AU683061B2 - Yeast strain and methods for expressing heterologous proteins in yeast - Google Patents

Description

WO 94/26873 PCT/SE94/00421 1 YEAST STRAIN AND METHODS FOR EXPRESSING HETEROLOGOUS PROTEINS IN YEAST.

Technical field: 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.

Technical background: In fungi 0-glycosylation is initiated in the endoplasmatic reticulum and consists of the transfer of a single mannose residue from dolichol-phosphate-mannose (Dol-P-Man) to the nascently secreted protein (Haselbeck and Tanner, FEBS lett. 158 (1983) 335- 338). In the yeast S. cerevisiae further additions of 3-4 mannose residues using GDP-Man as donor can occur in the Golgi system leading to maximal O-glycosyl chain lengths of 5 mannose residues (Sharma et al., Eur. J. Biochem. 46 (1974) 35-41). Linkages between mannose residues at positions 1, 2 and 3 are a 1-2, while al-3 linkages exist between mannose residues at positions 3, 4 and While the in vivo attachment sites for 0-glycosyl chains on yeast proteins have not been defined, sites on some heterologous proteins secreted by S. cerevisiae are known. Human gr 4 nulocytemacrophage colony-stimulating factor (hGM-CSF) is party 0glycosylated in yeast (Ernst et al., Eur. J. Biochem. 203 (1992) 663-667). O-glycosylated hGM-CSF species appear to rarry either an extended chain of up to 5 mannoses on serine-9, or single mannose residues simultaneously on serine-9 and Thus, serine-9 is the principal 0-glycosyl attachment site in hGM- CSF. In yeast-secreted human insulin-like growth factor (hlGF-I) about 50% of the secreted protein carries a dimannosyl chain on threonine-29 (Gellerfors et al., J. Biol. Chem. 19 (1989) 11444-49).

Serine and threonine attachment sites have also been identified in experiments in vitro using synthetic peptides as substrates for I I

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WO 94/26873 PCT/SE94/00421 yeast mannosyltransferases (Bause and Lehle, Eur. J. Biochem. 101 (1979) 531-40; Strahl-Bolsinger and Tanner, Eur. J. Biochem. 196 (1991) 185-90; Lorenz et Eur. J. Biochem. 205 (1992) 1163-67).

Like in hGM-CSF and hIGF-! the O-glycosylated serine or threonine residues in synthetic peptides may be directly flanked at their Nterminal by a proline residue. The glycosylation patterns in mammalian systems and in S. cerevisiae differ significantly, particularly with regard to 0-glycosylation attachment sites (serine and threonine) and types of carbohydrates involved.

Recently, Strahl-Bohlsinger et al (Yeast 8 (1992) S489, the 16th International Conference on Yeast Genetics and Molecular Biology, Vienna (Austria), August 15-21, 1992) described a gene in S. cerevisiae, whose mutation reduces the in vivo O-glycosylation by about 50%, while the in vitro O-glycosylation in the mutant extract is absent. The mntl mutation leads to shortened O-glycosyl chains consisting of only 2 mannose residues; in this mutant a specific a 1-2 mannosyltransferase is defective (Hausler et al., Proc. Natl. Acad. Sci. USA 89 (1992) 6846-50). The mntl mutation is also known as kre2 and renders S. cerevisiae resistant to the action of K1 killer toxin (Hausler et al., Proc. Natl. Acad. Sci. USA 89 (1992) 6846-50). In mnnl mutants, a defective a 1-3 mannosyltransferase does not add the terminal 1-3 linked mannose residues in and N-glycosyl chains (Ballou et al., Proc. Natl.

Acad. Sci. USA 88 (1991) 3209-12). Other mutations defective in the synthesis of glycosylation precursors, mannose, GDP-Man and Dol-P-Man, also affect both O-or N-glycosylation. Mutations of this type include defects in SEC59, DPM1, SEC53 and PMI genes encoding dolichol kinase, dolichol phosphate mannose synthase, phosphomannomutase and phosphomannose isomerase, respectively.

At present it is not clear, if O-glycosylation in yeast is essential for viability. Only mutations abolishing O- and N-glycosylation completely (SEC59, DPM1, SEC53 and PMI) lead to lethality (Heller et al., Proc. Natl. Acad. Sci. USA 89 (1992) 7013-16; Orlean et al., J. Biol. Chem. 163 (1988) 17499-507; Kepes and Schenkman, J. Biol.

Chem. 163 (1988) 9155-61; Smith et al., Mol. Cell. Biol. 12 (1992) 2924-30).

_L WO 94/26873 PCTISE94/00421 3 An Article in J. Protein Chem. 9:95-104 (1990) by Elliot et al.

describes the purification and properties of yeast-produced hlGF- 1.The hlGF-1 protein has been mutated to reduce O-glycosylation.

Site-directed mutagenesis was used to convert Thr 29 to Asn29 which reduced but not eliminated IGF-I glycosylation. It thus describes alterations in the IGF-I protein itself, and not the host cell.

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 0-glycosylation. This patent describes mutants of the expressed protein GM-CSF, and not the host mutants.

Problemrs solved by the invention: The unique glycosylation patterns of yeast strains have in many cases a negative impact on the yield obtained of the desired form of heterologously produced proteins. Moreover extra precautions during purification must be applied in order to remove undesired forms of the produced protein.

Figure 1 A and B shows reversed phase HPLC chromatograms.

Objectives of the invention: 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. In addition the invention will provide S. cerevisiae homologous proteins that are minimally Oglycosylated at a serine or threonine site.

The invention: The S. cerevisiae strain of the invention is characterized in that it is defective in initiating 0-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 -r r ~u WO 94/26873 PCT/SE94/00421 4 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 0-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. Thus, further attachment of saccharide units to a monomannosylated serine or threonine residue and/or Nglycosylation and/or in vivo synthesis of necessary carbohydrate intermediates are in most cases normal as determined by the procedures given in the experimental section.

Particularly important modes of the invention are transformed S. cerevisiae strains comprising an expression vector containing a gene for a heterologous, in particular mammalian, protein, such as hGM-CSF or hlGF-I. 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.

Best Mode at the priority date: The greatest advantage based on today's knowledge for the inventive S. cerevisiae strains and production method is obtained for proteins containing one or more serine or threonine residue(s) that are recognized as 0-glycosylation sites by wild-type strains of S.cerevisiae (containing a vector expressing such a protein) but not by mammalian systems such as in humans. In other words the most preferred S. cerevisiae strains of the invention contain an expression vector plasmid) for a mammalian, in particular human, protein that is not 0-glycosylated by its original species but O-glycosylated by for instance YE-449 containing the appropriate expression plasmid for the protein. The most preferred strain has the same mutation as the M195 strain (see below). The most preferred heterologous protein is hlGF-I.

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WO 94/26873 PCT/SE94/00421 EXPERIMENTAL PROCEDURES tranins and growth conditions: The starting strain for mutagenesis was S. cerevisiae YE-465 which consists of host strain YE-449 (Biogen) (MATa leu2 ura3-52 prbl-7122 pep4-3 cirO) carrying plasmid p539/12 (Gelierfors et al., J. Biol. Chem. 264 (1989) 11444-49). p539/12 is an expression plasmid for hlGF-l that leads to the secretion of biologically active hlGF-I into the growth medium of yeast transformants.

Mutaenesis: Ethyl methane sulfonate (EMS) 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 300C. One ml of the culture supernatant was analysed by concanavalin A blotting, or immunoblotting using an anti-hlGF-l antibody.

To lose the expression plasmid in putative mutants they were grown no;i-selectively in YPD medium (Sherman et al., Methods in Yeast Genetics (Cold Spring Harbor Lab.) Cold Harbor, N.Y. (1986) 163), followed by the analysis of single isolates for the Uraphenotype. While mutants are named cured mutants were given the designation Cured mutants (and YE-449 as control) were retransformed with p539/1 2, or with a secretion vector for hGM- CSF, pER545/4. 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 Genetics (Cold Spring Harbor Lab.) Cold Spring Harbor N.Y. (1986) 164); this culture was used to inoculate production medium containing 4% casamino acids (Ernst et al., Bio/Technology (1987) 831-34) and the culture was grown for 2-3 days at 30oC to an OD600nm=l 0. Cells were removed by centrifugation; for hlGF-l expressing transformants the culture supernatant was concentrated 25-fold by Trichloro acetic acid (TCA) precipitation WO 94/26873 PCTSE94/0421 6 prior to electrophoresis followed by concanavalin A and immunoblotting procedures.

Blotting procedures: Glycoproteins in the culture medium were identified by concanavalin A blotting (Clegg, Anal. Biochem. 127 (1982) 389-94). For this purpose 20 pl 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 583 diluted 1:1000, followed by treatment with peroxidase-coupled goat antimouse IgG antibody diluted 1:2000 (Jackson Immuno Research, USA). 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.

Chromatography procedures: Reversed phase high performance liquid chromatography (HPLC) was performed with a diphenyl silica analytical column equipped with a C4-silica precolUmn. The monomeric hlGF-I forms were eluted with an acetonitrile-trifluoro acetic acid (TFA) gradient containing 0.1% TFA. The hIGF-l forms were eluted at around 25 acetonitrile.

Other procedures: Standard procedures were used for genetic analyses and crosses of mutants to laboratory strains S150-2B (MATa leu2-3,112 ura3-52 trpl-289 his3-1) and BJ1991 (MATa ura3-52 leu2 trpl prbl-1 22 pep4-3 gal2) (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 pg/ml).

Resistance/sensitivity to killer toxin K1 was assayed as previously described (Sherman et al., Methods in Yeast Genetics (Cold Spring Harbor Lab.) Cold Spring Harbor N.Y. (1986) 57-60) using strain RC1777 (MATa ade his4C i, -I I WO 94/26873 PCT/SE94/00421

RESULTS

Screening for 0-lycosylation mutants: Out of 600 mutagenized isolates 8 putative mutants were isolated. To exclude the possibility that the mutagenesis had affected the hlGF-I gene or other genetic material present on the expression plasmid we obtained derivatives of the mutants that had lost their plasmid during non-selective growth. When these cured mutants were tran' -ned with the expression plasmid, an identical phenotype as in L..e original mutant was obtained. Two of the identified mutants, M195 and M38, were characterized in more detail, as described below.

Characteristics of M195: Mutant strain M195 secretes similar amounts of hIGF-I into the growth medium as compared to the parent production strain YE-465 according to SDS-PAGE and immunobiotting. In contrast, we consistently detected lower concanavalin A-reactivity with hlGF-l secreted by M195, than with hIGF-l secreted by YE-465 indicating reduced 0-glycosylation at threonine-29. This phenotype was not due to mutations in the expression vector since the cured retransformed mutant (CM195) also showed reduced hiGF-I O-glycosylation. SDS-PAGE also showed several minor bands of somewhat larger size than IGF-I.

These bands represent material O-glycosylated on serine-69, adding to the evidence that the mutation in strain CM195 affects O-linked threonine glycosylation but not serine glycosylation.

In order to quantify the mutant phenotype we purified and analyzed hIGF-I secreted by strains M195 and YE-465 by reversed phase HPLC. HPLC-results for culture media from strain YE-465 and M195 are represented in Figure 1 (For legends see after Results).

The chromatograms show four main peaks for monomeric hIGF-1 corresponding to 0-glycosylated misfolded hIGF-1, nonglycosylated misfolded hlGF-i, O-glycosylated correctly folded hIGF-I, non-glycosylated correctly folded hIGF-I the product).

Misfolding of hlGF-1 occurs due to improper disulfide formation from cysteine (Axelsson et al., Eur. J. Biochem. 206 (1992) 987- 994). The results clearly demonstrate that the amount of 0glycosylated hIGF-l is reduced and the amount of non-glycosylated hIGF-I is increased for M195 compared to YE-465.

47, Cori A r o WO 94/26873 PCTIS'E9410021 8 In order to test if strain M195 is also defective for protein 0" glycosylation at serine residues, we cured strain M195 of its h'

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I expression plasmid (resulting in strain CM195) and transfo': u an expression plasmid for hGM-CSF (pER545/4) into the cir-.

strain. No differences in the glycosylation pattern was fouind between a wild-type transformant (YE-449(pER545/4)) and the mutant transformant (CM195(pER545/4)) as revealed by SDS-PAGE followed by immunoblotting. This result suggests that serine glycosylation is not affected in M195. On the other hand, Nglycosylation of hGM-CSF also does not seem to be affected in M195, a finding that is confirmed for yeast invertase (see below).

Strain M195 does not show any morphological abnormality, nor does it display temperature sensitivity, or sensitivity to high osmolarities in the growth medium. M195 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 M195 is an increased sensitivity for the aminoglycoside antibiotic hygromycin B. While strain YE- 465 grows well in the presence of 60 pg/ml hygromycin B, M195 is completely inhibited. The mnn9 mutation that affects Nglycosylation also is hygromycin B sensitive but resistant to 5 mM vanadate (Ballou et al., Proc. Natl. Acad. Sci. USA 88 (1991) 3203- 12). To ascertain that the increased hygromycin B sensitivity in M195 was associated with the observed glycosylation defect we crossed M195 to the wild-type strain S150-2B and examined the haploid progeny. Although many of the segregants of this cross failed to grow we were able to examine hlGF-1 0-glycosylation in two hygromycin B sensitive haploid segregants by HPLC analysis and found reduced O-glycosylation in both strains. This finding suggests that reduced 0-glycosylation and hygromycin Bsensitivity are due to defects in the same gene.

Characteristics of M38: Secretion of hlGF-I by mutant strain M38 neither shows quantitative, nor qualitative differences compared to the parental strain YE-465; in particular hlGF-1 is modified by O-glycosylation to approximately the same extent in M38 and YE-465 as revealed by SDS-PAGE followed by immunoblotting. However, differences in the glycoprotein staining

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WO 94/26873 PCT/SE94/00421 9 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.

In a wild-type strain of S. cerevisiae transformed with an hGM-CSF expressing plasmid immunoblotting on SDS-PAGE of secreted forms of hGM-CSF give bands at 14.5 kDa (unglycosylated), 15.5 kDa (0-glycosylated) and 50 kDa (Nglycosylated) (Ernst et al., Eur. J. Biochem 203 (1992) 663-67). To assess if glycosylation of hGM-CSF is affected, we isolated a plasmid-free derivative of M38. 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).

However, 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 M195, although both defects affect aspects of O-glycosylation in S. cerevisiae.

O-glycosylation of chitinase: The O-glycosylation of a homologous yeast protein, chitinase, which is extensively 0glycosylated, was examined in the yeast strains. In YE-465 chitinase appeared on a SDS-PAGE as a band of an approximate molecular weight of 110 kDa. In the mutant strain M195 the migration of chitinase was unchanged, indicating no difference in the 0-glycosyation of this homologous protein.

N-glycosylation of invertase in mutant strains: To examine if any of the putative glycosylation mutants is deficient in Nglycosylation, 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 mnn invertase migrates further than for the strain YE-465 and the mutants. By immunoblotting after SDS-PAGE on the same extracts, the defect in the mnn9 strains is clearly detected leading to a relatively homogeneous protein due to the WO 94/26873 PCT/SE94/00421 lack of outer glycosyl chains, but the production strain and the mutants all express invertase as a heterogeneous glycoform. This result indicates that in the putative mutants including M195 and M38 N-glycosylation is not affected.

DISCUSSION

The mutant strains M1 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, DPM1 or PMI, which affect as well as Nglycosylation. It has been reported recently that defects in the mntl gene lead to a specific shortening of all 0-glycosyl chains to two mannose residues (HAusler et al Proc. Natl. Acad. USA 89 (1992) 6846-50). However, in M195 full-length 0-glycosyl chains are observed in hlGF-I (although at a low frequency) and in hGM- CSF; in M38 O-glycosylation of hGM-CSF is defective. Thus defects in the mntl gene appear not to be the reason for the mutant phenotype in M195, or M38, although M38 is resistant to the K1 killer toxin, as has been reported previously for mntl strains. A mannosyltransferase has recently been purified from S. cerevisiae (Strahl-Bolsinger et al., Yeast 8 (1992) S489, the 16th International Conference on Yeast Genetics and Molecular Biology, Vienna (Austria), August 15-21, 1992) b ,ed on in vitro 0glycosylation assays using synthetic peptides (Strahl-Bolsinger and Tanner, Eur. J. Biochem. 196 (1991) 185-90). A gene corresponding to the transferase was isolated, the disruption of which leads to loss of in vitro mannosyltransferase activity and to a reduction of in vivo 0-glycosylation to about 50% (Strahl- Bolsinger et al., Yeast 8 (1992) S489, the 16th International Conference on Yeast Genetics and Molecular Biology, Vienna (Austria), August 15-21, 1992). In addition, the disruptant strain shows normal cytology, but forms multiple adherent clumps of cells. Unlike the phenotype of this disruptant, strains M195 and M38 do not form clumps during growth; M195 has no morphological defects, while M38 tends to form rod-like, elongated cells at high

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WO 94/26873 PCT/SE94/00421 11 temperature. Also, cell extracts of strains M195 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 M195 and M38.

The genetic evidence obtained here and in previous studies strongly suggests that O-glycosylation in S. cerevisiae is a complex process that requires multiple cellular factors. The experiments by Strahi-Bolsinger et al., (Yeast 8 (1992) S849, the 16th International Conference on Yeast Genetics and Molecular Biology, Vienna (Austria), August 15-21, 1992) indicate that not all of the mannosyltransferases that are active in vivo can be assayed in vitro, a result that clearly demonstrates the necessity of a genetic approach. 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. Thus, M1 95 may be defective in mannosylation of threonine residues (as in hlGF-I), but not serine residues (as in hGM-CSF). On the other hand M38 may be defective in mannosylation of serine, but not threonine residues. Further analyses of the isolated mutants including gene cloning and their in vivo disruption promise to clarify details of the 0-glycosylation process in S. cerevisiae.

Legends to Figure 1 Reversed phase HPLC chromatograms. culture medium from M195; culture medium from YE-465. Four main peaks representing monomeric hIGF-I are visible in each chromatogram, from left (shorter retention time): O-glycosylated incorrectly folded hlGF-I (mismatched), nonglycosylated incorrectly folded hlGF-1 (mismatched), O-glycosylated correctly folded hlGF-I and nonglycosylated correctly folded hlGF-I (desired product). There is a remaining small peak in position for the M195 strain This peak might represent some remaining 0-glycosylation on serine-29 of hlGF-I or possibly a completely different form of hlGF-I with similar retention time.

Claims (9)

1. Saccharomyces cerevisiae strain, characterized in that it is a transformant containing a heterologous gene for human insulin-like growth factor I (hIGF-I) to be expressed and is defective in O-mannosylation of one hydroxyl group in at least one residue selected from serine and threonine in hIGF-I expressed by the strain.

2. Saccharomyces cerevisiae strain according to claim 1, ch,-acterized in that the strain is the mutant strain M195 as hereinbefore described.

3. Saccharomyces cerevisiae strain according to claim 1, characterized in that the strain is defective in O-mannosylation of a free hydroxyl group of at least one serine residue.

4. Saccharomyces cerevisiae strain according to any one of claims 1-3, characterized in that the strain is defective in the O-mannosylation of a free hydroxyl group i1i at least one threorine residue.

5. Saccharomyces cerevisiae strain according to any one of claims 1-4, characterized in that: the expressed protein is derived from a mammalian species, and the O-glycosylation of the protein occurring directly at a hydroxyl group of at least one threonine and serine attachment site differs between the mammalian species and a wild-type strain of S. cerevisiae, preferably the mammalian species is homo sapiens and the protein is a protein that is not substantially O-glycosylated in homo sapiens.

6 Saccharomyces cerevisiae strain according to any one of claims 1-5, characterized in thai the strain performs N-glycosylation that is essentially normal for Saccharomyces cerevisiae. P- i, 13

7. Saccharomyces cereviSiae strain according to any one of claims 1-6, character~ized in, that the strain performs essentially normal wild-type attachment of monosaccharide residues to an 0-attached monomarmosyl serine or 0- attach-d monomannosyl threonine residue.

8. Process for the production of a protein that normally is 0- glycosylated by S. cerevisiae, characterized in that a S. cerevisiae strain according to any one of claims 1-7 is used to express the protein. 0

9. Process according to claim 8, characterized in that the protein is huma ii insulin-like growth factor I (hIGF-I) and that the strain carries a gene for said protein. .4. *09 DATED this day of June 19 97 PHAPMACIA AB, By its Patent Attorneys, E. F. WEL.LINGTON CO., By: (Bruce Wellington) *4 4 44 4 .444 Tu