EP0108132A1 - Fabrication et expression de genes pour l'urogastrone et leurs analogues polypeptides - Google Patents

Fabrication et expression de genes pour l'urogastrone et leurs analogues polypeptides

Info

Publication number
EP0108132A1
EP0108132A1 EP83901992A EP83901992A EP0108132A1 EP 0108132 A1 EP0108132 A1 EP 0108132A1 EP 83901992 A EP83901992 A EP 83901992A EP 83901992 A EP83901992 A EP 83901992A EP 0108132 A1 EP0108132 A1 EP 0108132A1
Authority
EP
European Patent Office
Prior art keywords
ctg
gac
atg
tgc
tac
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP83901992A
Other languages
German (de)
English (en)
Inventor
Allen R. Banks
David L. Hare
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amgen Inc
Original Assignee
Applied Molecular Genetics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Applied Molecular Genetics Inc filed Critical Applied Molecular Genetics Inc
Publication of EP0108132A1 publication Critical patent/EP0108132A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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], i.e. 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

On décrit des séquences d'ADN comprenant des gènes structuraux de codages (1) un polypeptide possédant la séquence d'acides aminés et les propriétés de l'urogastrone et de (2) ses analogues polypeptides qui diffèrent en ce qui concerne l'identité et/ou l'emplacement d'un ou plusieurs acides aminés, par exemple, les analogues d'urogastrone ADAsp25 BD et AD Pro52, Pro53 BD. Les séquences de gènes structuraux peuvent présenter des séquences initiales et terminales qui facilitent la production de produits protéiques séparés par les micro-organismes hôtes sélectionnés, tout en permettant l'expression par les organismes hôtes de protéines de fusion, par exemple le beta-lactamase-urogastrone et le beta-galactosidase-uroastrone à partir duquel on peut isoler les produits désirés.
EP83901992A 1982-05-06 1983-05-02 Fabrication et expression de genes pour l'urogastrone et leurs analogues polypeptides Withdrawn EP0108132A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US37550082A 1982-05-06 1982-05-06
US48609183A 1983-04-25 1983-04-25
US486091 1983-04-25
US375500 1995-01-19

Publications (1)

Publication Number Publication Date
EP0108132A1 true EP0108132A1 (fr) 1984-05-16

Family

ID=27007082

Family Applications (1)

Application Number Title Priority Date Filing Date
EP83901992A Withdrawn EP0108132A1 (fr) 1982-05-06 1983-05-02 Fabrication et expression de genes pour l'urogastrone et leurs analogues polypeptides

Country Status (4)

Country Link
EP (1) EP0108132A1 (fr)
CA (1) CA1214739A (fr)
IT (1) IT1212984B (fr)
WO (1) WO1983004030A1 (fr)

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4652639A (en) * 1982-05-06 1987-03-24 Amgen Manufacture and expression of structural genes
IL71991A (en) 1983-06-06 1994-05-30 Genentech Inc Preparation of human FGI and FGE in their processed form through recombinant AND tranology in prokaryotes
US4710473A (en) * 1983-08-10 1987-12-01 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
EP0147178B1 (fr) * 1983-12-23 1991-08-14 Pfizer Inc. Plasmides d'expression pour la produciton de protéines héterologues dans des bactéries
US4745179A (en) * 1984-04-02 1988-05-17 Fujisawa Pharmaceutical Co., Ltd. 59 Valine insulin-like growth factor I and process for production thereof
JP2554459B2 (ja) * 1984-07-02 1996-11-13 アース製薬 株式会社 β−ウロガストロン遺伝子、対応プラスミド組換体及び対応形質転換体
CA1263619A (fr) * 1984-10-09 1989-12-05 Ryuji Marumoto Adn, production et utilisation
JP2549504B2 (ja) * 1984-12-21 1996-10-30 ア−ス製薬株式会社 Ｄｎａ塩基配列、ポリペプチド分泌発現ベクター及び形質転換微生物
US4743679A (en) * 1986-02-24 1988-05-10 Creative Biomolecules, Inc. Process for producing human epidermal growth factor and analogs thereof
GB2188933A (en) * 1986-04-10 1987-10-14 Bayer Ag Expression vectors for production of polypeptides, method for enhanced expression of polypeptides, hosts containing the expression vectors, products manufactured thereby
IN165717B (fr) * 1986-08-07 1989-12-23 Battelle Memorial Institute
EP0305500B1 (fr) * 1987-03-20 1994-11-09 Creative Biomolecules, Inc. Procede de purification de polypeptides recombinants
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
US5472702A (en) * 1987-08-26 1995-12-05 United States Surgical Corporation Sterilization of growth factors
US5366081A (en) * 1987-08-26 1994-11-22 United States Surgical Corporation Packaged synthetic absorbable surgical elements
IL89673A0 (en) * 1988-03-24 1989-09-28 Oncogen Novel polypeptides having growth factor activity and nucleic acid sequences encoding the polypeptides
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
CA2059245C (fr) * 1991-02-08 2004-07-06 Michael P. Chesterfield Methode et appareil permettant de laminer et d'enrober ou de remplir des jonctions
US5904716A (en) * 1995-04-26 1999-05-18 Gendler; El Method for reconstituting cartilage tissue using demineralized bone and product thereof
US20090192554A1 (en) 2008-01-29 2009-07-30 Confluent Surgical, Inc. Bioabsorbable block copolymer

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IE53166B1 (en) * 1980-08-05 1988-08-03 Searle & Co Synthetic urogastrone gene,corresponding plasmid recombinants,transformed cells,production thereof and urogastrone expression

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8304030A1 *

Also Published As

Publication number Publication date
WO1983004030A1 (fr) 1983-11-24
IT1212984B (it) 1989-12-07
CA1214739A (fr) 1986-12-02
IT8367501A0 (it) 1983-05-06

Similar Documents

Publication Publication Date Title
CA1214739A (fr) Fabrication et expression des genes codant pour l'urogastrone et analogues polypeptidiques
EP0136489B1 (fr) Analogues de l'Interleukin II humaine et leur préparation
EP0711303B2 (fr) Biotinylation de proteines
WO1985000831A1 (fr) Expression microbienne d'un facteur de croissance semblable a l'insuline
CA1200515A (fr) Production et expression de genes structuraux
EP0020147B1 (fr) Vecteur de transfert d'ADN pour la prohormone de croissance humaine, microorganisme transformé par celui-ci et procédé de clonage à cet effet
EP0150572A1 (fr) Expression microbienne de facteur à croissance de type I, polypeptides analogiques et polypeptides hybridisés de EGF/TGF
EP0108787A1 (fr) Production et expression de genes structuraux.
US5089406A (en) Method of producing a gene cassette coding for polypeptides with repeating amino acid sequences
KR920009543B1 (ko) β-유로가스트론 유전자, 상응하는 재조합 플라스미드, 상응하는 형질전환체 및 그의 제조방법, 및 β-유로가스트론의 제조방법
JP2506322B2 (ja) Ｇｒｆ前駆体をコ―ドするｄｎａ
EP0107710A1 (fr) Fabrication et expression de genes pour la calcitonine et ses analogues de polypeptide
US4764593A (en) Manufacture and expression of genes for urogastrone and polypeptide analogs thereof
AU6774887A (en) Human placenta angiogenic factor capable of stimulating capillary endothelial cell protease synthesis, dna synthesis and migration
JP2545377B2 (ja) Ｄｎａ配列体
US5545564A (en) Vector for expression and secretion of polypeptide microorganism transformed by the vector and production of polypeptide with the same microorganism
WO1988005082A1 (fr) Production microbienne d'oligomeres peptidiques
US5464770A (en) DNA encoding (ASP 113) and (LYS 46, ASP 113) thaumatin I
KR840002107B1 (ko) 재조합 미생물 클로닝 베히클로부터 폴리펩티드를 제조하는 방법
KR840002090B1 (ko) 폴리펩티드 헵텐을 함유하는 면역성 물질인 폴리펩티드를 제조하는 방법
JPS61500250A (ja) タイプｉ形質転換増殖因子、そのポリペプチド類似物、及び雑種ｅｇｆ／ｔｇｆポリペプチドの微生物による発現
JPS63202387A (ja) Ｄｎａ配列、これを含むベクタ−、該ベクタ−を保有する宿主細胞及びポリペプチドの製造法
JPS62190083A (ja) ポリペプチド発現ベクタ−、該ベクタ−で形質転換した宿主及び該宿主によるポリペプチドの製造法
JPH02195895A (ja) Ｂｃｇ菌由来免疫タンパクｍｐｂ７０
JPS59501095A (ja) カルシトニンおよびその類似体のための遺伝子の製造と発現

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Designated state(s): AT BE CH DE FR GB LI LU NL SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19840409

RIN1 Information on inventor provided before grant (corrected)

Inventor name: HARE, DAVID L.

Inventor name: BANKS, ALLEN R.