EP0119247A1 - Herstellung reifer proteine in transformierter hefe - Google Patents

Herstellung reifer proteine in transformierter hefe

Info

Publication number
EP0119247A1
EP0119247A1 EP19830903055 EP83903055A EP0119247A1 EP 0119247 A1 EP0119247 A1 EP 0119247A1 EP 19830903055 EP19830903055 EP 19830903055 EP 83903055 A EP83903055 A EP 83903055A EP 0119247 A1 EP0119247 A1 EP 0119247A1
Authority
EP
European Patent Office
Prior art keywords
yeast
protein
precursor
calcitonin
bovine
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Withdrawn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
EP19830903055
Other languages
English (en)
French (fr)
Inventor
Walter C. Mahoney
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.)
Immuno Nuclear Corp
Original Assignee
Immuno Nuclear Corp
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 Immuno Nuclear Corp filed Critical Immuno Nuclear Corp
Publication of EP0119247A1 publication Critical patent/EP0119247A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • 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

  • BACKGROUND OP THE INVENTION 1. Field of the Invention. This invention relates to a method for producing a mature protein in transformed yeast and further relates to Saccharomyces cerevisiae transformed by a plasmid containing a preproparathyroid hormone cDNA insert. 2. Description of the Prior Art.
  • Recombinant DNA technology now makes it possible to isolate specific genes or portions thereof from higher organisms, such as man and other animals, and to transfer the genes or fragments to a microorganism species, such as ⁇ coli or yeast.
  • the transferred gene is replicated and propagated as the transformed microorganism may become endowed with the capacity to make whatever protein the gene or fragment encodes, whether it be an enzyme, a hormone, an antigen or an antibody, or a portion thereof.
  • the microorganism passes on this capability to its progeny, so that in effect, the transfer results in a new strain, having the described capability.
  • Recombinant DNA conventionally utilizes transfer vectors.
  • a transfer vector is a DNA molecule which contains genetic information which insures its own replication when transferred to a host microorganism strain. Plasmids are an example of a transfer vector commonly used in genetics. Although plasmids have been used as the
  • Plasmid is the term applied to any autonomously replicating DNA unit which might be found in a microbial cell, other than the genome of the host cell itself.
  • a plasmid is not usually genetically linked to the chromosome of the host cell.
  • Plasmid DNA exists as doublestranded ring structures generally on the order of a few million daltons molecular weight, although some are greater than 10 8 daltons in molecular weight. They usually represent only a small percent of the total DNA of the cell.
  • Transfer vector DNA is usually separable from host cell DNA by virtue of the great difference in size between them. Transfer vectors carry genetic information enabling them to replicate within the host cell.
  • Plasmid DNA exists as a closed ring. However, by appropriate techniques, the ring may be opened, a fragment of heterologous DNA inserted, and the ring reclosed, forming an enlarged molecule containing the inserted DNA segment.
  • Transfer is accomplished by a process known as transformation.
  • host cells mixed with plasmid DNA incorporate entire plasmid molecules into the cells. Once a cell has incorporated a plasmid, the latter is replicated within the cell and the plasmid replicas are distributed to the progeny cells when the cell divides.
  • heterologous DNA inserted into the plasmid, can in principle be expressed in the host cell.
  • the inserted heterologous DNA typically representing
  • OMP a single gene, is expressed when the protein product coded by the gene is synthesized by the organism.
  • the availability of the gene in substantial quantity is assured.
  • the gene replicates as the microorganism proliferates.
  • the vector containing the gene is easily purified from cultures of the host microorganism by known tech ⁇ niques and separable from the vector by restriction endonuclease cleavage followed by gel electrophoresis.
  • the protein product expressed by the heterologous gene can also be recovered in substantial quantities from cultures of the host microorganism by harvesting the culture and retrieving the protein product contained in the harvested cells.
  • Recombinant DNA thus holds great promise for economically producing substantial quantities of useful proteins that are difficult or costly to isolate in such quantitites from mammalian tissue.
  • mRNA Mature messenger RNA
  • cDNA complementary DNA
  • cDNA enzymatically prepared by reverse transcription has the potential to express a protein chain identical to the protein expressed by tissue from which the mRNA was extracted. This alone is not sufficient, however, for the expression of desired mature animal proteins because many animal proteins, represented by such diverse classes as hormones, binding proteins, enzymes, antibodies, and collagen, are produced in nature in the form of larger precursors that are subsequently modified by cleavage to smaller bioactive forms commonly designated mature proteins. Thus, expression of cDNA synthesized by reverse transcription only has the potential to express the precursor of the mature protein product.
  • bacteria such as E ⁇ coli can remove the "pre" portion of its own secreated proteins. Examples include the processing of pre-ribose binding protein, pre-galactose binding protein and pre-arabinose binding protein. (L. Randall, et al., Eur. J. Biochem., Vol. 92,. pp. 411-415 (1978); L. Randall, S. Hardy, and L. Josefsson, Proc. Natl. Acad. Sci. USA, Vol. 75, pp. 1209-1212 (1978)).
  • yeast shares the ability to remove "pre” sequences from its own pre-proteins. Furthermore, when an E_ ⁇ _ coli preprotein was genetically engineered into yeast, pre-B-lactamase was processed to B-lactamase. (Roggenkamp, et al., Proc. Natl. Acad. Sci. USA, Vol. 78, No. 7, pp. 4466-4470 (1981)).
  • preproteins will not process to mature proteins many of the mammalian hormone precursors and many of the other interesting mammalian protein precursors in ⁇ coli. These latter hormone and protein precursors contain a "pro" portion which is not processed by the enzymatic mechanism responsible for processing the "pre” portion of preproteins.
  • preproinsulin is processed in E ⁇ coli to form proinsulin.
  • Many investigators have been unable to express pre-proteins in yeast or E ⁇ coli, let
  • Insulin is the result of natural processing in human tissue involving cleaving two peptide chains, A and B, from the single large precursor preproinsulin and assembling the A and B chains to form the mature hormone insulin.
  • the A and B chains are located within proinsulin and hence E_ j _ coli which processes preproinsulin to proinsulin does not produce the mature hormone insulin.
  • An approach to obtaining mature insulin using E. coli employs chemically synthesized genes compatible with E_ ⁇ _ coli.
  • a double-stranded synthetic DNA-coding sequence for the insulin A chain was synthesized chemically from fundamental nucleotide units to yield the correct coding sequence. An extra amino acid (methionine) was added at one end.
  • the fused proteins are insoluble in water and readily isolated from broken cells.
  • the A and B chains of insulin are released from B-galactosidase at the extra methionine by cyanogen bromide cleavage and subsequently mixed together under conditions that allow formation of disulfide bonds between A
  • OMPI encoding the precursor was enzymatically prepared from mRNA isolated from human pituitary tissue.
  • the first useful cleavage site of the cDNA occurs at the site encoding amino acid residues 23-24 of HGH.
  • Treatment of the cDNA with restriction endonuclease Hae III gives a DNA fragment of 551 base pairs which includes coding sequences for amino acids 24-191 of HGH.
  • a gene fragment having coding sequences for residues 1-23 of HGH (and an initiation codon) was chemically synthesized. The two DNA fragments were combined to form a synthetic-natural hybrid gene which when inserted into a plasmid vector directed expression of mature HGH in ⁇ . coli. (D.
  • OMPI are not "real" mature proteins.
  • FIG. 1 shows inferred protein cleavage sites within the precursor of yeast ⁇ -f ctor, where "K” designates lysine and “R” designates arginine amino acid residues.
  • FIG. 2 shows the cDNA sequence encoding preproparathyroid hormone and the unique Pvu II and Hinf 1 cleavage sites.
  • FIG. 3 shows certain portions of the nucleotide sequence of the pYEM-1 plasmid.
  • a method for producing protein in yeast transformed to express a corresponding precursor containing a pair or triplet of basic amino acid residues located proximally and/or distally adjacent to the protein portion of the precursor sequence comprising proteolytic processing by the yeast of the precursor at the site of such pairs or triplets of basic amino acid residues.
  • the method comprises proteolytic processing by transformed yeast which contains an endopeptidase, designated herein as a trypsin-like enzyme or enzymes.
  • the trypsin-like enzyme or enzymes proteolytically process the precursor at the site of such pairs or triplets of basic amino acid residues by cleaving at the distal side of such pairs or triplets.
  • the method further comprises proteolytic processing by transformed yeast that contains an exopeptidase, designated herein a carboxypeptidase-B-like enzyme or enzymes.
  • the carboxypeptidase-B-like enzyme or enzymes proteolytically process the precursor at the site of such pairs or triplets of basic amino acid residues by degrading- such pairs or triplets of basic amino acid residues remaining distally adjacent to the protein portion of the precursor sequence after the cleavage by the trypsin-like enzyme or enzymes.
  • Proto-proteins defined with greater specificity infr-a, consist generally of precursor proteins in which the
  • -g ⁇ fcEaA OMPI protein portion of the precursor sequence is identical in structure .to .the mature protein except for the abscence of the amino terminal and the carboxyl terminal in the precursor sequence.
  • the above method is also disclosed for proteolytic processing of certain non-proto-proteins.
  • the above method is disclosed for proteolytic processing of preproinsulin or proinsulin to mature insulin.
  • the above method is disclosed for producing mammalian insulin generally as well as human, bovine, and porcine insulin specifically. .
  • preprocalcitonin and procalcitonin may be proteolytically processed by transformed yeast to form mature calcitonin or a calcitonin relative in the case of animal calcitonin generally and human, bovine, and porcine calcitonin specifically.
  • a recombinant DNA plasmid transfer vector useful for transforming yeast comprising a DNA sequence comprising the preproparathyroid gene cDNA sequence is disclosed as well as the plasmid pYEM-1 and yeast transformed ' by a plasmid comprising the above transfer vector and yeast transformed by the plasmid pYEM-1.
  • Proto-proteins may consist of precursors for which DNA and mRNA encoding the precursors naturally occur in animals. This type of proto-protein is designated source natural proto-proteins. Proto-proteins may also consist of precursors in which synthetic DNA encodes the precursor. This type of proto-protein is designated source synthetic proto-protein. For example, by chemical synthesis, or alternatively by enzymatic cleavage, rearrangement and subsequent fusion, DNA can be synthesized so that the precursor which
  • OMPI it encodes has the cleavage properties discussed below. Production of mature protein might be enhanced by transforming yeast with synthetic DNA encoded for a precursor having repetitive sequences of the mature protein, each sequence being flanked by appropriate cleavage sites.
  • Source natural proto-proteins are illustrated by, but not limited to, certain hormone precursors, including preproparathyroid (J. Habener & J. Potts, The New England Journal of Medicine (Second Part) , Vol. 299, No. 12, pp. 635-643 (Sept. 1978)), preprosomatostatin (P. Hobart, et al.. Nature, Vol. 288, pp. 137-139 (November 1980)), AVP-NpII precursor to arginine vasopressin and its corresponding neurophysin (H. Land, et al.. Nature, Vol. 295, pp.
  • preproparathyroid J. Habener & J. Potts, The New England Journal of Medicine (Second Part) , Vol. 299, No. 12, pp. 635-643 (Sept. 1978)
  • preprosomatostatin P. Hobart, et al.. Nature, Vol. 288, pp. 137-139 (November 1980)
  • cortitropin B-lipotropin precursor to corticotopin (ACTH) and B-lipotropin (B-LPH) S. Nakanishi, et al., Nature, Vol. 278, pp. 423-427 (March 1979)
  • preproglucagon P. Lund, et al.. Pro. Natl. Acad. Sci. USA, Vol. 79, pp. 345-349 (January 1982)
  • POMC pro-opiomelanocortin precursor to B-endorphin and Met- and Leu-enkephalin precursor
  • Source natural proto-proteins are also illustrated by melittin precursor (G. Suchanek, et al., Eur. J. Biochemistry, Vol. 60, pp. 309-315 (1975); G. Suchanek, et al., Proc. Natl. Acad. Sci. USA, Vol. 75, pp. 701-704 (1978)) and serum albumin precursors (R. Lawn, et al., Nucleic Acids Research, Vol 9, No.22, pp. 6103-6114 (1981)).
  • these precursors contain within their sequence at least one mature protein sequence. Where there is a single mature protein sequence contained in the precursor it is flanked proximally by a pair or
  • triplet of basic amino acid residues consisting of lysine and/or arginine and is flanked distally by either the carboxyl-terminal of the precursor or a pair or triplet of basic amino acid residues lysine and/or arginine. If there are several mature protein sequences contained in the precursor, at least one of the mature protein sequences is flanked proximally by a pair or triplet of such basic amino acid residues and is flanked distally by either the carboxyl-terminal of the precursor or a pair or triplet of such basic amino acid residues. Any precursor protein falling within this description is defined herein as a proto-protein, whether it be source natural or source synthetic.
  • the mature protein in preproparathyroid hormone the mature protein is flanked proximally by the basic triplet lysine-lysine-arginine and is flanked distally by the carboxyl-terminal of the precursor.
  • a single cleavage by a trypsin-like enzyme is sufficient to produce the mature hormone.
  • two mature glucagon proteins are flanked both proximally and distally by a basic pair lysine- arginine. Combined cleavage by a trypsin-like enzyme and degradation of the resulting carboxyl- terminal by a carboxypeptidase-B-like enzyme are required to produce the mature proteins.
  • the method of the present invention comprises preteolytic processing by yeast of proto-proteins to mature proteins.
  • transformed yeast naturally containing a trypsin-like enzyme or enzymes and a carboxypeptidase-B-like enzyme or enzymes, proteolytically release mature proteins from larger precursors.
  • These enzymes will effectively cleave and degrade proto-proteins to mature proteins. This is confirmed by a trypsin-like cleavage, discussed infra, of preproparathyroid hormone yielding mature parathyroid hormone. This is further confirmed by yeast processing its own mating factor, ⁇ -factor. (T. Tanaka, et al., J. Biochemistry, Vol. 82, pp. 1681-1687 (1977)). As shown in FIG.
  • the nucleotide sequence of ⁇ -factor shows that yeast naturally expresses a precursor containing four distinct codings for mature ⁇ -factor. Three of the four ⁇ -factors in the precursor are flanked distally by a pair of basic amino acids residues. A trypsin-like cleavage in combination with a carboxypeptidase- B-like degrading naturally yields correctly processed C-termini for these three. ⁇ -factors. After a trypsin-like cleavage, N-termini of the four ⁇ -factors are flanked proximally by a series of several glutamic acid and alanine amino acid residues. These latter residues are in turn removed by an a inopeptidase.
  • insulin-A-chain and insulin-B-chain may be considered mature proteins and preproinsulin and proinsulin without disulfide bonds may be considered a proto-protein according to the above discussion of proto-proteins.
  • Mature calcitonin contains disulfide bonds between the cysteines located at positions 1 and 7 of the sequence, contains a carbohydrate attached at the sequence at position 3, and the proline at position 32 has been amidated to pro-amide while the glycine at position 33 has been removed.
  • Preprocalcitonin and procalcitonin will contain the requisite disulfide bonds. (cf. the numerous examples of disulfide bond formation in yeast as disclosed in Dayhoff, supra) . A carbohydrate will be attached at position 3 in calcitonin.
  • Preprocalcitonin and procalcitonin will undergo proteolytic processing in yeast transformed to express the preprocalcitonin or procalcitonin.
  • a pair of basic amino residues are located proximally adjacent to the 33 amino acid sequence, while a triplet is located distally adjacent to the 33 amino acid sequence. It is expected that amidation of the proline located at 32 will occur in yeast after the cleavage distall to and degradation of the triplet. (cf. numerous examples of amidation in yeast as disclosed by Dayhoff, supra) .
  • the calcitonin relative containing the differing carbohydrate may be converted to mature calcitonin by conventional means.
  • the calcitonin relative lacking the amidation may also be converted to mature calcitonin by conventional means.
  • cDNA By reverse transcription, cDNA can be prepared encoding any proto-protein of interest by isolating mRNA from tissues expressing the protein. Although many hormone and other protein genes have
  • yeast has heretofore not been the host of choice.
  • cDNA not previously cloned in yeast can be rendered compatible with a yeast host by proper codon selection (J. Bennetzen & B. Hall, J. Bio Che . , Vol. 257, pp. 3026 (1982)) and by site specific mutagenesis of the cDNA (G. Simmons, et al., Nucleic Acid Research, Vol. 10, pp. 821 (1982)) .
  • the plasmid YEp-13 was obtained from Dr. Steven Henekoff, Fred Hutchinston, Dept. of Developmental Biology, Seattle, Washington, and can be constructed according to J. Broach, et al., Gene, Vol. 8, pp. 121-133, (1979) .
  • the gene which encodes yeast alcohol deh ' ydrogenase 1 was modified according to Hitzelman, et al.. Nature (London) , vol. 293, pp. 717-722 (1981), allowing the isolation of the transcription signals. These sequences, including the cloning site, were provided by Dr. G. Ammera.
  • the plasmid YEp-13 was modified so that the tet gene of YEp-13 was interrupted at the Bam HI site with the yeast alcohol dehydrogenase 1 gene promotor and RNA polymerase
  • FIG. 3 shows certain portions of the nucleotide sequence of pYEM-1.
  • yeast cells were transformed with the plasmid using the methods of Beggs, Nature (London) , Vol. 275, pp. 104-109 (1978) and Hinnen, et al., ' Proc. Natl. Acad. of
  • pYEM-1 has the yeast leu 2 gene
  • the use of a leu 2 negative strain of east was used in the transformation for the purposes of selecting successful transformants.
  • Yeast strain, X106.9-2D a strain of Saccharomyces : cerevisiae defective in leu-2 function, was obtained from the Yeast Genetic Stock Center,. Univ. of Califorinia- Berekeley. Of course any other defective yeast strain, including strains within ' Saccharomyces pombe and other species, could be used. All that is required is that a complementation system be established between the yeast strain and the cloning/expression vector and that the vector be stabily maintained in yeast.
  • Trp 1 strain could be used if the Trp 1 gene was on the vector.
  • Trp 1 gene was on the vector.
  • several stable transformation systems have been described. (A. Hinnen and B. Meyhack, Current Topics in Microbiology and Immunology, Vol. 96, pp. 101-117 (1981); C. Hollenberg,- Current Topics in Microbiology and Immunology, Vol. 96, pp. 119-144 (1981)).
  • the transformed yeast cells containing plasmid pYEM-1 were grown in a leucine deficient media containing 5% glucose, yeast extract, yeast nitrogen base and other nutrients suitable for yeast strain X1069-2D. After 24 hours of growth at 30°C, the media was collected and the yeast cells lysed. Bioassay was performed according to conventional techniques and PTH
  • OMPI radioimmunoassay was performed using Immuno Nuclear Corporation (Stillwater, MN) assays specific to the N-terminal, mid-molecule, and C-terminal regions of parathyroid hormone.
  • MN Immuno Nuclear Corporation
  • preproparathyroid hormone expressed by the yeast was left unprocessed, we would expect 35S methionine in cycles 1, 2, 7, 11, 14, 49, and 59 reflecting the appearance of methionine at positions -31, -30, -25, -21, -18, +8, +18 in the preproparathyroid sequence.
  • novel microorganism yeast strain X1069-2D transformed by novel plasmid pYEM-1 designated Xl069-2D-pYEM-l
  • Xl069-2D-pYEM-l The novel microorganism yeast strain X1069-2D transformed by novel plasmid pYEM-1, designated Xl069-2D-pYEM-l, was placed on permanent deposit in the Northern Regional Research Center, U.S. Dept. of Agriculture, Peoria, Illinois 61604 on September 8> 1982.
  • the NRRL number for Xl069-2D-pYEM-l is Y-15153.
  • the plasmid pYEM-1 and the transfer vector contained therein may be removed from this novel yeast strain by known means.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Endocrinology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP19830903055 1982-09-15 1983-09-09 Herstellung reifer proteine in transformierter hefe Withdrawn EP0119247A1 (de)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US41853782A 1982-09-15 1982-09-15
US45233982A 1982-12-22 1982-12-22
US452339 1982-12-22
US418537 1999-10-14

Publications (1)

Publication Number Publication Date
EP0119247A1 true EP0119247A1 (de) 1984-09-26

Family

ID=27024171

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19830903055 Withdrawn EP0119247A1 (de) 1982-09-15 1983-09-09 Herstellung reifer proteine in transformierter hefe

Country Status (2)

Country Link
EP (1) EP0119247A1 (de)
WO (1) WO1984001173A1 (de)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3312928A1 (de) * 1983-04-11 1984-11-22 Gesellschaft für Biotechnologische Forschung mbH (GBF), 3300 Braunschweig Human-parathormon produzierende hybridvektoren und human-parathormongen
US4870008A (en) * 1983-08-12 1989-09-26 Chiron Corporation Secretory expression in eukaryotes
GB8412517D0 (en) * 1984-05-16 1984-06-20 Nagai K Recombinant fusion proteins
US5010010A (en) * 1986-10-22 1991-04-23 Selmer-Sande, A.S. Production of human parathyroid hormone from microorganisms
US5420242A (en) * 1986-10-22 1995-05-30 Kaare M. Gautvik Production of human parathyroid hormone from microorganisms
AU627147B2 (en) * 1986-10-22 1992-08-20 Peter Alestrom Production of human parathyroid hormone from microorganisms
SE9702401D0 (sv) 1997-06-19 1997-06-19 Astra Ab Pharmaceutical use
CN103275223B (zh) * 2013-06-04 2015-06-17 福建省洪诚生物药业有限公司 降钙素原抗体的制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2068969B (en) * 1980-02-05 1983-07-27 Upjohn Co Gene expression

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8401173A1 *

Also Published As

Publication number Publication date
WO1984001173A1 (en) 1984-03-29

Similar Documents

Publication Publication Date Title
US4916212A (en) DNA-sequence encoding biosynthetic insulin precursors and process for preparing the insulin precursors and human insulin
JP2930984B2 (ja) Ｅ．Ｃｏｌｉからの異種蛋白質の排泄
EP0128733B1 (de) Menschlicher, insulinähnlicher Wachstumsfaktor (IGF)hergestellt aus einem rekombinanten Wirt, Verfahren, Expressionsvektor und rekombinanter Wirt hierfür und IGF enthaltende pharmazeutische Zusammensetzung
CA2087261C (en) Purification directed cloning of peptides
US4431740A (en) DNA Transfer vector and transformed microorganism containing human proinsulin and pre-proinsulin genes
US6500645B1 (en) N-terminally extended proteins expressed in yeast
CA2033176C (en) Growth hormone fusion proteins
US5707826A (en) Enzymatic method for modification of recombinant polypeptides
EP0195691A1 (de) Verfahren zum Herstellung von Insulin-Vorläufern und Verfahren zur Herstellung von Humaninsulin
IE840975L (en) Human insulin - like growth factor.
JPH10501695A (ja) イーストにおいて発現されたｎ末端に伸長した蛋白質
CA1180289A (en) Human proinsulin and preproinsulin genes
EP0119247A1 (de) Herstellung reifer proteine in transformierter hefe
WO1988010299A1 (en) A process for preparing a protein or polypeptide, a dna sequence coding for the polypeptide, a microorganism containing the dna sequence as well as the polypeptide and its use as a pharmaceutical preparation
JP4180112B2 (ja) 酵母細胞におけるｎ末端を伸長されたタンパクの発現のためのベクター
RU2144957C1 (ru) РЕКОМБИНАНТНАЯ ПЛАЗМИДНАЯ ДНК pPINS07, КОДИРУЮЩАЯ ГИБРИДНЫЙ ПОЛИПЕПТИД, СОДЕРЖАЩИЙ ПРОИНСУЛИН ЧЕЛОВЕКА, И ШТАММ БАКТЕРИЙ Escherichia coli - ПРОДУЦЕНТ ГИБРИДНОГО ПОЛИПЕПТИДА, СОДЕРЖАЩЕГО ПРОИНСУЛИН ЧЕЛОВЕКА
CA2357072C (en) Expression of a human insulin precursor in p. pastoris
EP1169461B1 (de) Verwendung von pankreatischer Procarboxypeptidase B zur Herstellung von Insulin
EP0534705A2 (de) Expression von niedermolekularen rekombinanten Polypeptiden
JP4668414B2 (ja) 形質転換した酵母細胞におけるタンパク質の製造方法
EP0361956A2 (de) Expression von rekombinanten Proteinen mit niedrigem Molekulargewicht
Wengenmayer Synthesis of peptide hormones using Recombinant DNA techniques
JP2666069B2 (ja) 組換え型鳥類プロラクチン又は組換え型鳥類プレプロラクチン，組換え型ニワトリプロラクチン，組換え型ニワトリプロラクチン構造遺伝子，組換え型ニワトリプレプロラクチン，組換え型ニワトリプレプロラクチン構造遺伝子，組換えプラスミド，組換えプラスミドを含む微生物
RU2167939C2 (ru) Способ продуцирования полипептида в дрожжах
CA1302321C (en) Preparation of polypeptides

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: 19841227

RIN1 Information on inventor provided before grant (corrected)

Inventor name: MAHONEY, WALTER, C.