy sequencing technique.
  • Restriction endonuclease Bell cleaves plasmid pHEGFl at its unique Bell site lying 7 nucleotides 5' to the translation initiation codon of the urogas- trone gene. Approximately 750 nucleotides 5' to this restriction site is a unique restriction endonuclease EcoRl site. Cleavage of pHEGFl with EcoRl and Bell permitted the insertion of a ⁇ P R promoter under control of lac repressor between these restriction sites by in vitro recombination to create pHEGFS. Cloning the ⁇ P_ promoter using this approach insured correct orientation of the ⁇ P R -lac promoter-operator relative to the urogastrone structural gene. The ⁇ P R promoter under lac control used for this construction was an 84 base pair EcoRl BamHl excisable synthetically derived DNA segment in E ⁇ coli cloning vector pBR322. The ⁇ P R promoter under lac control used for this construction was an 84 base
  • BamHl restriction site of the promoter lies one nucleo ⁇ tide 3' to the Shine-Dalgarno sequence. Consequently, fusion of the ⁇ P... lac promoter with the urogastrone structural gene at their BamHl - Bell cohesive termini junction creates a ribosome binding site with eight nucleotides between the Shine-Dalgarno sequence and the HEGF-URO translation initiation codon. This is close to optimal relative positioning for these two elements.
  • the insertion of the ⁇ P R promoter in the correct orientation has been verified fay restriction enzyme analysis and molecular weight sizing using polyacrylamide gel electrophoresis.
  • the ⁇ P_-lac-HEGF 259 base pair segment was excised from pHEGF5 using EcoRl and BamHl restric- tion endonuclease digestion. This fragment was inserted into EcoRl-BamHl digested pBR322 to construct pHEGFlO. This construction was performed because pBR322- expressed proteins are more easily analyzed in a maxicell system than pBR325-expressed proteins. In addition, pBR322 is a higher copy number plasmid than pBR325, conse ⁇ quently urogastrone should be expressed in greater amounts in pBR322.
  • E. coli containing pHEGF5 and pHEGFlO are being examined for expression of urogastrone polypeptide products using the maxicell system.
  • Polypeptide products can be characterized using immunoprecipitation and/or radioi munoassay techniques with rabbit IgG to mouse EGF.
  • the following example illustrates the prepara ⁇ tion of a DNA sequence which comprises a gene coding for [Met- ] urogastrone and which includes terminal base sequences facilitative of insertion of the sequence into DNA plasmid restriction sites as well as internal 10 base sequences facilitative of disassembly and reconstruc ⁇ tion of selected portions of the gene.
  • the oligonucleotides were combined to form 35 duplexes and sequentially annealed as in Example 2 to yield the structural gene set out in Table IV, having bases forming the "sticky end" of a Ba Hl restric ⁇ tion site (prior to the polypeptide coding region) and a Sail site (following the transcription termination codons). While the codon usage generally involved selection based on projected use of an E.coli bacterial expression system, the codons employed in this gene also resulted in generation of internal recognition sites for cleavage by, e.g., Hinfl (5'-GATTC-3') , SphI (5 1 - GCATGC-3 • ) and Nrul (5'-TCGCGA-5') .
  • Table IV The assembled sequence of Table IV was ampli ⁇ fied by insertion into a Ba HI/Sall cleaved M13 mp9 vector and then ligated to an EcoRI/BamHI DNA "linker" constructed with an internal Xbal recognition site, as set out in Table V.
  • the construction was excised from an ampli ⁇ fication plasmid with Xbal and Sail and inserted into a pBR322-derived plasmid (pINT- ⁇ -TXb4) at a anufac- tured Xbal site following the trp promoter/regulator
  • the resulting vector designated pADH25, was employed as an expression vector in a E_ ⁇ coli host to generate a polypeptide including a "pro" sequence of 8 amino acids, as set out below, prior to urogastrone polypeptide:
  • the following example relates to presently preferred procedures for enhancing the levels of expres ⁇ sion of products of the invention.
  • Plasmid pADH25 was treated with EcoRl and Sail to isolate the entire urogastrone protein coding region (including the DNA sequence coding for the eight residue "pro" sequence) and the entire trp pro ⁇ moter/regulator DNA sequence.
  • the EcoRI/Sall fragment was inserted in a DNA vector containing a temperature sensitive mutation in the copy control region.
  • the host cells normally contain a low copy number of the vector when grown at temperatures of less than 34°C.
  • the plasmid copy number increases 50-fold (i.e., "runs away") within the host cell upon elevation of culture temperature above 34°C. Growth at 37°C or above will ordinarily be lethal to the transformed host cells.
  • the new plasmid containing the above-noted EcoRI/Sall insert from pADH25 was designated pADH59.
  • the plasmid was employed to transform ___ coli K-12 JM103 cells (Bethesda Research Labs.) and samples of the strain harboring pADH59 have been deposited under contract with the American Type Culture Collec ⁇ tion, Rockville Maryland as A.T.C.C. 393335.
  • the level of expression of urogastrone analog product by A.T.C.C 393335 was on the order of fifty milligrams per O.D. liter as determined by SDS-PAGE.
  • the following example relates to a bioassay employed to assess the levels of microbial expression of polypeptides of the present invention.
  • a radioreceptor bioassay was developed to assay for biological activity of microbially-expressed products of the invention and was generally patterned on the procedures of Fabricant, et al., P.N.A.S. U.S.A., 74, pp. 565-569 (1977). Briefly put, the assay is a competitive receptor binding assay wherein the amount of urogastrone activity in an unknown sample is deter- mined by the ability to displace radiolabelled urogas ⁇ trone from bound association with cells in culture.
  • cells of human epider oid carcinoma cell line A-431 are grown in culture and incubated with fixed quantities of I 125-labelled urogastrone (Collaborative Research, Boston, MA.) which binds to specific URO-EGF receptors on the cell surface. The cells are washed to remove excess, unbound labelled materials. Microbial cells transformed for production of urogastrone and urogastrone analog products of the invention are lysed and centrifuged and the super ⁇ natant is applied to the culture of A-431 cells and incubated. The culture medium is then assayed for the presence of I 125-labelled urogastrone displaced from bound association with cell surface receptors by products of the invention present in the microbial cell lysate supernatant.
  • Polypeptide products of the invention which include amino terminal residues in addition to the native urogastrone sequence may be processed, if desired, to remove- the additional residues.
  • the above-noted [Met ⁇ ]urogastrone may be suitably treated with cyanogen bromide to yield polypeptides commencing with an amino terminal asparagine residue characteristic of the naturally occurring urogastrone products. if such procedures are to be employed, it may be expec- ted that the [Met 21 ] residue of urogastrone polypeptide products might provide an additional site for cyanogen bromide cleavage or the methionine may be chemically transformed to a homoserine residue.
  • the methionine residue at position 21 may be replaced by another amino acid, such as valine, through reconstruc ⁇ tion of the DNA sequence to delete the methionine- specifying codon, ATG, and replace it with an alternate codon, such as GTA which specifies valine.
  • this process would involve an initial variation in construction of oligonucleotide segments 3 and 10.
  • the modification could be effected by excising the Hinfl/Sphl fragment from plasmid pADH59 and replacing it with a manufactured sequence including the desired codon change.
  • the cyanogen bromide cleavage product of microbial expression of such an altered gene would itself be an analog of urogastrone, e.g., [Val 21]urogas ⁇ trone.
  • Products of the present invention and/or antibodies thereto may be suitably "tagged", for example radiolabelled (e.g., with I 125) conjugated with enzymes or fluorescently labelled, to provide reagent materials useful in assays and/or diagnostic test kits, for the qualitative and/or quantitative determination of the presence of such products and/or said antibodies in fluid samples.
  • Such antibodies may be obtained from the innoculation of one or more animal species (e.g., mice rabbit, goat, human, etc.) or from mono ⁇ clonal antibody sources. Any of such reagent materials may be used alone or in combination with a suitable substrate, e.g., coated on a glass or plastic particle or bead.

Abstract

DNA sequences comprising structural genes coding for (1) a polypeptide having the amino acid sequence and properties of urogastrone and for (2) polypeptide analogs thereof which differ in terms of the identity and/or location of one or more amino acids, e.g., ADAsp25 BD and ADPro52, Pro53 BD analogs of urogastrone. Structural gene sequences may be provided with initial and terminal sequences which facilitate production of discrete protein products by selected host microorganisms as well as for expression by host organisms of fusion proteins, e.g., beta-lactamase-urogastrone and beta-galactosidase-urogastrone from which the desired products may be isolated.

Description

THE MANUFACTURE AND EXPRESSION OF GENES FOR UROGASTRONE AND POLYPEPTIDE ANALOGS THEREOF
This is a continuation-in-part of prior co-pending U.S. Patent Application Serial No. 375,500, filed May 6, 1982.
BACKGROUND
The present invention relates generally to the manipulation of genetic materials and, more particularly, to the manufacture of specific DNA sequen¬ ces useful in recombinant procedures to secure the production of urogastrone and polypeptide analogs thereof.
Incorporated by reference herein for the purpose of providing information pertinent to the prior art with respect to recombinant DNA techniques is co-owned, co-pending U.S. Patent Application Serial No. 375,493, filed May 6, 1982, by Yitzhak Stabinsky, entitled "Manufacture and Expression of Structural Genes".
A component in human urine which inhibits gastric acid secretion was first described by Gray in 1939 [Gray, et al., Science, 89, 489 (1939)]. This component, named "urogastrone" was completely sequenced and its structure was published in 1975 - 2 -
[N. Gregory, Nature (London) , 257, 325 (1975)]. Earlier, the isolation and characterization of a factor. from mouse salivary glands which promotes the growth of epidermal tissue had been published [Cohen, J. Biol. Chem. , 237, 1555 (1962)]. This compound was called
"epidermal growth factor". When the amino acid composi¬ tion of epidermal growth factor was compared with that of urogastrone, it was found that the two peptides were closely related. It is now known that these compounds, mouse and human epidermal growth factor- urogastrone (EGF-URO) , are examples of a large class of "growth factors" and are widespread in animals and man.
EGF-URO like the other growth factors such as insulin, nerve growth factor, the insulin-like growth factors, and the like, is synthesized in mammals as part of a larger "pro-peptide" molecule from which it is cleaved by specific proteases to liberate the active form of the protein [Frey, et al., Proσ. Nat. Acad. Sci., 76, 6294 (1979)]. When cleaved from its pro-peptide, EGF-URO, in both the mouse and in man, is composed of 53 amino acids. Further processing in the body also gives rise to a 51 amino acid-contain¬ ing form which lacks the two amino acid residues at the carboxyl terminus of the peptide. The 53 and 51 amino acid forms of the peptide are called beta- and gamma- EGF-URO, respectively. Both forms have shown high activity as inhibitors of gastric acid secretion and as stimulators of growth of epidermoid tissue. High gastric secretion inhibitory activi¬ ties have also been reported for the 46 and 47 amino acid products of selective enzymatic degradation. [See, U.S. Patent Nos. 4,032,633 and 4,035,485.]
Receptors for EGF-URO have been found in various tissues of the human, mouse, rat, chicken, rabbit, cow, monkey, dog, cat, mink and hamster [Adamson, et al., Mol. Cell. Biochem. , 34, 129 (1981)]. Work - 3 -
done with mouse and human EGF-URO has shown that they have identical activities in both species, the best documented of which are the abilities to virtually stop gastric acid secretion and to cause prolifera- tion of epidermal and other epithelial tissues. [See, e.g, Starkey, et al., Science, 189, pp. 800-803 (1974) and Carpenter, Birth Defects; Original Article Series, 16, pp. 61-72 (1980)] .
Despite its significant biological activities, little has been done to explore the full clinical potential of urogastrone and synthetic analogs thereof. This is due in large part to lack of large quantities of the substance. EGF-URO is presently isolated in small quantities by purification from mouse salivary glands or by a complex purification from human urine [Hollenberg, Vitamins and Hormones, 37, 69 (1979); Gregory et al., U.S. Patent No. 3,883,497].
The polypeptide substance is too large to be readily synthesized by the well-known Merrifield procedure. Recombinant DNA techniques for the manufac¬ ture, cloning and expression of a structural gene for urogastrone and genes for polypeptide analogs which differ therefrom in terms of the identity and/or location of one or more amino acids have not been brought to bear on this problem.
BRIEF SUMMARY
Provided by the present invention is a manu- factured gene capable of directing synthesis in a selected host microorganism of urogastrone. In a preferred form of manufactured gene, the base sequence includes one or more codons selected from among alterna¬ tive codons specifying the same amino acid on the basis of preferential expression characteristics of the codon in a projected host microorganism, e.g., Α- coli« Other preferred forms of manufactured genes - 4 -
include those wherein: (1) a base codon specifying additional amino acid in the polypeptide synthesized which facilitates direct expression in E. coli organisms (e.g., an initial Met residue) and/or (2) base codons specifying urogastrone are preceded and/or followed by a sequence of bases comprising a portion of a base sequence which provides for restriction endonuclease cleavage of a DNA sequence (e.g., a Bell or BamHI site) and consequently facilitates formation of expres- sion vectors.
Also provided by the present invention are: (1) a manufactured gene capable of directing the synthe¬ sis in a selected host microorganism of a urogastrone polypeptide analogs which differ from urogastrone in terms of the identity and/or location of one or more amino acids (e.g., [Asp 25] urogastrone and [Pro52, Pro 53] urogastrone) ? and (2) a fusion gene comprising a manufactured gene accordi'ng to the invention fused to a second gene capable of directing synthesis of a second polypeptide (e.g., β-lactamase and β-galacto- sidasε) in a manner permitting the synthesis of a fused polypeptide including urogastrone polypeptide or. a urogastrone analog.
In practice of the invention to generate polypeptide products, DNA sequences including manufac¬ tured genes are inserted into a viral or circular plasmid DNA vector to form a hybrid vector and the hybrid vectors are employed to transform host micro¬ organisms such as bacteria (e.g., E. coli) or yeast cells. The transformed microorganisms are thereafter grown under appropriate nutrient conditions and express the polypeptide products of the invention.
Novel DNA sequences of the invention are preferably synthesized from nucleotide bases according to the methods disclosed in the aforementioned co- owned, concurrently-filed U.S. Patent Application Serial No. 375,493, by Yitzhak Stabinsky, entitled "Manufacture and Expression of Structural Genes". Briefly summarized, the general method comprises the steps of:
(1) preparing two or more different, linear, duplex DNA strands, each duplex strand including a double stranded region of 12 or more selected complemen¬ tary base pairs and further including a top single stranded terminal sequence of from 3 to 7 selected bases at one end of the strand and/or a bottom single stranded terminal sequence of from 3 to 7 selected bases at the other end of the strand, each single stranded terminal sequence of each duplex DNA strand comprising the entire base complement of at most one single stranded terminal sequence of any other duplex DNA strand prepared; and
(2) annealing each duplex DNA strand prepared in step (1) to one or two different duplex strands prepared in step (1) having a complementary single stranded terminal sequence, thereby to form a single continuous double stranded DNA sequence which has a duplex region of at least 27 selected base pairs including at least three base pairs formed by complemen¬ tary association of single stranded terminal sequences of duplex DNA strands prepared in step (1) and which has from 0 to 2 single stranded top or bottom terminal regions of from 3 to 7 bases.
In the preferred general process of manufac¬ ture, at least three different duplex DNA strands are prepared in step (1) and all strands so prepared are annealed concurrently in a single annealing reaction mixture to form a single continuous double stranded DNA sequence which has a duplex region of at least 42 selected base pairs including at least two non- adjacent sets of 3 or more base pairs formed by comple- mentary association of single stranded terminal sequen¬ ces of duplex strands prepared in step (1) . The duplex DNA strand preparation step (1) of the DNA sequence manufacturing process noted above preferably comprises the steps of:
(a) constructing first and second linear deoxyoligonucleotide segments having 15 or more bases in a selected linear sequence, the linear sequence of bases of the second segment comprising the total complement of the sequence of bases of the first segment except that at least one end of the second segment shall either include an additional linear sequence of from 3 to 7 selected bases beyond those fully comple¬ menting the first segment, or shall lack a linear sequence of from 3 to 7 bases complementary to a ter¬ minal sequence of the first segment, provided, however, that the second segment shall not have an additional sequence of bases or be lacking a sequence of bases at both of its ends; and,
(b) combining the first and second segments under conditions conducive to complementary association between segments to form a linear, duplex DNA strand. The sequence of bases in the double stranded DNA subunit sequences formed preferably includes one or more triplet codons selected from among alternative codons specifying the same amino acid on the basis of preferential expression characteristics of the codon in a projected host microorganism, such as yeast cells or bacteria, especially E. coli bacteria.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof.
DETAILED DESCRIPTION
As employed herein, the term "manufactured" as applied to a DNA sequence or gene shall designate a product either totally chemically synthesized by assembly of nucleotide bases or derived from the bio¬ logical replication of a product thus chemically synthe¬ sized. As such, the term is exclusive of products "synthesized" by cDNA methods or genomic cloning method- ologies which involve starting materials which are initially of biological origin.
The following abbreviations shall be employed herein to designate amino acids: Alanine, Ala; Arginine, Arg; Asparagine, Asn; Aspartic acid. Asp; Cysteine, Cys; Glutamine, Gin;. Glutamic acid, Glu; Glycine, Gly; Histidine, His; Isoleucine, lie; Leucine, Leu; Lysine, Lys; Methionine, Met; Phenylalanine, Phe; Proline, Pro; Serine, Ser; Threonine, Thr; Tryptophan, Trp; Tyrosine, Tyr; Valine, Val. The following abbrevia- tions shall be employed for nucleotide bases: A for adenine; G for guanine; T for thymine; U for uracil; and C for cytosine.
For ease of understanding of the present invention, Table I below provides a tabular correlation between the 64 alternate triplet nucleotide base codons of DNA and the 20 amino acids and transcription termina¬ tion ("stop") function specified thereby.
TABLE I
FIRST POSITION SECOND POSITION THIRD POSITION
T C A G
Phe Ser Tyr Cys T
Phe Ser Tyr Cys C
T Leu Ser Stop Stop A
Leu Ser Stop Trp G
Leu Pro His Arg T
Leu Pro His Arg C
C Leu Pro Gin Arg A
Leu Pro Gin Arg G
He Thr Asn Ser T
He Thr Asn Ser C
A He Thr Lys Arg A
Met Thr Lys Arg G
Val Ala Asp Gly T
Val Ala Asp Gly C
G Val Ala Glu Gly A
Val Ala Glu Gly G
The following example illustrates a preferred general procedure for preparation of deoxyoligonucleo- tides for use in the manufacture of DNA sequences of the invention.
EXAMPLE 1
Oligonucleotide fragments were synthesized using a four-step procedure and several intermediate washes. Polymer bound dimethoxytrityl protected nucleo- side in a sintered glass funnel was first stripped of its 5'-protecting group (dimethoxytrityl) using 3% trichloroacetic acid in dichloromethane for 1-1/2 minutes. The polymer was then washed with methanol, tetrahydrofuran and acetonitrile. The washed polymer was then rinsed with dry acetonitrile, placed under argon and then treated in the condensation step as follows. 0.5 ml of a solution of 10 mg tetrazole in acetonitile was added to the reaction vessel contain- ing polymer. Then 0.5 ml of 30 mg protected nucleoside phosphoramidite in acetonitrile was added. This reaction was agitated and allowed to react for 2 minutes. The reactants were then removed by suction and the polymer rinsed with acetonitrile. This was followed by the oxidation step wherein 1 ml of a solution contain¬ ing 0.1 molar I2 in 2-6-lutidine/H20/THF, 1:2:2, was reacted with the polymer bound oligonucleotide chain for 2 minutes. Following -a THF rinse capping was done using a solution of dimethylaminopyridine (6.5 g in 100 ml THF) and acetic anhydride in the proportion 4:1 for 2 minutes. This was followed by a methanol rinse and a THF rinse. Then the cycle began again with a trichloroacetic acid in CH2C12 treatment. The cycle was repeated until the desired oligonucleotide sequence was obtained.
The final oligonucleotide chain was treated with thiophenol dioxane, triethylamine 1:2:2, for 45 minutes at room temperature. Then, after rinsing with dioxane, methanol and diethylether, the oligonucleo- tide was cleaved from the polymer, with concentrated ammonia at room temperature. After decanting the solution from the polymer, the concentrated ammonia solution was heated at 60°C for 16 hours in a sealed tube. Each oligonucleotide solution was then extrac¬ ted four times with 1-butanol. The solution was loaded into a 20% polyacrylamide 7 molar urea electrophoresis gel and, after running, the appropriate product band was isolated.
The following example illustrates the prepara¬ tion of a DNA sequence which comprises a gene coding for [Met" ] urogastrone and which includes terminal base sequences facilitative of insertion of the sequence into DNA plasmid restriction sites.
EXAMPLE 2
The following deoxyoligonucleotides were synthesized according to the procedures of Example 1.
1. 5 « '-GG TGG GAA CTG CGT TAA TAG-
2. 5 '•-CAG TAC CGT GAT CTG AAA T
3. 5 ''-GT TAC ATC GGT GAA CGT TGC
4. 5 *•-GCT TGC AAC TGC GTA GTT G
5. 5 «•-GTA ACC AAC TAC GCA GTT G
6. 5 ''-TACTG GCA ACG TTC ACC GAT
7 . 5 ''-CCA CCA TTT CAG ATC ACGG
8 . 5 *•-GATC CTA TTA ACG CAG TTC
9. 5 ''-G TAT ATC GAA GCT CTG GAC AAA TAC
10. 5 ''-AT TGT CTG CAC GAC GGT GTT TGC AT
11. 5 "'-AA TGC CCG CTG TCC CAC GAC GGT T
12. 5 ''-G ATC ACA ATC AAC TCT GAT TCC G
13. 5 ''-GCA TTC GGA ATC AGA GTT CAT TGT
14. 5 ''-ACA ATA^ACC TGC GTG GGA CAG CGG
15 . 5 ''-AT ATA CAT GCA AAC ACC GTC GTG CAG
16. 5 ''-CA AGC GTA TTT TGC CAG AGC TTC G
The oligonucleotide sequences purified by polyacrylamide gel electrophoresis were phosphorylated at the 5' ends using ATP and T4 polynucleotide kinase in a standard reaction using one nanomole of DNA, a two fold excess of ATP and 1 unit of T. kinase in 20 μl of buffer made with 50 mM hydroxyethylpiperazine ethane sulfonic acid, 10 mM MgCl2, 10 mM dithiothreitol, pH 7.6. After reaction, the kinase was destroyed by boiling for 5 minutes. These phosphorylated oligo- nucleotides in the buffer were then used directly for ligation. These sequences are shown in Table 1.
The oligonucleotides in 20 μl standard buffer were combined to form short duplexes. Each duplex was formed by combining two complementary sequences in equimolar amounts, boiling the mixture, then slow cooling over a 1/2 hour period to room temperature. In this way, the duplexes in Table II were formed.
TABLE II
(12) G ATC ACA ATG AAC TCT GAT TCC G
TGT TAC TTG AGA CTA AGG CTT ACG . (13)..
(ID AA TGC CCG CTG TCC CAC GAC GGT T GGC GAC AGG GTG CTG CCA ATA ACA
(14)
(10) AT TGT CTG CAC GAC GGT GTT TGC AT
GAC GTG CTG CCA CAA ACG TAC ATA TA
(15) (9)
G TAT ATC GAA GCT CTG GAC AAA TAC
G CTT CGA GAC CTG TTT ATG CGA AC
(16)
(4) GCT TGC AAC TGC GTA GTT G G TTG ACG CAT CAA CCA ATG
(5)
(3) GT TAC ATC GGT GAA CGT TGC
TAG CCA CTT GCA ACG GTC AT
(6) (2) CAG TAC CGT GAT CTG AAA T
G GCA CTA GAC TTT ACC ACC
(7)
(1) GG TGG GAA CTG CGT TAA TAG CTT GAC GCA ATT ATC CTA G
(8)
These 8 duplexes were combined sequentially, annealing each set of duplexes at 37°C for 5 minutes until the final structural gene was in a single tube ready for ligation. . The oligonucleotide mixture was then made 150 μmolar in ATP and treated with 84 units of T.DNA ligase for 16 hours at 4°C. The fully ligated structural gene was then purified by polyacrylamide gel electrophoresis. The final structural gene with appropriate restriction sites is shown in Table III.
This was a 175 base pair duplex having Bel I restriction site at the amino terminal end and a Bam HI site at the carboxy terminal end.
TABLE III
-1 1 2 3 4 5 6 7 8 9 Bel I -Met-Asn-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser- G ATC ACA ATG AAC TCT GAT TCC GAA TGC CCG CTG TCC- TGT TAC TTG AGA CTA AGG CTT ACG GGC GAC AGG-
10 11 12 13 14 15 16 17 18 19 20 21 His-Asp-Gly-Tyr-Cys-Leu-His-Asp-Gly-Val-Cys-Met- CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG- GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-
22 23 24 25 26 27 28 29 30 31 32 33 Tyr-He-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys.-Asn-Cys- TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC- ATA TAG CTT CGA GAC CTG TTT ATG CGA ACG TTG ACG-
34 35 36 37 38 39 40 41 42 43 44 45
Val-Val-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gln-Try-Arg- GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT- CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-
46 47 48 49 50 51 52 53 Asp-Leu-Lys-Trp-Trp-Glu-Leu-Arg-Stop Stop- GAT CTG AAA TGG TGG GAA CTG CGT TAA TAG- CTA GAC TTT ACC ACC CTT GAC GCA ATT ATC CTA G-
Bam HI
Mutant genes coding for polypeptide sequences different from the natural sequence were also prepared. This was done by changing selected segments and repeat¬ ing the ligation step to obtain the new genes. By altering segments 9 and 16, the alanine at residue 25 was changed to aspartic acid. The codon modification was from GCT to GAT. This changes a neutral amino acid residue to an acidic residue and may produce a peptide with novel characteristics. Another mutant gene was prepared by changing codons in segments 1
52 tr-3 and 8. Specifically, codons for the Leu -Arg resi- dues (5'-CTG CGT-3') were replaced by those coding for Pro52, Pro53 (5'-CCG CCA-3'). This gene should code for a peptide resistant to enzyme degradation, but still retaining its other desirable properties. The following example relates to cloning of the [Met ] urogastrone gene prepared in Example 2.
EXAMPLE 3
The 175 base pair HEGF-URO synthetic gene was inserted into the E. coli cloning vector pBR325 using the restriction endonuclease sites Bell and BamHl. Because the restriction sites have the same cohesive termini, the gene was insertable in both orientations. However, because both restriction sites are destroyed by insertion of the gene in the incorrect orientation, only those clones which contained the gene in the correct orientation were excisable with Bell and BamHl. Those clones with the gene in the. correct orientation were 'characterized by polyacrylamide gel electrophoresis to verify the estimated molecular weight for the urogastrone structural gene.
To further characterize the cloned synthetic DNA segment, the 175 base pair fragment was excised from the chimeric pBR325 plasmid (pHEGFl) and inserted into single-strand bacteriophage M13mp8 relicative form DNA at its BamHl site. Clones with the inserted
DNA in a defined orientation were isolated and character¬ ized by polyacrylamide gel eletrophoresis. Single- strand phage for one orientation were isolated and the DNA sequence for the urogastrone structural gene has been determined using the Sanger Dideoxy sequencing technique.
Restriction endonuclease Bell cleaves plasmid pHEGFl at its unique Bell site lying 7 nucleotides 5' to the translation initiation codon of the urogas- trone gene. Approximately 750 nucleotides 5' to this restriction site is a unique restriction endonuclease EcoRl site. Cleavage of pHEGFl with EcoRl and Bell permitted the insertion of a λ PR promoter under control of lac repressor between these restriction sites by in vitro recombination to create pHEGFS. Cloning the λ P_ promoter using this approach insured correct orientation of the λ PR-lac promoter-operator relative to the urogastrone structural gene. The λ PR promoter under lac control used for this construction was an 84 base pair EcoRl BamHl excisable synthetically derived DNA segment in E^ coli cloning vector pBR322. The
BamHl restriction site of the promoter lies one nucleo¬ tide 3' to the Shine-Dalgarno sequence. Consequently, fusion of the λ P... lac promoter with the urogastrone structural gene at their BamHl - Bell cohesive termini junction creates a ribosome binding site with eight nucleotides between the Shine-Dalgarno sequence and the HEGF-URO translation initiation codon. This is close to optimal relative positioning for these two elements. The insertion of the λ PR promoter in the correct orientation has been verified fay restriction enzyme analysis and molecular weight sizing using polyacrylamide gel electrophoresis.
The λ P_-lac-HEGF 259 base pair segment was excised from pHEGF5 using EcoRl and BamHl restric- tion endonuclease digestion. This fragment was inserted into EcoRl-BamHl digested pBR322 to construct pHEGFlO. This construction was performed because pBR322- expressed proteins are more easily analyzed in a maxicell system than pBR325-expressed proteins. In addition, pBR322 is a higher copy number plasmid than pBR325, conse¬ quently urogastrone should be expressed in greater amounts in pBR322. The insertion of the 'λ Pp-lac- HEGF DNA segment has been verified by restriction enzyme analysis and polyacrylamide gel electrophoresis. E. coli containing pHEGF5 and pHEGFlO are being examined for expression of urogastrone polypeptide products using the maxicell system. Polypeptide products can be characterized using immunoprecipitation and/or radioi munoassay techniques with rabbit IgG to mouse EGF. 5 The following example illustrates the prepara¬ tion of a DNA sequence which comprises a gene coding for [Met- ] urogastrone and which includes terminal base sequences facilitative of insertion of the sequence into DNA plasmid restriction sites as well as internal 10 base sequences facilitative of disassembly and reconstruc¬ tion of selected portions of the gene.
EXAMPLE 4
15 The following deoxyoligonucleotides were synthesized according to the general procedures of Example 1.
1. GATCCAA ATG AAC TCT GAT TCC GAA T
20 2. GC CCG CTG TCT CAT GAC GGT TAC T
3. GC CTG CAT GAT GGC GTA TGC ATG TA
4. C ATC GAA GCT CTG GAC AAA TAC GCA
5. TGC AAC TGT GTT GTA GGT TAC ATC G
6. GC GAA CGT TGC CAG TAT CGC GAC CT 25 7. G AAA TGG TGG GAA CTG CGT TAA TAG
8. GG GCA TTC GGA ATC AGA GTT CAT TTG
9. CAG GCA GTA ACC GTC ATG AGA CAG C
10. C GAT GTA CAT GCA TAC GCC ATC ATG
11. TT GCA TGC GTA TTT GTC CAG AGC TT 30 12. TTC GCC GAT GTA ACC TAC AAC ACA G
13. A TTT CAG GTC GCG ATA CTG GCA ACG
14. TCGA CTA TTA ACG CAG TTC CCA CC
The oligonucleotides were combined to form 35 duplexes and sequentially annealed as in Example 2 to yield the structural gene set out in Table IV, having bases forming the "sticky end" of a Ba Hl restric¬ tion site (prior to the polypeptide coding region) and a Sail site (following the transcription termination codons). While the codon usage generally involved selection based on projected use of an E.coli bacterial expression system, the codons employed in this gene also resulted in generation of internal recognition sites for cleavage by, e.g., Hinfl (5'-GATTC-3') , SphI (51- GCATGC-3•) and Nrul (5'-TCGCGA-5') .
TABLE IV
-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Ba Hl Met Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val
Figure imgf000020_0001
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Cys Met Tyr He Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr He Gly Glu Arg
Figure imgf000020_0002
42 43 44 45 46 47 48 49 50 51 52 53 Sal I
Cys Gin Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg Stp Stp
Figure imgf000020_0003
Figure imgf000020_0004
The assembled sequence of Table IV was ampli¬ fied by insertion into a Ba HI/Sall cleaved M13 mp9 vector and then ligated to an EcoRI/BamHI DNA "linker" constructed with an internal Xbal recognition site, as set out in Table V.
TABLE V
EcoRl Xbal BamHl
GAA TTC TCT AGA ATG AAG AAA TAT TG
AGA T T TAC TTC TTT ATA ACC TAG
Thus provided with an adenosine-rich series of bases prior to the urogastrone polypeptide-specifying sequences, the construction was excised from an ampli¬ fication plasmid with Xbal and Sail and inserted into a pBR322-derived plasmid (pINT-γ-TXb4) at a anufac- tured Xbal site following the trp promoter/regulator
DNA sequence. The resulting vector, designated pADH25, was employed as an expression vector in a E_^ coli host to generate a polypeptide including a "pro" sequence of 8 amino acids, as set out below, prior to urogastrone polypeptide:
-8 -7 -6 -5 -4 -3 -2 -1 NH2-Met-Lys-Lys-Tyr-Trp-He-Gln-Met-[Urogastrone] .
The icrobially expressed polypeptide dis¬ played the biological activity of naturally-occurring human urogastrone. The levels of expression of the product as determined by bioassay procedures discussed infra were on the order of fifteen micrograms per O.D. liter. The following example relates to presently preferred procedures for enhancing the levels of expres¬ sion of products of the invention.
EXAMPLE 5
Plasmid pADH25 was treated with EcoRl and Sail to isolate the entire urogastrone protein coding region (including the DNA sequence coding for the eight residue "pro" sequence) and the entire trp pro¬ moter/regulator DNA sequence. The EcoRI/Sall fragment was inserted in a DNA vector containing a temperature sensitive mutation in the copy control region. After transformation with the vector, the host cells normally contain a low copy number of the vector when grown at temperatures of less than 34°C. The plasmid copy number increases 50-fold (i.e., "runs away") within the host cell upon elevation of culture temperature above 34°C. Growth at 37°C or above will ordinarily be lethal to the transformed host cells.
The new plasmid containing the above-noted EcoRI/Sall insert from pADH25 was designated pADH59. The plasmid was employed to transform ___ coli K-12 JM103 cells (Bethesda Research Labs.) and samples of the strain harboring pADH59 have been deposited under contract with the American Type Culture Collec¬ tion, Rockville Maryland as A.T.C.C. 393335.
The level of expression of urogastrone analog product by A.T.C.C 393335 was on the order of fifty milligrams per O.D. liter as determined by SDS-PAGE. The following example relates to a bioassay employed to assess the levels of microbial expression of polypeptides of the present invention. EXAMPLE 6
A radioreceptor bioassay was developed to assay for biological activity of microbially-expressed products of the invention and was generally patterned on the procedures of Fabricant, et al., P.N.A.S. U.S.A., 74, pp. 565-569 (1977). Briefly put, the assay is a competitive receptor binding assay wherein the amount of urogastrone activity in an unknown sample is deter- mined by the ability to displace radiolabelled urogas¬ trone from bound association with cells in culture. More specifically, cells of human epider oid carcinoma cell line A-431 are grown in culture and incubated with fixed quantities of I 125-labelled urogastrone (Collaborative Research, Boston, MA.) which binds to specific URO-EGF receptors on the cell surface. The cells are washed to remove excess, unbound labelled materials. Microbial cells transformed for production of urogastrone and urogastrone analog products of the invention are lysed and centrifuged and the super¬ natant is applied to the culture of A-431 cells and incubated. The culture medium is then assayed for the presence of I 125-labelled urogastrone displaced from bound association with cell surface receptors by products of the invention present in the microbial cell lysate supernatant.
Polypeptide products of the invention which include amino terminal residues in addition to the native urogastrone sequence may be processed, if desired, to remove- the additional residues. For example, the above-noted [Met~ ]urogastrone may be suitably treated with cyanogen bromide to yield polypeptides commencing with an amino terminal asparagine residue characteristic of the naturally occurring urogastrone products. if such procedures are to be employed, it may be expec- ted that the [Met 21 ] residue of urogastrone polypeptide products might provide an additional site for cyanogen bromide cleavage or the methionine may be chemically transformed to a homoserine residue. Alternately, the methionine residue at position 21 may be replaced by another amino acid, such as valine, through reconstruc¬ tion of the DNA sequence to delete the methionine- specifying codon, ATG, and replace it with an alternate codon, such as GTA which specifies valine. Applied, e.g., to the construction of Example 4, this process would involve an initial variation in construction of oligonucleotide segments 3 and 10. Alternately, the modification could be effected by excising the Hinfl/Sphl fragment from plasmid pADH59 and replacing it with a manufactured sequence including the desired codon change. The cyanogen bromide cleavage product of microbial expression of such an altered gene would itself be an analog of urogastrone, e.g., [Val 21]urogas¬ trone.
Products of the present invention and/or antibodies thereto may be suitably "tagged", for example radiolabelled (e.g., with I 125) conjugated with enzymes or fluorescently labelled, to provide reagent materials useful in assays and/or diagnostic test kits, for the qualitative and/or quantitative determination of the presence of such products and/or said antibodies in fluid samples. Such antibodies may be obtained from the innoculation of one or more animal species (e.g., mice rabbit, goat, human, etc.) or from mono¬ clonal antibody sources. Any of such reagent materials may be used alone or in combination with a suitable substrate, e.g., coated on a glass or plastic particle or bead.
Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing illustrative examples. Consequently, the invention should be considered as limited only to the extent reflected by the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A manufactured gene capable of directing the synthesis in a selected host microorganism of urogastrone polypeptide.
2. A manufactured gene according to claim
1 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in a projected host microorganism.
3. A manufactured gene according to claim 1 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in E_. coli.
4. A manufactured gene according to claim
1 wherein the base sequence comprises one of the follow¬ ing:
5'-AAC TCT GAT TCC GAA TGC CCG CTG Tec¬ s'-TTG AGA CTA AGG CTT ACG GGC GAC AGG-
CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG- GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-
TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC- ATA TAG CTT CGA GAC CTG TTT ATG CGA ACG TTG ACG-
GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT- CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-
GAT CTG AAA TGG TGG GAA CTG CGT-3' CTA GAC TTT ACC ACC CTT GAC GCA-5'
and
Figure imgf000026_0001
5*-AAC TCT GAT TCC GAA TGC CCG CTG TCT CAT GAC GGT 3'-TTG AGA CTA AGG CTT ACG GGC GAC AGA GTA CTG CCA
TAC TGC CTG CAT GAT GGC GTA TGC ATG TAC ATC GAA GCT ATG ACG GAC GTA CTA CCG CAT ACG TAC ATG TAG CTT CGA
CTG GAC AAA TAC GCA TGC AAC TGT GTT GTA GGT TAC ATC GAC CTG TTT ATG CGT ACG TTG ACA CAA CAT CCA ATG TAG
GGC GAA CGT TGC CAG TAT CGC GAC CTG AAA TGG TGG GAA CCG CTT GCA ACG GTC ATA GCG CTG GAC TTT ACC ACC CTT
CTG CGT-3' GAC GCA-5'
5. A manufactured gene according to claim
1 wherein base codons specifying urogastrone include initial and/or terminal codons respectively specifying additional initial and/or terminal amino acids in the polypeptide synthesized.
6. A manufactured gene according to claim 1 wherein the base codons specifying urogastrone are preceded and/or followed by a sequence of bases compris¬ ing a portion of a base sequence which provides a recognition site for restriction endonuclease enzyme cleavage.
7. A manufactured gene capable of directing the synthesis in a selected host microorganism of a urogastrone polypeptide analog which differs from therefrom in terms of the identity and/or location of one or more amino acids.
8. A manufactured gene according to claim 7 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in a projected host microorganism.
9. A manufactured gene according to claim 8 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in E. coli.
10. A manufactured gene according to claim
7 wherein the base sequence comprises one of the follow¬ ing:
5'-ATG AAC TCT GAT TCC GAA TGC CCG CTG Tec¬ s'-TAC TTG AGA CTA AGG CTT ACG GGC GAC AGG-
CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG- GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-
TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC- ATA TAG CTT CGA GAC CTG TTT ATG CGA ACG TTG ACG-
GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT- CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-
GAT CTG AAA TGG TGG GAA CTG CGT-3' CTA GAC TTT ACC ACC CTT GAC GCA-5'
and
5'-ATG AAC TCT GAT TCC GAA TGC CCG CTG TCT CAT GAC GGT 3'-TAC TTG AGA CTA AGG CTT ACG GGC GAC AGA GTA CTG CCA
TAC TGC CTG CAT GAT GGC GTA TGC ATG TAC ATC GAA GCT ATG ACG GAC GTA CTA CCG CAT ACG TAC ATG TAG CTT CGA
CTG GAC AAA TAC GCA TGC AAC TGT GTT GTA GGT TAC ATC GAC CTG TTT ATG CGT ACG TTG ACA CAA CAT CCA ATG TAG GGC GAA CGT TGC CAG TAT CGC GAC CTG AAA TGG TGG GAA CCG CTT GCA ACG GTC ATA GCG CTG GAC TTT ACC ACC CTT
CTG CGT-3' GAC GCA-5'
and
5'-ATG AAG AAA TAT TGG ATC CAA 3'-TAC TTC TTT ATA ACC TAG GTT
ATG AAC TCT GAT TCC GAA TGC CCG CTG TCT CAT GAC GGT TAC TTG AGA CTA AGG CTT ACG GGC GAC AGA GTA CTG- CCA
TAC TGC CTG CAT GAT GGC GTA TGC ATG TAC ATC GAA GCT ATG ACG GAC GTA CTA CCG CAT ACG TAC ATG TAG CTT CGA
CTG GAC AAA TAC GCA TGC AAC TGT GTT GTA GGT TAC ATC GAC CTG TTT ATG CGT ACG TTG ACA CAA CAT CCA ATG TAG
GGC GAA CGT TGC CAG TAT CGC GAC CTG AAA TGG TGG GAA CCG CTT GCA ACG GTC ATA GCG CTG GAC TTT ACC ACC CTT
CTG CGT-3' GAC GCA-5'
and
5'-AAC TCT GAT TCC GAA TGC CCG CTG TCC- 3'-TTG AGA CTA AGG CTT ACG GGC GAC AGG-
CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG- GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-
TAT ATC GAA GAT CTG GAC AAA TAC GCT TGC AAC TGC- ATA TAG CTT CTA GAC CTG TTT ATG CGA ACG TTG ACG-
GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT- CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-
Figure imgf000029_0001
GAT CTG AAA TGG TGG GAA CTG CGT-3' CTA GAC TTT ACC ACC CTT GAC GCA-5'
and
5'-AAC TCT GAT TCC GAA TGC CCG CTG TCC- 3'-TTG AGA CTA AGG CTT ACG GGC GAC AGG-
CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG- GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-
TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC- ATA TAG CTT CGA GAC CTG TTT ATG CGA ACG TTG ACG-
GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT- CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-
GAT CTG AAA TGG TGG GAA CCG CCA-3* CTA GAC TTT ACC ACC CTT GGC GGT-5' .
11. A manufactured gene according to claim
7 wherein the base codons specifying urogastrone are preceded and/or followed by a sequence of bases compris¬ ing a portion of a base sequence which provides a recognition site for restriction endonuclease cleavage of a DNA sequence.
12. A fusion gene comprising a manufactured gene according to either of claims 1 to 7 fused to a second gene capable of directing synthesis of a second polypeptide in a manner permitting the synthesis of a fused polypeptide including urogastrone polypeptide or a urogastrone polypeptide analog.
13. A fusion gene according to claim 12 wherein said second gene is a gene directing synthesis of β-galactosidase enzyme.
14. A fusion gene according to claim 12 wherein said second gene is a gene directing synthesis of β-lactamase.
15. A biologically functional DNA microorgan¬ ism transformation vector including a manufactured gene according to claim 1.
16. A biologically functional DNA icroorgan- ism transformation vector including a manufactured gene according to claim 7.
17. A biologically functional DNA microorgan¬ ism transformation vector including a fusion gene according to claim 12.
18. A vector according to either of claims 15, 16 or 17 which is a.circular DNA plasmid.
19. A microorganism transformed with a vector according to either of claims 15, 16 or 17.
20. A process for the production of urogas¬ trone polypeptide comprising: growing, under appropriate nutrient condi¬ tions, microorganisms transformed with a biologically functional DNA including a manufactured gene according to claim 1, whereby said microorganisms express said gene and produce urogastrone polypeptide.
21. A process according to claim 20 wherein the microorganisms grown are E. coli microorganisms.
22. A process for the production of urogas- trone polypeptide analog comprising: growing, under appropriate nutrient conditions, microorganisms transformed with a biologically func¬ tional DNA including a manufactured gene according to claim 7, whereby said microorganisms express said gene and produce urogastrone polypeptide.
23. A process according to claim 22 wherein the microorganisms grown are E. coli microorganisms.
24. A polypeptide product of the expression in a microorganism of a manufactured gene according to claim 1.
25. A polypeptide product of the expression in a microorganism of a manufactured gene according to claim 7.
26. A product according to claim 25 which is [Asp 25] urogastrone.
27. A product according to claim 25 which is [Pro 52, Pro53] urogastrone.
28. A product according to claim 25 which is [Met ]urogastrone.
29. A product according to claim 25 which is [Met"8, Lys*"7, Lys-6, Tyr"5, Trp"4, He"3, Gin"2,
Met ]urogastrone.
30. A reagent material comprising a radio- labelled manufactured gene according to claim 1.
31. A reagent material comprising a radio- labelled polypeptide product of claim 24 or claim
25.
OMPI
32. A reagent material according to claim
125 30 or 31 wherein said radiolabel is I
33. A reagent material comprising a tagged antibody to a polypeptide according to claim 24 or claim 25, coated on the surface of a plastic bead.
OMPI_
PCT/US1983/000650 1982-05-06 1983-05-02 The manufacture and expression of genes for urogastrone and polypeptide analogs thereof WO1983004030A1 (en)

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EP0108787A1 (en) * 1982-05-06 1984-05-23 Applied Molecular Genetics Inc The manufacture and expression of structural genes.
EP0147178A2 (en) * 1983-12-23 1985-07-03 Pfizer Inc. Expression plasmids for improved production of heterologous protein in bacteria
EP0158892A2 (en) * 1984-04-02 1985-10-23 Fujisawa Pharmaceutical Co., Ltd. 59 Valine insulin-like growth factor I and process for production thereof
FR2566799A1 (en) * 1984-07-02 1986-01-03 Earth Chemical Co B-UROGASTRONE GENE AND SUBUNITS, PLASMIDS AND CORRESPONDING TRANSFORMANTS AND THEIR PREPARATION, PROCESS FOR THE PRODUCTION OF B-UROGASTRONE
EP0177915A2 (en) * 1984-10-09 1986-04-16 Takeda Chemical Industries, Ltd. Novel DNA, production and use thereof
EP0207165A1 (en) * 1984-12-21 1987-01-07 Earth Chemical Co., Ltd. Polypeptide secretion-causing vector, microorganisms transformed by said vector, and process for preparing polypeptide using said microorganisms
EP0244627A2 (en) * 1986-04-10 1987-11-11 Bayer Ag Expression vectors for the production of polypeptides
US4743679A (en) * 1986-02-24 1988-05-10 Creative Biomolecules, Inc. Process for producing human epidermal growth factor and analogs thereof
WO1988007085A1 (en) * 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Process for the purification of recombinant polypeptides
US4870008A (en) * 1983-08-12 1989-09-26 Chiron Corporation Secretory expression in eukaryotes
FR2632321A1 (en) * 1988-03-24 1989-12-08 Oncogen DNA SEQUENCE ENCODING A POLYPEPTIDE, EXPRESSION CASSETTE COMPRISING SUCH A SEQUENCE, HOST CELL COMPRISING THE EXPRESSION CASSETTE, AND PROCESS FOR PRODUCING SUCH A POLYPEPTIDE
US4935370A (en) * 1983-12-23 1990-06-19 Pfizer Inc. Expression plasmids for improved production of heterologous protein in bacteria
US5013653A (en) * 1987-03-20 1991-05-07 Creative Biomolecules, Inc. Product and process for introduction of a hinge region into a fusion protein to facilitate cleavage
US5087616A (en) * 1986-08-07 1992-02-11 Battelle Memorial Institute Cytotoxic drug conjugates and their delivery to tumor cells
US5102789A (en) * 1989-03-15 1992-04-07 The Salk Institute Biotechnology/Industrial Associates, Inc. Production of epideramal growth factor in pichia pastoris yeast cells
US5366081A (en) * 1987-08-26 1994-11-22 United States Surgical Corporation Packaged synthetic absorbable surgical elements
US5447100A (en) * 1991-02-08 1995-09-05 United States Surgical Corporation Apparatus for calendering sutures in orthogonal directions
US5472702A (en) * 1987-08-26 1995-12-05 United States Surgical Corporation Sterilization of growth factors
US5674746A (en) * 1983-08-10 1997-10-07 Amgen, Inc. DNA plasmids
US5904716A (en) * 1995-04-26 1999-05-18 Gendler; El Method for reconstituting cartilage tissue using demineralized bone and product thereof
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EP0108787A1 (en) * 1982-05-06 1984-05-23 Applied Molecular Genetics Inc The manufacture and expression of structural genes.
US5674746A (en) * 1983-08-10 1997-10-07 Amgen, Inc. DNA plasmids
US4870008A (en) * 1983-08-12 1989-09-26 Chiron Corporation Secretory expression in eukaryotes
US4935370A (en) * 1983-12-23 1990-06-19 Pfizer Inc. Expression plasmids for improved production of heterologous protein in bacteria
US5955297A (en) * 1983-12-23 1999-09-21 Pfizer Inc. Expression plasmids for improved production of heterologous protein in bacteria
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US4745179A (en) * 1984-04-02 1988-05-17 Fujisawa Pharmaceutical Co., Ltd. 59 Valine insulin-like growth factor I and process for production thereof
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EP0158892A3 (en) * 1984-04-02 1987-09-09 Fujisawa Pharmaceutical Co., Ltd. 59 valine insulin-like growth factor i and process for production thereof
GB2162851B (en) * 1984-07-02 1989-05-17 Earth Chemical Co Novel b-urogastrone gene, corresponding recombinant plasmids, corresponding transformants and preparations thereof and of b-urogastrone.
AU599003B2 (en) * 1984-07-02 1990-07-12 Earth Chemical Co. Ltd. Novel beta-urogastrone gene, corresponding recombinant plasmids, corresponding transformants and preparation thereof and of beta-urogastrone
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GB2162851A (en) * 1984-07-02 1986-02-12 Earth Chemical Co beta -urogastrone gene
EP0177915A2 (en) * 1984-10-09 1986-04-16 Takeda Chemical Industries, Ltd. Novel DNA, production and use thereof
EP0177915A3 (en) * 1984-10-09 1987-08-05 Takeda Chemical Industries, Ltd. Novel dna, production and use thereof
US4849350A (en) * 1984-10-09 1989-07-18 Takeda Chemical Industries, Ltd. Novel DNA, production and use thereof
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EP0207165A4 (en) * 1984-12-21 1988-07-04 Earth Chemical Co Polypeptide secretion-causing vector, microorganisms transformed by said vector, and process for preparing polypeptide using said microorganisms.
EP0207165A1 (en) * 1984-12-21 1987-01-07 Earth Chemical Co., Ltd. Polypeptide secretion-causing vector, microorganisms transformed by said vector, and process for preparing polypeptide using said microorganisms
US4743679A (en) * 1986-02-24 1988-05-10 Creative Biomolecules, Inc. Process for producing human epidermal growth factor and analogs thereof
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US7105638B1 (en) 1987-03-20 2006-09-12 Micromet Ag Product and process for the production, isolation, and purification of recombinant polypeptide
US5013653A (en) * 1987-03-20 1991-05-07 Creative Biomolecules, Inc. Product and process for introduction of a hinge region into a fusion protein to facilitate cleavage
WO1988007085A1 (en) * 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Process for the purification of recombinant polypeptides
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US5366081A (en) * 1987-08-26 1994-11-22 United States Surgical Corporation Packaged synthetic absorbable surgical elements
US5472702A (en) * 1987-08-26 1995-12-05 United States Surgical Corporation Sterilization of growth factors
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US5102789A (en) * 1989-03-15 1992-04-07 The Salk Institute Biotechnology/Industrial Associates, Inc. Production of epideramal growth factor in pichia pastoris yeast cells
US5447100A (en) * 1991-02-08 1995-09-05 United States Surgical Corporation Apparatus for calendering sutures in orthogonal directions
US5540773A (en) * 1991-02-08 1996-07-30 United States Surgical Corporation Apparatus for calendering and coating/filling sutures
US5904716A (en) * 1995-04-26 1999-05-18 Gendler; El Method for reconstituting cartilage tissue using demineralized bone and product thereof
EP2093241A2 (en) 2008-01-29 2009-08-26 Confluent Surgical Inc. Bioabsorbable block copolymer

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EP0108132A1 (en) 1984-05-16
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IT1212984B (en) 1989-12-07

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