WO1994026873A1 - Yeast strain and methods for expressing heterologous proteins in yeast - Google Patents
Yeast strain and methods for expressing heterologous proteins in yeast Download PDFInfo
- Publication number
- WO1994026873A1 WO1994026873A1 PCT/SE1994/000421 SE9400421W WO9426873A1 WO 1994026873 A1 WO1994026873 A1 WO 1994026873A1 SE 9400421 W SE9400421 W SE 9400421W WO 9426873 A1 WO9426873 A1 WO 9426873A1
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- WIPO (PCT)
- Prior art keywords
- protein
- strain
- glycosylation
- saccharomyces cerevisiae
- hlgf
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Classifications
-
- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
-
- 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
- C07K14/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
-
- 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
- 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 invention concerns a novel yeast (Saccharomyces cerevisiae) strain that performs O-glycosylation of at least one serine and/or at least one threonine residue at a reduced level.
- the invention also provides novel methods for the production of heterologous proteins, primarily eukaryotic such as mammalian proteins, in yeast cells.
- Linkages between mannose residues at positions 1 , 2 and 3 are ⁇ 1 -2, while ⁇ l -3 linkages exist between mannose residues at positions 3, 4 and 5. While the in vivo attachment sites for O-glycosyl chains on yeast proteins have not been defined, sites on some heterologous proteins secreted by S. cerevisiae are known. Human granulocyte- macrophage colony-stimulating factor (hGM-CSF) is partially 0- glycosylated in yeast (Ernst et al., Eur. J. Biochem. 203 (1992) 663-667).
- hGM-CSF Human granulocyte- macrophage colony-stimulating factor
- O-glycosylated hGM-CSF species appear to carry either an extended chain of up to 5 mannoses on serine-9, or single mannose residues simultaneously on serine-9 and threonine-10.
- serine-9 is the principal O-glycosyl attachment site in hGM- CSF.
- yeast-secreted human insulin-like growth factor (hlGF-l) about 50% of the secreted protein carries a dimannosyl chain on threonine-29 (Gellerfors et al., J. Biol. Chem. 19 (1989) 1 1444-49).
- Serine and threonine attachment sites have also been identified in experiments in vitro using synthetic peptides as substrates for yeast mannosyltransferases (Bause and Lehle, Eur. J. Biochem. 101 (1979) 531 -40; Strahl-Bolsinger and Tanner, Eur. J. Biochem. 1 96 (1991 ) 185-90; Lorenz et al., Eur. J. Biochem. 205 (1992) 1 163-67). Like in hGM-CSF and hlGF-l the O-glycosylated serine or threonine residues in synthetic peptides may be directly flanked at their N- terminal by a proline residue.
- Mutations of this type include defects in SEC59, DPMI , SEC53 and PMI genes encoding dolichol kinase, dolichol phosphate mannose synthase, phosphomannomutase and phosphomannose isomerase, respectively.
- O-glycosylation in yeast is essential for viability. Only mutations abolishing 0- and N-glycosylation completely (SEC59, DPMI , SEC53 and PMI) lead to lethality (Heller et al., Proc. Natl. Acad. Sci. USA 89 (1992) 7013-16; Orlean et al., J. Biol. Chem.
- EP 314096 concerns yeast mutants that are defective in the addition of outer N-glycosyl chains.
- EP 276846 discloses the biological activities of various forms of GM-CSF lacking sites for N- and/or O-glycosylation. This patent describes mutants of the expressed protein GM-CSF, and not the host mutants.
- Figure 1 A and B shows reversed phase HPLC chromatograms.
- One objective of the invention is to provide S. cerevisiae expressed proteins, in particular heterologous proteins, that are minimally O-glycosylated at one or more particular serine and/or threonine attachment site(s).
- An additional objective is to provide a production method for these proteins and a means (a mutated S.cerevisiae strain) for the method.
- the invention will provide S. cerevisiae homologous proteins that are minimally 0- glycosylated at a serine or threonine site.
- the S. cerevisiae strain of the invention is characterized in that it is defective in initiating O-mannosylation of the hydroxyl group of at least one residue selected from serine and threonine in a protein expressed by the strain. This is probably due to a defect in the recognition of serine, or threonine acceptor sites, or in the mannosyltransferase activity required to glycosylate these sites. A serine and/or threonine specific O-mannosyltransferase may be missing or altered in activity. Other aspects of glycosylation are similar to wild-type glycosylation, e.g glycosylation performed by strain YE-465.
- S. cerevisiae strains comprising an expression vector containing a gene for a heterologous, in particular mammalian, protein, such as hGM-CSF or hlGF-l. It follows that in these modes of the invention, the mutation causing decreased O-glycosylation is in a yeast chromosomal gene.
- a second main aspect of the invention is a method for producing a protein, often a heterologous, in particular mammalian, protein comprising culturing the inventive S. cerevisiae strain and recovering the protein from the fermentation medium or from the cells. It is conceivable that conventional S. cerevisiae culturing methods and conventional recovering methods may be used.
- the most preferred strain has the same mutation as the Ml 95 strain (see below).
- the most preferred heterologous protein is hlGF-l.
- p539/12 is an expression plasmid for hlGF-l that leads to the secretion of biologically active hlGF-l into the growth medium of yeast transformants.
- Ethyl methane sulfonate was used to mutagenize strain YE-465.
- Treatment of a log-phase culture for 120 min and 180 min with 2.5% EMS yielded killing rates of 81 % and 91 %, respectively.
- the 180 min culture was plated for single colonies on minimal medium. Single colonies were inoculated with a needle into 2 ml production medium containing 4% casamino acids in reagent tubes and grown for 3 days at 30°C.
- One ml of the culture supernatant was analysed by concanavalin A blotting, or immunoblotting using an anti-hlGF-l antibody.
- pER545/4 is a derivative of pER562 (Ernst et al., Bio/Technology 5 (1987) 831-34). Transformants were first grown in selective minimal medium (Sherman et al., Methods in Yeast
- Glycoproteins in the culture medium were identified by concanavalin A blotting (Clegg, Anal. Biochem. 127 (1982) 389-94). For this purpose 20 ⁇ of the TCA-concentrated medium was separated by SDS-PAGE (17,5% gel), transferred to nitrocellulose and stained as described. For immunoblottings proteins were transferred to membranes (Immobilon-P, Millipore) and reacted with the monoclonal anti-hlGF-l antibody 5B3 diluted 1 :1000, followed by treatment with peroxidase-coupled goat anti- mouse IgG antibody diluted 1 :2000 (Jackson Immuno Research, USA).
- hGM-CSF For the detection of hGM-CSF a rabbit polyclonal anti-hGM- CSF antibody (kindly supplied by Glaxo Institute for Molecular Biology, Geneva) was used as first antibody (diluted 1 :100), followed by alkaline phosphatase-coupled goat anti-rabbit IgG antibody (Dianova, Germany) diluted 1 :5000.
- Standard procedures were used for genetic analyses and crosses of mutants to laboratory strains SI 50-2B (MATa leu2-3, 1 12 ⁇ ra3-52 trpl-289 his3-l) and BJ1991 (MAT ⁇ ura3-52 Ieu2 trpl prbl-1 122 pep4-3 ga!2) (Sherman et al., Methods in Yeast Genetics (Cold Spring Harbor Lab.) Cold Spring Harbor, N.Y. (1986) 17-27). Hygromycin B-sensitivity was analyzed on antibiotic gradient plates (gradient from 0 to 200 ⁇ g/ml).
- Ml 95 Mutant strain M195 secretes similar amounts of hlGF-l into the growth medium as compared to the parent production strain YE-465 according to SDS-PAGE and immunoblotting. In contrast, we consistently detected lower concanavalin A-reactivity with hlGF-l secreted by Ml 95, than with hlGF-l secreted by YE-465 indicating reduced O-glycosylation at threonine-29. This phenotype was not due to mutations in the expression vector since the cured retransformed mutant (CM195) also showed reduced hlGF-l O-glycosylation. SDS-PAGE also showed several minor bands of somewhat larger size than IGF-I.
- the chromatograms show four main peaks for monomeric hlGF-1 corresponding to O-glycosylated misfolded hlGF-1 , non- glycosylated misfolded hlGF-1 , O-glycosylated correctly folded hlGF-l, non-glycosylated correctly folded hlGF-l (i.e. the product). Misfolding of hlGF-1 occurs due to improper disulfide formation from cysteine (Axelsson et al., Eur. J. Biochem. 206 (1 992) 987- 994).
- Strain Ml 95 does not show any morphological abnormality, nor does it display temperature sensitivity, or sensitivity to high osmolarities in the growth medium. Ml 95 is as sensitive as the parent YE-465 for killer toxin K1 and 5 mM vanadate. The only detectable phenotype other than its O-glycosylation defect that appears associated with strain Ml 95 is an increased sensitivity for the aminoglycoside antibiotic hygromycin B. While strain YE- 465 grows well in the presence of 60 ⁇ g/ml hygromycin B, Ml 95 is completely inhibited.
- the mnn9 mutation that affects N- glycosylation also is hygromycin B sensitive but resistant to 5 mM vanadate (Ballou et al., Proc. Natl. Acad. Sci. USA 88 (1991 ) 3209- 12).
- Ml 95 was associated with the observed glycosylation defect
- SI 50-2 B was associated with the observed glycosylation defect
- many of the segregants of this cross failed to grow we were able to examine hlGF-l O-glycosylation in two hygromycin B sensitive haploid segregants by HPLC analysis and found reduced O-glycosylation in both strains. This finding suggests that reduced O-glycosylation and hygromycin B- sensitivity are due to defects in the same gene.
- Characteristics of M38 Secretion of hlGF-l by mutant strain M38 neither shows quantitative, nor qualitative differences compared to the parental strain YE-465; in particular hlGF-l is modified by O-glycosylation to approximately the same extent in M38 and YE-465 as revealed by SDS-PAGE followed by immunoblotting.
- differences in the glycoprotein staining pattern by concanavalin A demonstrate that several secreted glycoproteins are reduced in size in M38. In particular, a prominent protein of 35 kDa is missing; instead, a 27 kDa glycoprotein of equal intensity is detected.
- S wild-type strain of S.
- CM38 This strain, CM38, was transformed with the hGM-CSF expression plasmid pER545/4 and the hGM-CSF secreted by this transformant was analyzed by immunoblotting.
- the unglycosylated 14.5 kDa form of hGM-CSF and the heterogeneous N-glycosylated 50 kDa form of hGM-CSF occur both in CM38(pER545/4) and control strain YE-449(pER545/4).
- the O-glycosylated 15.5 kDa form of hGM-CSF is missing in CM38(pER545/4). This result suggests that the defect in M38 is different from the defect in Ml 95, although both defects affect aspects of O-glycosylation in S. cerevisiae.
- O-glvcosylation of chitinase The O-glycosylation of a homologous yeast protein, chitinase, which is extensively 0- glycosylated, was examined in the yeast strains. In YE-465 chitinase appeared on a SDS-PAGE as a band of an approximate molecular weight of 1 10 kDa. In the mutant strain Ml 95 the migration of chitinase was unchanged, indicating no difference in the O-glycosyation of this homologous protein.
- N-olvcosylation of invertase in mutant strains To examine if any of the putative glycosylation mutants is deficient in N- glycosylation, we analyzed the homologous protein invertase, which is essentially only N-glycosylated. Extracts of the mutants were separated on a non-denaturing acrylamide gel and invertase activity was visualised by an activity stain. For two control strains (mnn9 and mnnl) invertase migrates further than for the strain YE-465 and the mutants.
- mutant strains Ml 95 and M38 differ from other mutants that have been shown to be defective in O-glycosylation in S. cerevisiae. Because protein N-glycosylation of hGM-CSF and invertase proceed normally in these mutants their genetic defect is different from "unspecific" mutations, such as mutations in SEC59, SEC53, DPMI or PMI, which affect 0-, as well as N- glycosylation. It has been reported recently that defects in the mntl gene lead to a specific shortening of all O-glycosyl chains to two mannose residues (HSusler et al Proc. Natl. Acad. USA 89
- M38 do not form clumps during growth; Ml 95 has no morphological defects, while M38 tends to form rod-like, elongated cells at high temperature. Also, cell extracts of strains Ml 95 and M38 contain wild-type levels of in vitro O-glycosylation (Strahl-Bolsinger, unpublished results). This evidence indicates that neither gene known to affect O-glycosylation is mutated in mutants Ml 95 and M38.
- the mutants isolated in the present study may be defective in the recognition of serine, or threonine acceptor sites, or in the mannosyltransferase activity required to glycosylate these sites.
- Ml 95 may be defective in mannosylation of threonine residues (as in hlGF-l), but not serine residues (as in hGM-CSF).
- M38 may be defective in mannosylation of serine, but not threonine residues.
- Four main peaks representing monomeric hlGF-l are visible in each chromatogram, from left (shorter retention time): O-glycosylated incorrectly folded hlGF-l (mismatched), nonglycosylated incorrectly folded hlGF-1 (mismatched), O-glycosylated correctly folded hlGF-l and nonglycosylated correctly folded hlGF-l (desired product).
Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU66626/94A AU683061B2 (en) | 1993-05-07 | 1994-05-06 | Yeast strain and methods for expressing heterologous proteins in yeast |
JP6525318A JPH08509867A (en) | 1993-05-07 | 1994-05-06 | Methods for expressing heterologous structural proteins in yeast strains and yeast. |
EP94915327A EP0698086A1 (en) | 1993-05-07 | 1994-05-06 | Yeast strain and methods for expressing heterologous proteins in yeast |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE19939301583A SE9301583D0 (en) | 1993-05-07 | 1993-05-07 | YEAST STRAIN AND METHODS FOR EXPRESSING HETEROLOGOUS PROTEINS IN YEAST |
SE9301583-2 | 1993-05-07 |
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WO1994026873A1 true WO1994026873A1 (en) | 1994-11-24 |
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PCT/SE1994/000421 WO1994026873A1 (en) | 1993-05-07 | 1994-05-06 | Yeast strain and methods for expressing heterologous proteins in yeast |
Country Status (7)
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EP (1) | EP0698086A1 (en) |
JP (1) | JPH08509867A (en) |
AU (1) | AU683061B2 (en) |
CA (1) | CA2161973A1 (en) |
NZ (1) | NZ266045A (en) |
SE (1) | SE9301583D0 (en) |
WO (1) | WO1994026873A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000044772A2 (en) * | 1999-01-30 | 2000-08-03 | Delta Biotechnology Limited | Human serum albumin |
WO2002046437A2 (en) * | 2000-12-05 | 2002-06-13 | The Penn State Research Foundation | Methods and compositions for highly efficient production of heterologous proteins in yeast |
WO2006028595A3 (en) * | 2004-09-02 | 2007-01-18 | Lilly Co Eli | Muteins of fibroblast growth factor 21 |
US7223561B2 (en) | 1995-05-25 | 2007-05-29 | Novozymes Delta, Limited | Process of high purity albumin production |
US8232377B2 (en) | 2006-05-16 | 2012-07-31 | National Institute Of Advanced Industrial Science And Technology | Method for high-level secretory production of protein |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0276846A2 (en) * | 1987-01-29 | 1988-08-03 | Zymogenetics, Inc. | Colony-stimulating factor derivatives |
EP0314096A2 (en) * | 1987-10-29 | 1989-05-03 | Zymogenetics, Inc. | Methods of regulating protein glycosylation |
-
1993
- 1993-05-07 SE SE19939301583A patent/SE9301583D0/en unknown
-
1994
- 1994-05-06 CA CA002161973A patent/CA2161973A1/en not_active Abandoned
- 1994-05-06 JP JP6525318A patent/JPH08509867A/en active Pending
- 1994-05-06 EP EP94915327A patent/EP0698086A1/en not_active Withdrawn
- 1994-05-06 WO PCT/SE1994/000421 patent/WO1994026873A1/en not_active Application Discontinuation
- 1994-05-06 NZ NZ266045A patent/NZ266045A/en unknown
- 1994-05-06 AU AU66626/94A patent/AU683061B2/en not_active Ceased
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0276846A2 (en) * | 1987-01-29 | 1988-08-03 | Zymogenetics, Inc. | Colony-stimulating factor derivatives |
EP0314096A2 (en) * | 1987-10-29 | 1989-05-03 | Zymogenetics, Inc. | Methods of regulating protein glycosylation |
Non-Patent Citations (1)
Title |
---|
Dialog Information Services, file 155, Medline, Dialog Accession No. 07346592, Medline Accession No. 90253592, ELLIOTT S. et al.: "Yeast-Derived Recombinant Human Insulin-Like Growth Factor I: Production, Purification, and Structural Characterization", J. Protein Chem., Feb. 1990, 9 (1), p 95-104. * |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7223561B2 (en) | 1995-05-25 | 2007-05-29 | Novozymes Delta, Limited | Process of high purity albumin production |
EP2272961A1 (en) * | 1999-01-30 | 2011-01-12 | Novozymes Biopharma DK A/S | Process for producing recombinant albumin with reduced mannosylation |
WO2000044772A3 (en) * | 1999-01-30 | 2000-11-30 | Delta Biotechnology Ltd | Human serum albumin |
US9555344B2 (en) | 1999-01-30 | 2017-01-31 | Albumedix A/S | Process for the purification of recombinant albumin |
WO2000044772A2 (en) * | 1999-01-30 | 2000-08-03 | Delta Biotechnology Limited | Human serum albumin |
EP2048234A1 (en) * | 1999-01-30 | 2009-04-15 | Novozymes Biopharma UK Limited | Chromatographic method for purification of albumin |
US9029102B2 (en) | 1999-01-30 | 2015-05-12 | Novozymes Biopharma Dk A/S | Process for the purification of recombinant albumin |
US7993877B2 (en) | 1999-01-30 | 2011-08-09 | Novozymes Biopharma Dk A/S | Process for the purification of recombinant albumin |
WO2002046437A2 (en) * | 2000-12-05 | 2002-06-13 | The Penn State Research Foundation | Methods and compositions for highly efficient production of heterologous proteins in yeast |
WO2002046437A3 (en) * | 2000-12-05 | 2003-11-06 | Penn State Res Found | Methods and compositions for highly efficient production of heterologous proteins in yeast |
EA011390B1 (en) * | 2004-09-02 | 2009-02-27 | Эли Лилли Энд Компани | Muteins of fibroblast growth factor 21 |
EP2161281A1 (en) * | 2004-09-02 | 2010-03-10 | Eli Lilly & Company | Muteins of fibroblast growth factor 21 |
US7582607B2 (en) | 2004-09-02 | 2009-09-01 | Eli Lilly And Company | Muteins of fibroblast growth factor 21 |
WO2006028595A3 (en) * | 2004-09-02 | 2007-01-18 | Lilly Co Eli | Muteins of fibroblast growth factor 21 |
US8232377B2 (en) | 2006-05-16 | 2012-07-31 | National Institute Of Advanced Industrial Science And Technology | Method for high-level secretory production of protein |
Also Published As
Publication number | Publication date |
---|---|
JPH08509867A (en) | 1996-10-22 |
AU683061B2 (en) | 1997-10-30 |
SE9301583D0 (en) | 1993-05-07 |
CA2161973A1 (en) | 1994-11-24 |
EP0698086A1 (en) | 1996-02-28 |
NZ266045A (en) | 1997-06-24 |
AU6662694A (en) | 1994-12-12 |
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