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WO1998001473A1 - Procede de production de precurseurs de l'insuline, precurseurs d'analogues de l'insuline et peptides insulinoides - Google Patents

Procede de production de precurseurs de l'insuline, precurseurs d'analogues de l'insuline et peptides insulinoides Download PDF

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WO1998001473A1
WO1998001473A1 PCT/DK1997/000297 DK9700297W WO9801473A1 WO 1998001473 A1 WO1998001473 A1 WO 1998001473A1 DK 9700297 W DK9700297 W DK 9700297W WO 9801473 A1 WO9801473 A1 WO 9801473A1
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insulin
yeast
precursor
yap3
promoter
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PCT/DK1997/000297
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English (en)
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Michi Egel-Mitani
Jakob Brandt
Knud Vad
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Novo Nordisk A/S
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Priority to AU32553/97A priority Critical patent/AU3255397A/en
Publication of WO1998001473A1 publication Critical patent/WO1998001473A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the present invention relates to a novel method for the production of precursors of insulin, precursors of insulin analogues, and insulin like peptides in genetically modified yeast cells, said genetically modified yeast cells, and a method for the preparation of said yeast cells.
  • heterologous proteins in yeast after transformation of yeast cells with suitable expression vectors comprising DNA sequences coding for said proteins has been successful for precursors of insulin, precursors of insulin analogues, and insulin like peptides.
  • Yeasts, and especially Saccharomyces cerevisiae are preferred host microorganisms for the production of pharmaceutically valuable polypeptides due to the stable yield and safety.
  • EP 341215 describes the use of a yeast strain that lacks carboxypeptidase ysc ⁇ activity for the expression of the heterologous protein hirudin. Wild-type yeast strains produce a mixture of desulphatohirudin species differing in the C-terminal sequence due to the post-translational action of endogeneous yeast proteases on the primary expression product.
  • yeast mutant strains lacking carboxypeptidase ysc ⁇ activity are unable to remove amino acids from the C-terminus of heterologous proteins and therefore give rise to integral proteins.
  • the use of yeast strains defective in ysc A, B, Y, and/or S activity can only partially reduce random proteolysis of foreign gene products.
  • Another problem encountered in production of heterologous proteins in yeast is low yield, presumably due to proteolytic processing both in intracellular compartments and at the plasma membrane caused by aberrant processing at internal sites in the protein, e.g. secretion of human parathyroid hormone (Gabrielsen et al. Gene 90: 255-262, 1990; Rokkones et al. J. Biotechnol. 33: 293-306, 1994), and secretion of ⁇ - endorphine by S. cerevisiae (Bitter et al. Proc. Natl. Acad. Sci. USA 81 : 5330-5334, 1984).
  • WO 95/23857 discloses production of recombinant human albumin (rHA) in yeast cells having a reduced level of yeast aspartyl protease 3 (Yap3p) proteolytic activity resulting in a reduction of undesired 45 kD rHA fragment, and in a 30 to 50% increased yield of recovered rHA produced by the haploid ⁇ yap3 strain compared to the rHA produced by the corresponding haploid YAP3 wild-type strain.
  • Yap3p yeast aspartyl protease 3
  • Bourbonnais et al. (Biochimie 76: 226-233, 1994), have shown that the YAP3 protease gene product has in vitro substrate specificity which is distinct though overlapping with the Kex2p substrate specificity, and shown that Yap3p cleaves exclusively C-terminal to arginine residues present in the prosomatostatin's putative processing sites.
  • Cawley et al. (J. Biol. Chem. 271 : 4168-4176, 1996) have determined the in vitro specificity and relative efficiency of cleavage of mono- and paired-basic residue processing sites by Yap3p for a number of prohormone substrates, such as bovine proinsulin.
  • the purpose of the present invention is to provide an improved method for the production of secreted precursors of insulin, precursors of insulin analogues, and insulin related peptides in a yeast expression system.
  • the production of precursors of insulin, precursors of insulin analogues, and insulin related peptides by the method of the invention is increased considerably, e. g. from about 60 to about 350%, more preferably from about 100 to about 300%, compared to the production of precursors of insulin, precursors of insulin analogues, and insulin like peptides in conventional yeast expression systems, and/or, preferably, the level of proteolysis of the secreted product resulting in an inhomogeneous product is decreased considerably.
  • the method according to the present invention comprises culturing a yeast which has reduced activity of Yap3p or a homologue thereof and has been transformed with a hybrid vector comprising a yeast promoter operably linked to a DNA sequence coding for a precursor of insulin, a precursor of an insulin analogue, or an insulin related peptide, and isolating said precursor of insulin, precursor an insulin analogue, or an insulin related peptide.
  • the yeast cells lack Yap3p activity through disruption of the YAP3 gene.
  • the yeast is S. cerevisiae which lacks a functional YAP3 gene.
  • other yeast genera may have equivalent proteases, i.e. homologues of Yap3p, e. g. the genera Pichia and Kluyveromyces as shown in WO 95/23857 and Clerc et al. (J.
  • a gene is regarded as a homologue, in general, if the sequence of the translation product has greater than 50% sequence identity to Yap3p.
  • Komano and Fuller Proc. Natl. Acad. Sci, USA 92: 10752-10756, 1995
  • Other aspartyl proteases of Saccharomyces include the gene products of PEP4, BAR1 , and of open reading frames, the sequences of which are partially homologous with the YAP3 open reading frame, such as YAP3-link (coded by GenBank ace. No.
  • yeast gene names YAP3, YAP3 link, YIV 9, NO 4, and MKC 7 used herein correspond to the yeast open reading frame YLR120C, YLR121C, YIR039C, YDR349C, and YDR144C, respectively.
  • the gene product of open reading frame YGL259W is included among the yeast aspartyl proteases.
  • yeasts examples include Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., and Geotrichum fermentans.
  • a suitable means of eliminating the activity of a protease is to disrupt the host gene encoding the protease, thereby generating a non-reverting strain missing all or part of the gene for the protease including regulatory and/or coding regions, or, alternatively, the activity can be reduced or eliminated by classical mutagenesis procedures or by the introduction of specific point mutations.
  • Other methods which may be suitable for down regulation of the protease include the introduction of antisense and/or ribozyme constructs in the yeast, e.g. Atkins et al. (Antisense and Development 5: 295-305, 1995) and Nasr et al. (Mol. Gen Genet 249: 51-57, 1995).
  • the precursors of insulin, precursors of insulin analogues, and insulin related peptides may be of human origin or from other animals and recombinant or semisynthetic sources.
  • the cDNA used for expression of precursors of insulin, precursors of insulin analogues, or insulin related peptides in the method of the invention include codon optimised forms for expression in yeast.
  • precursors of insulin or precursors of insulin analogues we include all precursors of human insulin, preferably precursors of des(B30) human insulin, porcine insulin, and insulin analogues wherein at least one Lys or Arg is present.
  • preferred insulin analogues among those in which a Lys or Arg is present are insulin analogues in which Phe B1 has been deleted, insulin analogues in which the A-chain and/or the B- chain have an N-terminal extension and insulin analogues in which the A-chain and/or the B-chain have a C-terminal extension.
  • a parent insulin may instead of Asn have an amino acid residue selected from the group comprising Ala, Gin, Glu, Gly, His, lie, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular an amino acid residue selected from the group comprising Gly, Ala, Ser, and Thr.
  • Precursors of insulin analogues produced according to the method of the present invention may also be modified by a combination of changes outlined above.
  • a parent insulin precursor may instead of Pro have an amino acid residue selected from the group comprising Asp or Lys and in position B29 a parent insulin precursor may instead of Lys have the amino acid Pro.
  • Futhermore by "precursors of insulin analogue” as used herein is meant a peptide having a molecular structure similar to that of human insulin precursor including the di- sulphide bridges between Cys A7 and Cys B7 and between Cys* 20 and Cys B19 and an internal disulphide bridge between Cys A6 and Cys A1 ⁇ and which can be processed to a polypeptide having insulin activity.
  • a codable amino acid residue designates an amino acid residue which can be coded for by the genetic code, i. e. a triplet ("codon") of nucleotides.
  • the insulin related polypeptides are IGF-1 (insulin " like growth factor-1) and insulin single-chain hybrids, such as the SC hybrid, which designates a polypeptide consisting of the insulin B- and A-chains connected by the IGF-I C-peptide, cf. Kristensen et al. (Biochem J. 305: 981-986, 1995), and WO95/16708, and the insulin single-chain hybrids described in EP 741188.
  • a second aspect of the invention provides a culture of yeast cells containing a polynucleotide sequence, preferably a first DNA sequence, encoding a precursor of insulin, a precursor of insulin analogues, or insulin related peptides, and a second polynucleotide sequence, preferably a second DNA sequence, encoding a secretion signal causing said precursor of insulin, precursor of insulin analogues, or insulin like peptides to be secreted from the yeast, characterized in that the yeast cells have reduced Yap3 protease activity.
  • the yeast cells are transformed with a hybrid vector comprising said first DNA sequence and said second DNA sequence, and, preferably, the yeast cells lack Yap3p activity, and this may conveniently be obtained through disruption of the YAP3 gene.
  • the culture of yeast cells according to the invention is haploid or polyploid, preferably diploid.
  • the DNA encoding the precursor of insulin, precursor of insulin analogue, or insulin related peptide may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration on host chromosome is desired.
  • the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • the vector is then introduced into the host through standard techniques and, generally, it will be necessary to select for transformed host cells.
  • the DNA is inserted into an yeast integration plasmid vector, such as pJJ215, pJJ250, pJJ236, pJJ248, pJJ242 (Jones & Prakash, Yeast 6: 363,1990) or pDP6 (Fleig et al. Gene 46:237, 1986), in proper orientation and correct reading frame for expression, which is flanked with homologous sequences of any non-essential yeast genes, transposon sequence or ribosomal genes.
  • the flanking sequences are yeast protease genes or genes used as a selective marker.
  • the DNA is then integrated on host chromosome(s) by homologous recombination occured in the flanking sequences, by using standard techniques shown in Rothstein (Method in Enzymol, 194: 281-301 , 1991) and Cregg et al. (Bio/Technol. 11 :905-910, 1993).
  • Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression and secretion of the precursor of insulin, precursor of insulin analogues, or insulin like peptides, which can then be recovered, as is known.
  • Useful yeast plasmid vectors include the POT (Kjeldsen et al. Gene 170: 107-112, 1996) and YEp13, YEp24 (Rose and Broach, Methods in Enzymol. 185: 234-279, 1990), and pG plasmids (Schena et al. Methods in Enzymol. 194: 289-398, 1991).
  • Methods for the transformation of S. cerevisiae include the spheroplast transformation, lithium acetate transformation, and electroporation, cf. Methods in Enzymol. Vol. 194 (1991 ). Pereferably the transformation is as described in the examples herein.
  • Suitable promoters for S. cerevisiae include the MF ⁇ l promoter, galactose inducible promoters such as GAL1 , GAL7 and GAL10 promoters, glycolytic enzyme promoters including TPI and PGK promoters, TRP1 promoter, CYCI promoter, CUP1 promoter, PH05 promoter, ADH1 promoter, and HSP promoter.
  • a suitable promoter in the genus Pichia is the AOXI (methanol utilisation) promoter.
  • the transcription terminal signal is preferably the 3' flanking sequence of a eucaryotic gene which contains proper signal for transcription termination and polyadenylation.
  • Suitable 3' flanking sequences may, e.g. be those of the gene naturally linked to the expression control sequence used, i.e. corresponding to the promoter.
  • the DNA constructs that are used for providing secretory expression of precursors of insulin, precursors of insulin analogues, or insulin related peptides according to the invention comprising a DNA sequence that includes a leader sequence linked to the polypeptide by a yeast processing signal.
  • the leader sequence contains a signal peptide ("pre-sequence") for protein translocation across the endoplasmic reticulum and optionally contains an additional sequence (“pro-sequence”), which may or may not be cleaved within yeast cells before the polypeptide is released into the surrounding medium.
  • Useful leaders are the signal peptide of mouse ⁇ -amylase , S cerevisiae MF ⁇ l , YAP3, BAR1 , HSP150 and S.
  • kluyveri MF ⁇ signal peptides and prepro-sequences of S. cerevisiae MF ⁇ l , YAP3, PRC, HSP150, and S. kluyveri MF ⁇ and synthetic leader sequences described in WO 92/11378, WO 90/10075 and WO 95/34666.
  • the precursor of insulin, precursor of insulin analogues, or insulin related peptides to be produced according to the the method of invention may be provided with an N-terminal extension as described in WO 95/35384.
  • the invention also relates to a method of preparing a yeast having reduced Yap3p activity comprising the steps of a) providing a hybrid plasmid containing a part of the YAP3 gene and suitable for transformation into a yeast cell, b) disrupting the YAP3 gene by deleting the fragment of YAP3 and inserting the URA3 gene instead to obtain a ⁇ yap3::URA3 gene disruption plasmid, c) providing a yeast ⁇ ura3 deletion mutant, d) transforming said mutant with said plasmid, and e) selecting the ⁇ yap3::URA3 deletion mutants on a medium without uracil. Further the invention relates to a method of preparing a yeast having reduced Yap3p activity using antisense technology.
  • the precursors of insulin, precursors of insulin analogues, or insulin related peptides to be produced according to the method of the invention may conveniently be expressed coupled to an N- or C-terminal tag or as a precursor or fusion protein.
  • Fig. 1 shows the construction of the pS194 plasmid.
  • Fig. 2 shows the construction of plasmids pME834 and pME1389.
  • Fig. 3 is a restriction map of a general expression plasmid used herein.
  • Fig. 4 is a restriction map of the pME973 plasmid, containing the genes encoding the
  • the ⁇ ura3 deletion mutation was constructed as follows: pJJ244 (pUC18 containing a 1.2 kb Hindlll fragment of the URA3 gene) was digested with Sty I and filled in with Klenow polymerase and self ligated. The resulting plasmid designated pS194 contains a 84bp of Styl-Styl fragment deletion of the URA3 gene, cf. Fig.1.
  • the ⁇ yap3::URA3 gene disruption plasmid pME1389 was constructed as follows: The 2.6kb Sacl-Pstl fragment which contains the YAP3 gene in pME768 (Egel-Mitani et al. Yeast 6: 127-137, 1990) was inserted in 2.6 kb of the Sacl-Pstl fragment of plC19R (Marsh et al. Gene 32: 481-485, 1984). The resulting plasmid is pME834.
  • pME834 was digested with Hindlll to form a deletion of the 0.7 kb YAP3 fragment and the 1.2 kb Hindlll fragment of the URA3 gene (Rose et al. Gene 29: 113-124, 1984) was inserted instead.
  • the resulting plasmid is pME1389.
  • the construction of plasmids pME834 and pME1389 is shown in Fig. 2 in diagram
  • S. cerevisiae strain E11-3C (MAT ⁇ YAP3 pep4-3 ⁇ tpi::LEU2 leu2 URA3), cf. ATCC 20727, US pat. 4766073, was transformed with linialized pS194 (Bsgl digested) to make ⁇ ura deletion mutation.
  • 5-FOA 5-fluoro-orotic acid
  • strain SY107 (MAT ⁇ YAP3 pep4-3 ⁇ tpi::LEU2 Ieu2 ⁇ ura3), was then trans- formed with pME1389 previously being cut by Sail and Sad, and 3kb fragment of
  • ⁇ yap3::URA3 was isolated for the transformation.
  • ⁇ yap3::URA3 deletion mutants were selected on minimal plates without uracil.
  • URA3 transformants were characterized by PCR and Southern hybridisation to confirm the correct integration of the ⁇ yap3::URA3 fragment in the YAP3 locus.
  • ME1487 was isolated as a ⁇ yap3::URA3 deletion mutant (MAT ⁇ ⁇ yap3::URA3 pep4-3 ⁇ tpi::LEU2 Ieu2 ⁇ ura3).
  • ME 1487 was mutagenized by using EMS (methane-sulfonic acid ethylester) and ura3 mutants were selected on plates containing 5-FOA.
  • EMS methane-sulfonic acid ethylester
  • ura3 mutants were selected on plates containing 5-FOA.
  • One of the selected isolates, ME1656 was then subjected to mating type switch (Herskowitz and Jensen, Methods in Enzymol. 194: 132-146,1991) by transient transformation with pME973 shown in Fig. 4.
  • pME973 contains the genes encoding the HO (homothallism) endo- nuclease and URA3 inserted into the YEp13 plasmid (Rose and Broach, Methods in Enzymol. 185: 234-279, 1990).
  • ME1695 was selected as the haploid strain, which had switched from MAT ⁇ to MATa, and have the following genetic background: MATa ⁇ yap3::ura3 pep4-3 ⁇ tpi::LEU2 Ieu2 ⁇ ura3.
  • ME1695 was then crossed with ME1487 by micromanipulation (Sherman and Hicks, Methods in Enzymol. 194: 21-37, 1991) in order to get ⁇ yap3/ ⁇ yap3 diploids. From the resulting diploids, ME 1719 was selected as the strain with the following genetic background: MATa/ ⁇ ⁇ yap3::ura3/ ⁇ yap3::URA3 pep4-3/pep4-3 ⁇ tpi::LEU2/ ⁇ tpi::LEU2 Ieu2/leu2 ⁇ ura3/ ⁇ ura.
  • P1 and P2 each contains 40 nucleotides corresponding to sequences within the coding region of YLR121C and YAP3, respectively, as well as a Hindlll site (AAGCTT) and 12 nucleotides corresponding to sequences flanking the URA3 gene (YEL021W).
  • P1 and P2 were used for PCR using the URA3 gene as template.
  • the resulting 1248bp PCR fragment contains the URA3 selective marker flanked with 40 nucleotides derived from the YAP3 or YLR121C encoding regions.
  • the PCR fragment was then transformed into ME1655, and ⁇ yap3::URA3:: ⁇ ylr121c deletion mutants were selected and characterized as described in Example 1.
  • ME1684 was isolated as a ⁇ yap3::URA3:: ⁇ ylr121c mutant with the following genetic background: MAT ⁇ ⁇ yap3::URA3:: ⁇ ylr121c pep4-3 ⁇ tpi::LEU2 Ieu2 ⁇ ura3.
  • YPGGE medium 1% yeast extract, 2% peptone, 2% glycerol, 2% galactose, 1% ethanol
  • Cells were harvested by centrifugation and resuspended in 10ml SCE (1 M sorbitol, 0.1 M Na-citrate, 10mM Na 2 EDTA, pH5.8) + 2mg Novozyme SP234 and incubated at 30°C for 30 min. After cells were harvested by centrifugation and washed once by 10ml 1.2M sorbitol and subsequently by 10ml CAS (1M sorbitol 10mM CaCI 2 , 10mM Tris-HCI pH7.5), cells were harvested by centrifugation and resuspended finally in 2ml CAS. Competent cells were frozen in portion of 100 ⁇ l per Eppendorf tube at -80°C.
  • Transformation was made as follows: Frozen competent cells (100 ⁇ l) were warmed up quickly and 1 ⁇ g plasmid DNA were added. Cells were incubated at room temp, for 15 min. and 1ml PEG solution (20% polyethyleneglycol 4000, 10mM CaCI 2 , 10mM Tris-HCI pH7.5) was added. After 30min. at room temperature, cells were har- vested by centrifugation at 2000rpm for 15min. and resuspended in 100 ⁇ l SOS (1M sorbitol, 1/2 vol. YPGGE, 0.01% uracil, 7mM CaCI 2 ).
  • Yeast-E.coli shuttle vector (Fig. 3) used in the following examples contains a heterologous protein expression cassette, which includes a DNA sequence encoding a leader sequence followed by the heterologous polypeptide in question operably placed in between the TPI promoter and TPI terminator of S. cerevisiae in a POT plasmid (Kjeldsen et al. 1996, op. cit). Examples are shown as follows: Table 2
  • Insulin precursor EEAEPK-B chain(1-29)-AAK-A chain(1-21) (referred to as "EEAEPK-MI3" below) expression plasmid pAK729 equivalent to the plasmid shown in Fig. 3, in which the leader sequence-polypeptide is YAP3(1-21)-LA19KR- EEAEPK-MI3), was transformed into ME1487 ( ⁇ yap3), ME1719 ( ⁇ yap3/ ⁇ yap3) and SY107 (YAP3 WT). Transformants were selected by glucose utilization as a carbon source on YPD plates (1% w/v yeast extract, 2% w/v peptone, 2% glucose, 2% agar).
  • ME1541 and YES1729 are pAK729 transformants obtained from ME1487 ( ⁇ yap3) and ME1719 ( ⁇ yap3/ ⁇ yap3), respectively, whereas ME1540 is the pAK729 transformant obtained from SY107 ( ⁇ yap3).
  • Transformants were inoculated in 5ml YPD + 5mM CaCI 2 liquid medium and incubated at 30°C for 3 days with shaking at 200rpm. Culture supernatants were obtained after centrifugation at 2500rpm for 5 min. and 1ml supernatants were analyzed by reverse phase HPLC. Production levels shown in Table 3 were average value of cultures from 2 independently isolated transformants (Exp. 1) or values from a mixculture of 3 transformants, and were normalized so that YAP3 wild type level was taken as 100%. HPLC analyses showed that ME1541 and YES 1729 produced 1.7 to 2.5 times more insulin precursor than ME1540.
  • HPLC settings for analysis of precursors of insulin HPLC-Column: 4 x 250 mm Novo Nordisk YMC-OdDMeSi C18 5 urn
  • Insulin precursor is eluated from the HPLC columns with the following acetonitrile gradient:
  • Insulin precursor EEAEAEAK-B chain(1-29)-AAK-A chain(1-21) (refered to as "EEAEAEAK-MI3" below) expression plasmid pJB152 equivalent to the plasmid shown in Fig. 3, in which the leader sequence-polypeptide is MF ⁇ 1(1-81)MAKR- EEAEAEAK-MI3 (Kjeldsen et al. 1996, op. Cit ) was transformed into ME 1487 ( ⁇ yap3), ME1719 ( ⁇ yap3/ ⁇ yap3) and SY107 (YAP3 WT) and transformants were selected and analysed as desribed in Example 4.
  • ME1600 is the pJB152 transfor- mant obtained from ME1487( ⁇ yap3), whereas ME1599 is the pJB152 transformant obtained from SY107 (YAP3 WT).
  • Production levels shown in Table 4, were an average value from 2 independently isolated transformants, and were normalised so that the haploid YAP3 wild-type level of EEAEAEAK-MI3 insulin precursor was taken as 100%.
  • HPLC analyses showed that ME1600 produced 3.7-fold more EEAEAEAK- MI3 insulin precursor than ME1599.
  • the insulin precursor produced from ME1600 was homogeneous compared to that from ME1599, which produced 32% N- terminal trunkated insulin precursor in form of B chain(1-29)-AAK-A chain(1-21) (designated "MI3" in Tabel 4)

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Abstract

L'invention concerne un nouveau procédé de production de précurseurs de l'insuline, des précurseurs d'analogues de l'insuline et des peptides insulinoïdes, dans des cellules de levure modifiées génétiquement ayant une activité réduite de protéase YAP3. On décrit les cellules de levure modifiées génétiquement et un procédé pour leur préparation.
PCT/DK1997/000297 1996-07-05 1997-07-04 Procede de production de precurseurs de l'insuline, precurseurs d'analogues de l'insuline et peptides insulinoides WO1998001473A1 (fr)

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AU32553/97A AU3255397A (en) 1996-07-05 1997-07-04 Method for the production of precursors of insulin, precursors of insulin analogues, and insulin like peptides

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999037758A3 (fr) * 1998-01-27 1999-09-30 Univ Community College Sys Nev Expression de peptides sensibles a la proteolyse
WO2008034881A1 (fr) * 2006-09-22 2008-03-27 Novo Nordisk A/S Analogues de l'insuline résistants à une protéase
EP1975182A1 (fr) 2000-02-01 2008-10-01 PanGenetics B.V. Molécules d'activation des APC se liant au CD40
WO2013098651A1 (fr) * 2011-12-30 2013-07-04 Oxyrane Uk Limited Procédés et des matériels de réduction de la dégradation de protéines recombinantes
WO2013191369A1 (fr) * 2012-06-19 2013-12-27 한국생명공학연구원 Souche de levure au gène hpgas1 interrompu et procédé de production d'une protéine recombinante à l'aide de celle-ci
US8710001B2 (en) 2006-07-31 2014-04-29 Novo Nordisk A/S PEGylated, extended insulins
US9206408B2 (en) 2007-04-03 2015-12-08 Oxyrane Uk Limited Microorganisms genetically engineered to have modified N-glycosylation activity
US9249399B2 (en) 2012-03-15 2016-02-02 Oxyrane Uk Limited Methods and materials for treatment of pompe's disease
US9260502B2 (en) 2008-03-14 2016-02-16 Novo Nordisk A/S Protease-stabilized insulin analogues
US9347050B2 (en) 2010-09-29 2016-05-24 Oxyrane Uk Limited Mannosidases capable of uncapping mannose-1-phospho-6-mannose linkages and demannosylating phosphorylated N-glycans and methods of facilitating mammalian cellular uptake of glycoproteins
US9387176B2 (en) 2007-04-30 2016-07-12 Novo Nordisk A/S Method for drying a protein composition, a dried protein composition and a pharmaceutical composition comprising the dried protein
US9481721B2 (en) 2012-04-11 2016-11-01 Novo Nordisk A/S Insulin formulations
US9598682B2 (en) 2009-09-29 2017-03-21 Vib Vzw Hydrolysis of mannose-1-phospho-6-mannose linkage to phospho-6-mannose
US9688737B2 (en) 2008-03-18 2017-06-27 Novo Nordisk A/S Protease stabilized acylated insulin analogues
US9689015B2 (en) 2010-09-29 2017-06-27 Oxyrane Uk Limited De-mannosylation of phosphorylated N-glycans
US9896496B2 (en) 2013-10-07 2018-02-20 Novo Nordisk A/S Derivative of an insulin analogue
US10265385B2 (en) 2016-12-16 2019-04-23 Novo Nordisk A/S Insulin containing pharmaceutical compositions
US10287557B2 (en) 2009-11-19 2019-05-14 Oxyrane Uk Limited Yeast strains producing mammalian-like complex N-glycans
CN114380903A (zh) * 2021-12-28 2022-04-22 上海仁会生物制药股份有限公司 一种胰岛素或其类似物前体

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DIALOG INFORMATION SERVICES, File 154, Medline, Dialog Accession No. 08582247, Medline Accession No. 96200865, KJELDSEN T. et al., "A Removable Spacer Peptide in an Alpha-Factor-Leader/Insulin Precursor Fusion Protein Improves Processing and Concomitant Yield of the Insulin Precursor in Saccharomyces Cerevisiae"; & GENE, *

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999037758A3 (fr) * 1998-01-27 1999-09-30 Univ Community College Sys Nev Expression de peptides sensibles a la proteolyse
EP1975182A1 (fr) 2000-02-01 2008-10-01 PanGenetics B.V. Molécules d'activation des APC se liant au CD40
US8710001B2 (en) 2006-07-31 2014-04-29 Novo Nordisk A/S PEGylated, extended insulins
EP2404934A1 (fr) * 2006-09-22 2012-01-11 Novo Nordisk A/S Analogues d'insuline résistants à la protéase
EP2074141B1 (fr) 2006-09-22 2016-08-10 Novo Nordisk A/S Analogues de l'insuline resistants a une protease
US9018161B2 (en) 2006-09-22 2015-04-28 Novo Nordisk A/S Protease resistant insulin analogues
WO2008034881A1 (fr) * 2006-09-22 2008-03-27 Novo Nordisk A/S Analogues de l'insuline résistants à une protéase
US10023854B2 (en) 2007-04-03 2018-07-17 Oxyrane Uk Limited Microorganisms genetically engineered to have modified N-glycosylation activity
US9206408B2 (en) 2007-04-03 2015-12-08 Oxyrane Uk Limited Microorganisms genetically engineered to have modified N-glycosylation activity
US9222083B2 (en) 2007-04-03 2015-12-29 Oxyrane Uk Limited Microorganisms genetically engineered to have modified N-glycosylation activity
US9387176B2 (en) 2007-04-30 2016-07-12 Novo Nordisk A/S Method for drying a protein composition, a dried protein composition and a pharmaceutical composition comprising the dried protein
US9260502B2 (en) 2008-03-14 2016-02-16 Novo Nordisk A/S Protease-stabilized insulin analogues
US10259856B2 (en) 2008-03-18 2019-04-16 Novo Nordisk A/S Protease stabilized acylated insulin analogues
US9688737B2 (en) 2008-03-18 2017-06-27 Novo Nordisk A/S Protease stabilized acylated insulin analogues
US9598682B2 (en) 2009-09-29 2017-03-21 Vib Vzw Hydrolysis of mannose-1-phospho-6-mannose linkage to phospho-6-mannose
US10392609B2 (en) 2009-09-29 2019-08-27 Oxyrane Uk Limited Hydrolysis of mannose-1-phospho-6-mannose linkage to phospho-6-mannose
US10287557B2 (en) 2009-11-19 2019-05-14 Oxyrane Uk Limited Yeast strains producing mammalian-like complex N-glycans
US11225646B2 (en) 2009-11-19 2022-01-18 Oxyrane Uk Limited Yeast strains producing mammalian-like complex n-glycans
US9689015B2 (en) 2010-09-29 2017-06-27 Oxyrane Uk Limited De-mannosylation of phosphorylated N-glycans
US10011857B2 (en) 2010-09-29 2018-07-03 Oxyrane Uk Limited Mannosidases capable of uncapping mannose-1-phospho-6-mannose linkages and demannosylating phosphorylated N-glycans and methods of facilitating mammalian cellular uptake of glycoproteins
US10344310B2 (en) 2010-09-29 2019-07-09 Oxyrane Uk Limited De-mannosylation of phosphorylated N-glycans
US9347050B2 (en) 2010-09-29 2016-05-24 Oxyrane Uk Limited Mannosidases capable of uncapping mannose-1-phospho-6-mannose linkages and demannosylating phosphorylated N-glycans and methods of facilitating mammalian cellular uptake of glycoproteins
WO2013098651A1 (fr) * 2011-12-30 2013-07-04 Oxyrane Uk Limited Procédés et des matériels de réduction de la dégradation de protéines recombinantes
US10648044B2 (en) 2012-03-15 2020-05-12 Oxyrane Uk Limited Methods and materials for treatment of Pompe's disease
US9249399B2 (en) 2012-03-15 2016-02-02 Oxyrane Uk Limited Methods and materials for treatment of pompe's disease
US9481721B2 (en) 2012-04-11 2016-11-01 Novo Nordisk A/S Insulin formulations
WO2013191369A1 (fr) * 2012-06-19 2013-12-27 한국생명공학연구원 Souche de levure au gène hpgas1 interrompu et procédé de production d'une protéine recombinante à l'aide de celle-ci
US9896496B2 (en) 2013-10-07 2018-02-20 Novo Nordisk A/S Derivative of an insulin analogue
US10265385B2 (en) 2016-12-16 2019-04-23 Novo Nordisk A/S Insulin containing pharmaceutical compositions
US10596231B2 (en) 2016-12-16 2020-03-24 Novo Nordisk A/S Insulin containing pharmaceutical compositions
CN114380903A (zh) * 2021-12-28 2022-04-22 上海仁会生物制药股份有限公司 一种胰岛素或其类似物前体
CN114380903B (zh) * 2021-12-28 2023-07-25 上海仁会生物制药股份有限公司 一种胰岛素或其类似物前体

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