USH245H - Plasmid for producing human insulin - Google Patents

Plasmid for producing human insulin Download PDF

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Publication number
USH245H
USH245H US06/625,176 US62517684A USH245H US H245 H USH245 H US H245H US 62517684 A US62517684 A US 62517684A US H245 H USH245 H US H245H
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United States
Prior art keywords
plasmid
preproinsulin
human
partial
product
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Abandoned
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US06/625,176
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English (en)
Inventor
Chander P. Bahl
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Novartis Vaccines and Diagnostics Inc
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Cetus Corp
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Priority to US06/625,176 priority Critical patent/USH245H/en
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Publication of USH245H publication Critical patent/USH245H/en
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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

  • This invention relates to molecular biology and more particularly to the so-called art of recombinant DNA. Specifically the invention relates to recombinant plasmids for producing human insulin.
  • the invention describes novel genetically engineered plasmids that carry a gene coding for human proinsulin.
  • Representative plasmid, pJW2172 illustrates the invention and has been deposited with the American Type Culture Collection, Rockville, Md., 20852; it has been awarded ATCC number 31891. Applicant has directed that the plasmid be freely available to the general public upon the issuance of a U.S. patent.
  • insulin is a peptide hormone that emphasizely regulates a variety of vital metabolic events.
  • insulin is synthesized as a single large precursor polypeptide, preproinsulin.
  • a transport sequence and an internal peptide sequence are excised from preproinsulin during biosynthesis, yielding a biologically active molecule consisting of two peptide chains linked together by disulfide bonds. Excision of only the transport sequence yields a peptide molecule known as proinsulin.
  • Proinsulins have now been isolated and characterized from a number of animals. See Steiner, D., "Peptide Hormone Precursors" in Peptide Hormones, Parsons, J. A., Editor, McMillan Press Limited, London (1976) at pages 49-64.
  • the beta-cells of the islets of Langerhans produce insulin in vivo.
  • insulin is expressed as a fused polypeptide comprised of the proinsulin sequence plus a pre-sequence known as the transport or signal sequence.
  • This transport sequence facilitates passage of the insulin molecule through the cellular membranes.
  • the preproinsulin is synthesized on ribosomes that are associated with the rough endoplasmic reticulum. See Permutt, M. and Kipnis, D. M., Proc. Nat. Acad. Sci., USA, 69:506-509 (1972).
  • the newly translated peptide When it is produced in vivo, the newly translated peptide is first transported intracellularly to the Golgi apparatus where it becomes incorporated within newly forming secretion granules. Exactly how the cell converts proinsulin to insulin is not known, but, conversion is believed to be initiated in either the Golgi apparatus or in the newly formed secretion granules.
  • the secretion granules contain zinc and membrane bound proteases which are believed to participate in the conversion mechanism.
  • the types of proteases can vary from species to species, perhaps to accomodate species specific amino acid substitutions in the insulin peptide. See Steiner, supra at 54.
  • Goedell et al technique provides one method of producing human insulin
  • a more natural approach involves the biosynthesis of proinsulin, which is the immediate precursor of insulin.
  • An advantage of producing insulin from proinsulin is the economy of fermenting and processing only one recombinant organism.
  • the Goeddell et al technique requires the use of two recombinant organisms. Numerous researchers have shown that proinsulin will oxidize spontaneously to form the correct disulfide bonds and can be quantitatively converted to insulin by controlled digestion with trypsin and carboxypeptidase B. See Steiner, D. and Clark, J. Proc. Nat. Acad. Sci., USA 60:622-629 (1968); and Kemmler, W. et al. J. Biol.
  • a further object of the present invention is to create a recombinant plasmid capable of transforming a host organism so that the host will express human proinsulin as a preproinsulin product that will be processed to human proinsulin by the host and then transported across the host's cell membrane.
  • a further object of the present invention is to demonstrate actual expression of the preproinsulin product by a host organism transformed by a recombinant plasmid carrying the gene for human proinsulin.
  • Another object of the present invention is to demonstrate that the preproinsulin product expressed by a host organism transformed by a recombinant plasmid carrying the gene for human proinsulin is processed to human proinsulin within the host and then transported across the host's cell membrane.
  • FIG. 1 shows the distribution of immunoreactivity after gel filtration of osmotic shockates from bacterial cultures containing plasmid pJW2172. Elution positions of standard proteins are also indicated.
  • FIG. 2 shows the distribution of radioactivity measured in slices obtained from tube gel SDS electrophoresis of an aliquot of 125 I-labeled immunoprecipitated plasmid pJW2172 product.
  • the vertical bar indicates the position of the stained band of bovine proinsulin.
  • FIG. 3 shows the distribution of radioactivity obtained from automated sequential Edman degradation of 125 I-labeled proinsulin product of plasmid pJW2172.
  • FIG. 4 shows the distribution of radioactivity obtained from automated sequential Edman degradation of 35 S-labeled proinsulin product of plasmid pJW2172.
  • FIG. 5 shows the binding of 125 I-labeled plasmid pJW2172 immunoprecipitated proinsulin to a human C-peptide antiserum. Samples were tested with normal rabbit serum (N) and human C-peptide antiserum (C).
  • the invention involves construction of novel recombinant plasmids carrying the human proinsulin structural gene in expressible form.
  • a functional transport presequence Immediately preceeding the eukaryotic proinsulin structural gene on the plasmid is a functional transport presequence.
  • the presequence can be comprised entirely of a prokaryotic bacterial transport presequence, entirely of a eukaryotic transport presequence, or of any functional combination of the two presequence types.
  • the eukaryotic proinsulin structural gene sequence is oriented in the plasmid so that it is in correct translational reading frame with respect to the initiation codon of the functional transport presequence. Such an orientation allows a preproinsulin product to be synthesized by actively metabolizing transformed host organisms.
  • the preproinsulin product is correctly processed to human proinsulin by the transformed host organisms, which then transport the human peptide across the cell membrane.
  • the human proinsulin In a transformed E. coli host, the human proinsulin accumulates in the bacterial periplasmic space. Following recovery, in vitro methods can be used to convert the human proinsulin to human insulin. See Steiner and Clark, supra, and Kemmler et al., supra.
  • the recombinant plasmids could be used to transform host organisms that are not capable of correctly processing preproinsulin to human proinsulin in vivo. In that case the preproinsulin peptide would be recovered by disrupting the transformed hosts' cell walls. The transport sequence portion of the preproinsulin peptide could be removed enzymatically.
  • Plasmid pJW2172 was constructed by modifying a parental plasmid, plasmid pHn677, which in turn was derived from the well-characterized plasmid pBR322 (ATCC 37017).
  • Parental plasmid pHn677 which contains human preproinsulin cDNA cloned into the Pst I site of the ampicillinase gene of plasmid pBR322, was modified with a restriction endonuclease and a double stranded exonuclease. Large portions of the ampicillinase coding region were excised by these enzymes, resulting in the production of a variety of fused gene segments. Many of these generated proteins were detectable with insulin or C-peptide antisera. One such fused gene segment generating these detectable proteins is carried by plasmid pJW2172.
  • Plasmid pJW2172 contains a perfect fused hybrid gene segment consisting of the amino-terminal coding half of the leader sequence of the prokaryotic ampicillinase (residues -23 to - 12) fused to the eukaryotic human preproinsulin prepeptide beginning at residue -13. Expression of the fused segment results in the synthesis of a preproinsulin product, in vivo processing of the preproinsulin product and finally, and secretion of human proinsulin into the periplasmic space of E. coli host organisms transformed by plasmid pJW2172. Characterization of the recovered human proinsulin product shows that it contains the A and B chain regions of insulin as well as specific C-peptide immunodeterminants. Tryptic digestion can be used in vitro to convert proinsulin to insulin. See Steiner and Clark, supra, and Kemmler et al., supra.
  • Parental plasmid, pHn677 was obtained originally by cDNA cloning of mRNA isolated from human insulinoma. The cDNA was inserted into the Pst I site of pBR322 by means of dC-dG tailing. Plasmid pHn677 contains all of the proinsulin sequence plus the coding sequence for 16 amino acids of the presequence. Although the DNA sequence in the parental plasmid was in the right orientation with respect to the beta-lactamase promoter, it was in the wrong reading frame with respect to the initiation codon of the beta-lactamase gene. As a result there was no expression of the DNA sequence coding for the human proinsulin gene.
  • Plasmid pHn677 was digested with restriction endonuclease Pvu I.
  • the DNA was then treated with double stranded exonuclease Bal 31 to digest approximately 150 nucleotides from each end.
  • This DNA was further treated with restriction endonuclease Hind III. After subjecting the resulting DNA to gel electrophoresis on agarose gels, the larger set of DNA fragments was isolated from the gels by electroelution.
  • Hinc II digested plasmid pBR322 DNA was subjected to Bal 31 to digest approximately 180 nucleotides from each end of the linearized plasmid. This DNA was then further digested with Hind III and Bam H1. Finally the fragment containing the beta-lactamase promoter region was isolated from an acrylamide gel. This fragment was ligated to the Pvu 1/Bal 31/Hind III treated large fragment. The ligation mixture was used to transform E. coli K12 strain CS412.
  • Transformants were first screened for tetracycline resistance. Some 350 clones exhibiting such resistance were further examined for insulin expression using the in situ radioimmunoassay procedure described by Broom, S. and Gilbert W., Proc. Nat. Acad. Sci., USA 75:2746-2749 (1978). This procedure showed that 118 of the 350 clones might be expressing insulin. Therefore, these 118 clones were further assayed by means of a radioimmunoassay that utilized both the anti-insulin and the anti-C-peptide antibodies. The radioimmunoassays showed that at least 23 of these clones showed significant, i.e. greater than 1 ⁇ unit per ml of insulin activity.
  • Plasmid pJW2172 From the total series of plasmids containing a variety of hybrid bacterial-eukaryotic transport sequences attached to the human proinsulin sequence, one plasmid, designated as plasmid pJW2172, was selected for further study. As described in Chan, S. et al Proc. Nat. Acad. Sci., USA 78:5401-5405 (1981), and illustrated here in FIG.
  • the proinsulin material generated by plasmid pJW2172 was characterized in the following manner.
  • a sample of an osmotic shockate, from cultures of bacteria transformed by plasmid pJW2172, containing about 0.05 nM of the peptide was extracted with acid ethanol.
  • the extract was then subjected to gel filtration on a column of Biogel P60 eluted with 2.5 M propionic acid. See generally, Steiner, D., et al., in Cell Biology: A Comprehensive Treatise 4:175-201 (1980).
  • the second aliquot was used to determine whether cleavage of the presequence had occurred at the first residue of human proinsulin.
  • the 5.0 pM aliquot of the immunoprecipitate was first reduced and carboxymethylated, and then subjected to automated Edman degradation. See Chan et al., supra. The results are shown in FIG. 3. They show that significant amounts of radioactively labeled tyrosine were found only at positions 16 and 26, as expected for human proinsulin. There was no indication of heterogeneity at the N-terminus. Further corroboration of this point was obtained when the osmotic shockate from a culture grown in the presence of 35 SO 4 was similarly immunoprecipitated, gel filtered, reduced, carboxymethylated and then sequenced. These results are shown in FIG. 4; they demonstrate the presence of the sulphur labeled B7 and B19 S-carboxymethyl-cysteine residues in correct register.
  • the plasmid pJW2172 proinsulin product was treated with trypsin and carboxypepsidase B. This treatment converted the product to a component eluting at the position of insulin on gel filtration. There was no indication of the release of free A or B chain material.
  • the invention involves construction of novel recombinant plasmids carrying the human proinsulin structural gene in expressible form.
  • a functional transport presequence Immediately preceeding the eukaryotic structural gene on each plasmid is a functional transport presequence.
  • the presequence can be comprised entirely of a prokaryotic bacterial transport presequence, entirely of a eukaryotic transport presequence, or of any functional combination of the two.
  • Representative plasmid pJW2172 carries the human proinsulin structural gene attached to a functional fused hybrid transport presequence.
  • the hybrid transport presequence is comprised of roughly half of the prokaryotic bacterial presequence fused to roughly half of the eukaryotic human presequence.
  • the bacterial presequence codes for the amino-terminal end of the hybrid transport peptide; the human presequence codes for the carboxy-terminal end.
  • E. coli host becteria transformed by plasmid pJW2172 express a preproinsulin peptide product that is correctly processed to human proinsulin by the bacteria. This human proinsulin is transported by the bacteria across the cell membrane to the periplasmic space, where it accumulates. Following its recovery, in vitro methods such as tryptic digestion can be used to convert the human proinsulin to human insulin.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
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  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Endocrinology (AREA)
  • Microbiology (AREA)
  • Diabetes (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US06/625,176 1981-06-29 1984-06-27 Plasmid for producing human insulin Abandoned USH245H (en)

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US06/625,176 USH245H (en) 1981-06-29 1984-06-27 Plasmid for producing human insulin

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US27833181A 1981-06-29 1981-06-29
US06/625,176 USH245H (en) 1981-06-29 1984-06-27 Plasmid for producing human insulin

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US (1) USH245H (da)
EP (1) EP0070632A3 (da)
JP (1) JPS58501227A (da)
AU (1) AU556980B2 (da)
CA (1) CA1201074A (da)
DK (1) DK84583A (da)
ES (1) ES513522A0 (da)
IL (1) IL66102A0 (da)
NO (1) NO830682L (da)
PT (1) PT75075B (da)
WO (1) WO1983000164A1 (da)
ZA (1) ZA824218B (da)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4767709A (en) 1984-06-28 1988-08-30 The Johns Hopkins University Growth-related hormones
US5496924A (en) * 1985-11-27 1996-03-05 Hoechst Aktiengesellschaft Fusion protein comprising an interleukin-2 fragment ballast portion
US6001604A (en) * 1993-12-29 1999-12-14 Bio-Technology General Corp. Refolding of proinsulins without addition of reducing agents
US6348327B1 (en) * 1991-12-06 2002-02-19 Genentech, Inc. Non-endocrine animal host cells capable of expressing variant proinsulin and processing the same to form active, mature insulin and methods of culturing such cells
CN107794471A (zh) * 2016-08-31 2018-03-13 通用电气公司 使用Laves相析出在IN706中的晶粒细化

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983003413A1 (en) * 1982-03-31 1983-10-13 Genetics Inst Creation of dna sequences encoding modified proinsulin precursors
DE3245665A1 (de) * 1982-05-04 1983-11-10 Boehringer Mannheim Gmbh, 6800 Mannheim Verfahren zur herstellung permanenter tierischer und humaner zellinien und deren verwendung
US5670371A (en) * 1983-07-15 1997-09-23 Bio-Technology General Corp. Bacterial expression of superoxide dismutase
DK58285D0 (da) * 1984-05-30 1985-02-08 Novo Industri As Peptider samt fremstilling og anvendelse deraf
PH25772A (en) * 1985-08-30 1991-10-18 Novo Industri As Insulin analogues, process for their preparation
DE3752347T2 (de) * 1986-05-20 2002-09-26 The General Hospital Corp., Boston Verfahren zur pharmakokinetischen Studie der Insulin-Expression mit nicht-menschlichem Transgen-Säugetier

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0026598A1 (en) 1979-09-12 1981-04-08 The Regents Of The University Of California DNA transfer vectors for human proinsulin and human pre-proinsulin, microorganisms transformed thereby, and processes involving such substances
EP0036258A2 (en) 1980-03-14 1981-09-23 Cetus Corporation Process for producing aspartame

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5210872B2 (da) * 1973-07-14 1977-03-26
US4082613A (en) * 1976-04-23 1978-04-04 The Regents Of The University Of Minnesota Process for the production of insulin by genetically transformed fungal cells
US4411994A (en) * 1978-06-08 1983-10-25 The President And Fellows Of Harvard College Protein synthesis
IE48385B1 (en) * 1978-08-11 1984-12-26 Univ California Synthesis of a eucaryotic protein by a microorganism
DE3001928A1 (de) * 1980-01-19 1981-08-06 Hoechst Ag, 6000 Frankfurt Genprodukt eines hoeheren organismus aus einem dieses gen enthaltenden mikroorganismus
US4338397A (en) * 1980-04-11 1982-07-06 President And Fellows Of Harvard College Mature protein synthesis
GR76959B (da) * 1981-01-02 1984-09-04 Univ New York State Res Found

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0026598A1 (en) 1979-09-12 1981-04-08 The Regents Of The University Of California DNA transfer vectors for human proinsulin and human pre-proinsulin, microorganisms transformed thereby, and processes involving such substances
EP0036258A2 (en) 1980-03-14 1981-09-23 Cetus Corporation Process for producing aspartame

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Bell et al., Nature, vol. 282, 525-527 (1979).
Chan et al., Proc. Natl. Acad. Sci. (US) 78 5401-5405 (1981).
Talmadge et al., Proc. Natl. Acad. Sci. (US) 77 3369-3373 (1980).

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4767709A (en) 1984-06-28 1988-08-30 The Johns Hopkins University Growth-related hormones
US5496924A (en) * 1985-11-27 1996-03-05 Hoechst Aktiengesellschaft Fusion protein comprising an interleukin-2 fragment ballast portion
US6348327B1 (en) * 1991-12-06 2002-02-19 Genentech, Inc. Non-endocrine animal host cells capable of expressing variant proinsulin and processing the same to form active, mature insulin and methods of culturing such cells
US6001604A (en) * 1993-12-29 1999-12-14 Bio-Technology General Corp. Refolding of proinsulins without addition of reducing agents
CN107794471A (zh) * 2016-08-31 2018-03-13 通用电气公司 使用Laves相析出在IN706中的晶粒细化

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NO830682L (no) 1983-02-28
PT75075A (en) 1982-07-01
DK84583D0 (da) 1983-02-24
EP0070632A3 (en) 1983-06-29
ZA824218B (en) 1983-04-27
DK84583A (da) 1983-02-24
PT75075B (en) 1983-12-28
IL66102A0 (en) 1982-09-30
JPS58501227A (ja) 1983-07-28
WO1983000164A1 (en) 1983-01-20
CA1201074A (en) 1986-02-25
AU556980B2 (en) 1986-11-27
AU8762382A (en) 1983-02-02
EP0070632A2 (en) 1983-01-26
ES8304430A1 (es) 1983-03-01
ES513522A0 (es) 1983-03-01

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