GB2249096A - Saccharomyces cerevisiae strains lacking carboxypeptidase yscY activity for expression of proteins at high yields - Google Patents

Abstract

Saccharomyces cerevisiae strains lacking carboxypeptidase yscY activity, disposing of protease yscA and carboxypeptidase ysc alpha activities, lacking endogenous two-micron DNA and transformed with an expression vector based on the two-micron plasmid were constructed. Such strains facilitate the expression and optionally the secretion of a wide variety of heterologous and/or homologous proteins including enzymes, hormones, growth factors, growth hormones, enzyme inhibitors, antigens, plasminogen activators and blood clotting factors.

Description

Improved methods for the production of proteins Field of the invention The invention pertains to the field of recombinant DNA technology and provides an improved method for the production of proteins by use of novel transformed yeast strains.

Background of the invention During the past years, recombinant yeast strains have become an excellent tool for the expression of homologous and heterologous proteins. Production of proteins requires the transformation of yeast cells with suitable expression vectors comprising DNA sequences encoding said proteins. Examples of successfully expressed proteins in yeast are a-interferon [IFNa, Hitzeman et al. (1981) Nature 294, 717-722], lysozyme [Oberto et al.

(1985) Gene 40, 57-65], a-amylase [Sato et al. (1986) Gene 50, 247-257], tissue-type plasminogen activator [t-PA, European Patent Application No. 143 081] or desulphatohirudin [European Patent Application No. 225 633].

However, protein production in yeast is often associated with the degradation of the primary expression product due to post-translational modification by endogenous yeast proteases. As a result of said proteolytic activity, a heterogeneous product mixture is obtained containing partially degraded such as N- or C-terminally shortened proteins in addition to the specific full length protein actually desired.

A variety of proteolytic enzymes has been found in yeast. Best characterized are the proteases of Saccharomyces cerevisiae, among them two endopeptidases yscA and yscB, several exopeptidases including carboxypeptidases yscY and yscS and arninopeptidases, e.g. yscI, yscCo and dipeptidylaminopeptidase yscV, as well as carboxypeptidase ysca [Achstetter, T., Wolf, D.H. (1985) Yeast 1, 139-157]. Mutants specifically lacking one or more of said proteolytic enzymes have been isolated and studied biochemically [e.g. Wolf, D.H., Weiser, U. (1977) Eur. J. Biochem. 73 553-556; Wolf, D.H., Ehmann, C. (1981) J. Bacteriol. 147 418-426].

In order to avoid C-terminal product degradation preparation of proteins which are susceptible to posttranslational shortening by carboxypeptidase ysca is performed by using yeast mutant strains lacking carboxypeptidase ysca [European Patent Application No. 341 215].

Apart from carboxypeptidase ysca, which is most likely localized to the Golgi apparatus of the yeast cell, the listed proteases reside in the vacuolar compartment (for review see Rendueles, P.S., Wolf, DS. (1988) FEMS MicrobioL Rev. 54 17-46). Detailed examination of the biosynthesis of some of the vacuolar proteases (i.e. yscA, yscB and yscY) revealed that they are synthesized as inactive precursor molecules and that they are part of a complex activation cascade triggered by protease yscA tHemmings et al. (1981) Proc. Natl. AcadL Sci. USA 78 435-439; Mechler et al. (1982) Biochem. Biophys. Res Commun. 107, 770-778; Stevens et al. (1982) Cell 30, 439-448; Mechler et al. (1987) EMBO J. 6, 2157-2163].Protease ysc is the expression product of the PEP4 gene [Ammerer et al. (1986) Mol. Cell. Biol. 6,2490-2499; Woolford et al. (1986) Mol. Cell.

Biol. 6, 2500-2510] which has been shown to be allelic to the PRA1 gene [Mechler et al.

(1987) EMBO J. 6,2157-2163]. Strains deficient in protease yscA, the pep4 mutants, accumulate inactive protease yscB and yscY precursor proteins and show reduced activities in a number of other vacuolar enzymes, including vacuolar species of ribonuclease, alkaline phosphatase, trehalase and an aminopeptidase [Jones et al. (1982) Genetics 102, 665-677; Trumbly and Bradley (1983), J. Bacteriol. 156, 3648; Harris and Cotter (1987) Curr. Micro. 15, 247-249]. Therefore, expression of proteins in S. cerevisiae is commonly achieved by use of transformed pep4 mutant strains in order to eliminate, in addition to protease yscA activity, a variety of other different proteolytic activities that require protease yscA for activation.As a consequence, random proteolysis is much less pronounced in such strains and laborious isolation and purification operations of the individual components resulting from proteolytic degradation processes are reduced. For instance, connective tissue activating peptide-E (CTAP-III) [Mullenbach, G.T. et al.

(1986), J. Biol. Chem. 261, 719-722], human epidermal growth factor (hEGF) [Brake, A.J.

et al. (1984) Proc. Natl. Acas. Sci. USA 81, 4662-4646] and echistatin [Jacobsen, M.J. et al. (1989) Gene 85 511-516] have been successfully prepared by employing transformed S. cerevisiae mutant strains deficient in yscA activity (for more examples see Hirsch et al.

(1989) in: Molecular and Cell Biology of Yeasts (E.F. Walton and G.T. Yarranton, end.), Blackie, Glasgow and London, 134-200).

However, strains lacking proteinase yscA are severely impaired in their ability to undergo the differentiation process of sporulation and show a dramatically increased loss of viability under nutritional stress conditions such as nitrogen starvation [Teichert, U. et al.

(1989), J. Biol. Chem. 264, 16037-16045]. This observation suggests that even though the elimination of proteinase yscA activity and the consequent elimination of other proteolytic activities is not lethal to the cell, the protease exerts major functions in the stress response and might be indispensable for the optimal performance of a yeast strain used for protein production.

Considering furthermore the facts that the use of transformed mutants deficient in yscA activity merely diminishes the degree of C-terminal product degradation due to random proteolysis and that protein yields frequently are still unsatisfactory there is a need for improved methods of preparing proteins in yeast.

Obiect of the invention It is an object of the present invention to provide improved methods for the production of proteins by supplying novel recombinant yeast strains.

Detailed description of the invention Surprisingly, it has now been found that the protein yield can be drastically increased by use of recombinant yeast strains which are deficient in yscY activity, dispose of active proteases yscA (PRA 1, PEP4) and ysca and are devoid of endogenous two-micron DNA.

Carboxypeptidase yscY is the expression product of the PRC1 gene [Stevens et al. (1986) J. Cell Biol. 102, 1551-1557] and carboxypeptidase ysca is encoded by the KEX1 gene [Wagner and Wolf (1987) FEBS Lett. 221, 423-426]. Additionally, direct inactivation of the yscY encoding gene PRC1 has the unexpected advantage of significantly decreasing the formation of C-terminally shortened derivatives.

Accordingly, the invention concerns a yeast strain lacking carboxypeptidase yscY activity, disposing of protease yscA and carboxypeptidase ysca activities, lacking endogenous two-micron DNA and which has been transformed with a hybrid vector comprising a yeast promoter operably linked to a DNA sequence coding for a protein homologous or heterologous to yeast, a DNA sequence containing yeast transcription termination signals and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites, and optionally an intact D gene.

A preferred embodiment of the invention relates to a yeast strain lacking carboxypeptidase yscY activity, disposing of protease yscA and carboxypeptidase ysca activities, lacking endogenous two-micron DNA and which has been transformed with a hybrid vector comprising a yeast promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding a homologous or heterologous protein, a DNA sequence containing yeast transcription termination signals and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites.

Yeast strains lacking carboxypeptidase vscY activitv, disposing of protease vscA and carboxypeptidase vsca activities, and lacking two-micron DNA Every proteaseaeficient mutant strain referred to in the following is equipped with proteases yscA and ysca, free of endogenous two-micron DNA and has been transformed with a hybrid vector as described below.

In addition to the lack of carboxypeptidase yscY the recombinant yeast strain according to the invention may be defective in further peptidase activity selected from the group consisting of yscB, yscS and vacuolar aminopeptidases, e.g. yscI, yscCo and dipeptidylaminopeptidase yscV. Accordingly, said yeast strain may be a single, double, triple or quadruple mutant.

Since spontaneous mutation frequencies are low, the efficient preparation of protease-deficient mutants is usually achieved by treating yeast with mutagens such as X-ray or U.V. radiation or chemical mutagens which can induce mutations at a rate of 1.10 to 10-3 per gene without a great deal of killing. The proteases which are lacking in the yeast strains according to the invention do not perform indispensible functions in the cell metabolism; therefore mutations which completely destroy the activity of these proteins are not lethal. Each protease-deficient mutant type can be isolated separately after mutagenesis. Isolation and selection is based on colony screening assays which are well-known in the art.

A second and even more efficient method to introduce the desired single or multiple protease deficiencies into the yeast genome is the site-directed mutagenesis or gene-disruption or gene replacement [cf. H. Rudolph et al., Gene, 36 (1985) 87 - 95].

When the genetic sequence is known, as it is, for example, the case for carboxypeptidase yscY the genomic protease gene can be made defective by insertion, substitution or deletion making use of the well-known site-directed mutagenesis procedure [see, for example, M.J. Zoller and M. Smith (1983) Methods Enzymol. 100, 468] which involves the preparation of an appropriately devised mutagenic oligodeoxyribonucleotide primer.

Alternatively, the genomic protease gene can be replaced by foreign DNA or said foreign DNA can be inserted into a suitable restriction site of the protease gene. For example, in order to prepare a yeast mutant lacking peptidase yscY activity (prcl-mutant) foreign DNA is inserted into a suitable restriction site occurring in the genomic PRC1 gene. In case the yeast strain used has a defect in a chromosomal gene coding for an enzyme of amino acid or purine biosynthesis a corresponding intact gene can be inserted into the chromosomal PRC1 gene thus providing for prototrophy in the auxotrophic yeast strain and changing the genotype at the same time from PRC1 to prcl. The gene replacement or directed mutagenesis procedures are commonly applied in the art and are absolutely reproducible.

The current method to compose multiple protease-defective strains, such as strains lacking yscY and yscB activity, consists in meiotic crossing and subsequent tetrad analysis. The tetrads, which derive from the diploid cells, are dissected according to standard genetic techniques. Random assortment among the four spores of a tetrad allows the construction of double and multiple mutants in subsequent crosses. Random spore analysis can also be used as an alternative system.

Since mutants devoid of individual proteases and even double mutants are available from yeast genetic stock centers, triple and quadruple mutants can reproducibly be combined by known successive meiotic crossing techniques.

Suitable starting strains of Saccharomyces cerevisiae include, for example, yeast peptidase B (yscB) negative strains HT246, H426 and H449 (the latter is, in addition, cur") deposited at the Deutsche Sammlung von Mikroorganismen, Braunschweig, FRG, under accession numbers 4084, 4231 and 4413, respectively and yeast peptidases B, Y and S (yscB, yscY and yscS) negative strain BYS232-31-42 deposited under accession number DSM 4583.

As mentioned above, the yeast strains according to the invention are devoid of endogenous two-micron DNA, so-called cairo strains. The two-micron plasmid is a high copy number, self-replicating, extrachromosomal DNA element, contained in most strains of Saccharomvces cerevisiae. The most striking structural features of the two-micron plasmid are two inverted repeats (1R1 and IR2) of 559 bp each dividing the plasmid in two DNA regions of different length. The homologous recombination between these two identical IR sequences results in the formation of two molecular isomers (form A and form B). Stability of the two-micron plasmid is given by three plasmid encoded functions.

The REP1 and REP2 gene products are trans-acting proteins that are required for the stable partitioning of the two-micron plasmitL Of these two, REP1 is possibly the more important, in that efficiency of partitioning is dependent on the gene dosage of the REP1 gene product [A. Cashmore et al. (1986) Mol. Gen. Genet. 203, 154]. These two proteins act through and on the STB (REP3) site, an important cis-acting element on the plasmid [M. Jayaram et al. (1985) Mol. Cell. Biol. 5,2466-2475; B. Viet et al. (1985) Mol. Cell.

Biol. 5 2190-2196].

Such cir" strains of Saccharomvces cerevisiae are known or can be prepared by methods known in the art [see, for example, C.P. Hollenberg (1982) Curr. Top. Microbiol. Immun.

96, 119]. The following alternative procedure for the preparation of cairo strains is based on the presumption that curing of the two-micron plasmid by a second plasmid involves increasing the dosage of the STB site to titrate out the REP1 and REP2 proteins. This relative reduction of the REP1 and REP2 proteins would lead to an instability of the endogenous two-micron plasmid.

Preferably, the second plasmid used has a defect in or lacks the REP1 gene. An example of such a plasmid is pDP38 european Patent Application No. 340 170] which apart from the REP1 gene lacks an inverted repeat (TR2). This makes its high copy number expression dependent on the complementation of REP1 protein by the endogenous two-micron plasmid It contains two yeast selective markers: URA3, used in both high and low copy number situations, and dLEU2, applicable only in high copy number situations [E.Erhart et al. (1968) J. Bacteriol. 625].

A yeast strain which is ura and leu (i.e. Ura~ and Leu~) is transformed with plasmid pDP38 and selected for Ura+ colonies. The selection on uracile free plates (Ura selection) gives a much better transformation frequency than the selection on leucine free plates (Leu selection), as the URA3 gene is much better expressed than the defective dLEU2 gene. A single colony is selected and streaked onto a Leu selection plate which gives colonies of varying sizes and form. Some of the smallest colonies are restreaked onto Ura selection plates and replica-plated onto Leu selection plates. Those colonies are selected that can grow under Ura selection but only very slowly under Leu selection.Growth on Ura selection plates shows that the plasmid pDP38 is still present and that the merely slow growth under Leu selection is not due to the loss of this plasmid, and the failure of growth under Leu selection implies that pDP38 is not able to complement this marker. The latter fact can be explained in two ways: A. The LEU2 gene on pDP38 is mutated, or B: The plasmid cannot complement leu2 because it cannot raise its copy number, implying that the two-micron plasmid is not available (i.e. lost) to complement the REP1 gene product.

These two possibilities can be distinguished very easily. In the first place, the minimal growth seen with said colonies (as against the absolute zero growth of cells without pDP38) shows that some LEU2 expression is present. The second point can be directly tested, as in the absence of the two-micron plasmid pDP38 will act only as an ARS type plasmid, i.e. it will be very unstable so that most of the colonies will lose it after a few generations. Accordingly, when a single colony is streaked onto a YPD plate, and single colonies taken and replica-plated onto uracile free plates, then only a few will grow under Ura selection. Non growing colonies are checked by hybridization for pUC and two-micron sequences. Colonies which show no hybridization signals are free of plasmid pDP38 and of endogenous two-micron plasmids (cur" strains).The cairo strains obtained can be treated as described above to yield yeast mutant strains according to the invention which lack peptidase yscY activity and, in addition, are devoid of two-micron DNA.

The recombinant yeast strains according to the invention can advantageously be used for the production of a homologous or heterologous protein. It was found that PRA1, KEX1, prcl, cir" strains according to the invention carrying a hybrid vector containing a gene coding for a protein homologous or heterologous to yeast exhibit a greatly increased product titer in combination with a significant reduction of C-terminal degradation.

Transformation of yeast strains and their production The invention concerns a yeast strain as described above and a method for the production thereof.

Suitable recombinant yeast host strains include strains of Saccharomyces cerevisiae lacking carboxypeptidase yscY activity provided with proteases yscA and ysca activities and devoid of endogenous two-micron DNA which optionally lack additional peptidase activities.

The method for the production of said transformed yeast strain comprises transforming a yeast strain which lacks carboxypeptidase yscY activity, disposes of protease yscA and carboxypeptidase ysca activities and is free of endogenous two-micron DNA with a hybrid vector composed as specified below.

The transformation of yeast with the hybrid vectors according to the invention may be accomplished according to the method described by Hinnen et al. [Proc. Natl. Acad. Sci.

USA 75 1929 (1978)]. This method can be divided into three steps: (1) Removal of the yeast cell wall or parts thereof using various preparations of glucosidases, such as snail gutjuices (e.g. GlusulaseE) or KelicaseB) or enzym mixtures obtained from microorganisms (e.g. Zymolyase) in osmotically stabilized solutions (e.g.

1 M sorbitol).

(2) Treatment of "naked" yeast cells (spheroplasts) with the DNA vector in the presence of PEG (polyethyleneglycol) and Ca2+ ions.

(3) Regeneration of the cell wall and selection of the transformed cells in a solid layer of agar. This regeneration is conveniently done by embedding the spheroplasts into agar. For example, molten agar (about 500C) is mixed with the spheroplasts. Upon cooling the solution to yeast growth temperatures (about 300C), a solid layer is obtained. This agar layer is to prevent rapid diffusion and loss of essential macro-molecules from the spheroplast and thereby facilitates regeneration of the cell wall. However, cell wall regeneration may also be obtained (although at lower efficiency) by plating the spheroplasts onto the surface of preformed agar layers.

Preferably, the regeneration agar is prepared in a way to allow regeneration and selection of transformed cells at the same time. Since yeast genes coding for enzymes of amino acid or nucleotide biosynthetic pathways are generally used as selective markers (infra), the generation is preferably performed in yeast minimal medium agar. If very high efficiencies of regeneration are required the following two step procedure is advantageous: (1) regeneration of the cell wall in a rich complex medium, and (2) selection of the transformed cells by replica plating the cell layer onto selective agar plates.

The transformed yeast strains are cultured using methods known in the art.

Thus, the transformed yeast strains according to the invention are cultured in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts.

Various carbon sources are usable. Example of preferred carbon sources are assimilable carbohydrates, such as glucose, maltose, mannitol, fructose or lactose, or an acetate such as sodium acetate, which can be used either alone or in suitable mixtures. Suitable nitrogen sources include, for example, amino acids, such as casamino acids, peptides and proteins and their degradation products, such as tryptone, peptone or meat extracts, furthermore yeast extract, malt extract, corn steep liquor, as well as ammonium salts, such as ammonium chloride, sulphate or nitrate which can be used either alone or in suitable mixtures. Inorganic salts which may be used include, for example, sulphates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium. Additionally, the nutrient medium may also contain growth promoting substances.Substances which promote growth include, for example, trace elements, such as iron, zinc, manganese and the like, or individual amino acids.

Yeast cells containing hybrid plasmids with a constitutive promoter (e.g. ADHI, GAPDH) express the DNA encoding a homologous or heterologous protein controlled by said promoter without induction. However, if said DNA is under the control of a regulated promoter (e.g. PGK or PHOS) the composition of the growth medium has to be adapted in order to obtain maximum levels of mRNA transcripts, i.e. when using the PHOS promoter the growth medium must contain a low concentration of inorganic phosphate for derepression of this promoter.

The cultivation is carried out by employing conventional techniques. The culturing conditions, such as temperature, pH of the medium and fermentation time are selected in such a way that maximal levels of the heterologous protein are produced. A chosen yeast strain is preferably grown under aerobic conditions in submerged culture with shaking or stirring at a temperature of about 250 to 350C, preferably at about 28"C, at a pH value of from 4 to 7, for example at approximately pH 5, and for at least 1 to 3 days, preferably as long as satisfactory yields of protein are obtained.

The transformed yeast host cells according to the invention can be prepared by recombinant DNA techniques comprising the steps of - preparing a hybrid vector comprising a yeast promoter operably linked to a DNA sequence coding for a protein homologous or heterologous to yeast, a DNA sequence containing yeast transcription termination signals, and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites, - providing a mutant yeast strain which lacks carboxypeptidase yscY activity, disposes of protease yscA and carboxypeptidase ysca activities, and is free of endogenous two-micron DNA, - transforming the mutant yeast strain obtained with said hybrid vector, - and selecting transformed yeast cells from untransformed yeast cells.

Expression vectors The yeast hybrid vectors used in the invention comprise a yeast promoter operably linked to a DNA sequence coding for a homologous or heterologous protein. Preferred hybrid vectors comprise a yeast promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding a homologous or, preferably, heterologous protein such as desulphatohirudin and a DNA sequence containing yeast transcription termination signals.

The yeast promoter is a regulated promoter such as the PHOS, MFal or GALl promoter, or a constitutive promoter. In case of the expression of desulphatohirudin, a constitutive promoter is preferred. The constitutive yeast promoter is preferably derived from a highly expressed yeast gene, such as a gene encoding a glycolytic enzyme, such as the promoter of the enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase gene, furthermore the ADHI or TRPI promoter and a shortened acid phosphatase PHOS promoter which has been deprived of its upstream activation sites.Especially preferred is the GAPDH promoter and functional fragments thereof starting at nucleotide between -550 and -180, in particular at nucleotide -540, -263 or -198, and ending at nucleotide -5 of the GAPDH gene, and the PH05 promoter and functional fragments thereof starting at nucleotide between -200 and -150, in particular at -173, and ending at nucleotide -9 of the PH05 gene.

The DNA sequence encoding a signal peptide ("signal sequence") is preferably derived from a yeast gene coding for a polypeptide which is ordinarily secreted. Yeast signal sequences are, for example, the signal and prepro sequences of the yeast invertase, a-factor, pheromone peptidase (KEX1), "killer toxin" and repressible acid phosphatase (PH05) genes and the glucoamylase signal sequence from Asperaillus awamori.

Alternatively, fused signal sequences may be constructed by ligating part of the signal sequence (if present) of the gene naturally linked to the promoter used (for example PH05), with part of the signal sequence of the heterologous or homologous protein. Those combinations are favoured which allow a precise cleavage between the signal sequence and the protein amino acid sequence. Additional sequences, such as pro- or spacersequences which may or may not carry specific processing signals can also be included in the constructions to facilitate accurate processing of precursor molecules. Alternatively, fused proteins can be generated containing internal processing signals which allow proper maturation in vivo or in vitro.The preferred signal sequences used in to the present invention are those of the yeast PH05 gene coding for a signal peptide having the formula Met Phe Lys Ser Val Val Tyr Ser Ile Leu Ala Ala Ser Leu Ala Asn Ala, and of the yeast invertase gene coding for a signal peptide having the formula Met Leu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys Ile Ser Ala.

Genomic DNA sequences coding for a homologous or heterologous protein can be isolated from natural sources, or a copy DNA (cDNA) can be produced from the corresponding complementary mRNA or by means of chemical and enzymatic processes, in a manner known per se.

For example, the DNA sequence coding for desulphatohirudin is known or can be isolated from genomic leech DNA or a complementary double-stranded desulphatohirudin DNA (desulphatohirudin ds cDNA) is produced from desulphatohirudin mRNA, or a gene coding for the amino acid sequence of desulphatohirudin is produced by means of chemical and enzymatic processes.

A DNA sequence containing yeast transcription termination signals is preferably the 3' flanking sequence of a yeast gene which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking sequences are for example those of the yeast gene naturally linked to the promoter used. The preferred flanking sequence is that of the yeast PHO5 gene.

The yeast promoter, the optional DNA sequence coding for the signal peptide, the DNA sequence coding for a homologous or heterologous protein and the DNA sequence containing yeast transcription termination signals are operably linked to each other, i.e.

they are juxtaposed in such a manner that their normal functions are maintained. The array is such that the promoter effects proper expression of the specific gene coding for the protein to be produced (optionally preceded by a signal sequence), the transcription termination signals effect proper termination of transcription and polyadenylation and the optional signal sequence is linked in the proper reading frame to said gene in such a manner that the last codon of the signal sequence is directly linked to the first codon of the gene coding for the homologous or heterologous protein and secretion of the protein occurs. If the promoter and the signal sequence are derived from different genes, the promoter is preferably joined to the signal sequence between the major mRNA start and the ATG of the gene naturally linked to the promoter.The signal sequence should have its own ATG for translation initiation. The junction of these sequences may be effected by means of synthetic oligodeoxynucleotide linkers carrying the recognition sequence of an endonuclease.

The hybrid vectors used in the invention contain the complete two-micron DNA in an uninterrupted form, i.e. two-micron DNA is cleaved once with a restriction endonuclease, the linearised DNA is linked with the other components of the vector prior to recircularization. The restriction site is chosen such that normal function of the REP1, REP2 and FLP genes and of the ORI, STB, IR1 and 1R2 sites of two-micron DNA is maintained. Optionally, the restriction site is chosen such that the D gene of two-micron DNA too is kept intact. Preferred restriction sites are the unique PstI site located within the D gene and the unique HpaI and SnaBI sites located outside of all said genes and sites.

Preferably, the hybrid vectors include one or more, especially one or two, selective genetic markers for yeast and such a marker and an origin of replication for a bacterial host, especially Escherichia coli.

As to the selective gene markers for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene.

Suitable markers for yeast are, for example, those expressing antibiotic resistance or, in the case of auxotrophic yeast mutants, genes which complement host lesions.

Corresponding genes confer, for example, resistance to the antibiotics G418, hygromycin or bleomycin or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, T LEU2, LYS2 or TRP1 gene.

As the amplification of the hybrid vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli replication origin are included advantageously. These can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid, for example pUC18 or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.

The hybrid vectors according to the invention are prepared by methods known in the art.

The process comprises cleaving two-micron plasmid DNA with a restriction endonuclease such that the normal function of the REP1, REP2 and FLP genes and of the ORI, STB, IR1 and IR2 sites is maintained, linking the linearized plasmid obtained to the expression cassette comprising a yeast promoter operably linked to DNA sequence coding for a protein homologous or heterologous to yeast, a DNA sequence containing yeast transcription termination signals, and optionally to DNA segments containing one or more selective genetic markers for yeast and a selective genetic marker and a replication origin for a bacterial host, and recircularising the hybrid vector obtained.

Use of transformed yeast strains for the production of proteins The invention also relates to an improved method for the production of a protein homologous or heterologous to yeast comprising culturing a yeast mutant strain which lacks carboxypeptidase yscY activity, disposes of protease yscA and carboxypeptidase ysca activities, is free of endogenous two-micron DNA and which has been transformed with a hybrid vector comprising a yeast promoter operably linked to a DNA sequence coding for said protein, a DNA sequence containing yeast transcription termination signals and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites, and isolating said protein.

A preferred embodiment of the present invention concerns an improved method for the production of a protein comprising culturing said mutant yeast strain which has been transformed with a hybrid vector comprising a yeast promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence coding for said protein, a DNA sequence containing yeast transcription termination signals and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites, and isolating said protein.

A protein which can be produced by the improved method according to the invention is homologous or, especially, heterologous to yeast The method is particularly useful for proteins which are susceptible to posttranslational C-terminal degradation by yeast proteases, e.g. carboxypeptidase yscY.

A homologous protein which can be prepared according to the invention comprises any protein originating from the particular yeast host used for transformation or a mutant of said protein.

A heterologous protein which can be produced according to the invention includes any protein or a mutant thereof that originates from a virus, a prokaryotic cell or, in particular, a eukaryotic organism which differs in species from the specific yeast host used for transformation.

Such a heterologous protein is in particular of higher eukaryotic such as mammalian (including animal and human) origin and is, for example, an enzyme which can be used, for example, for the production of nutrients and for performing enzymatic reactions in chemistry or molecular biology, or a protein which is useful and valuable for the treatment of human and animal diseases or for the prevention thereof, for example a hormone, polypeptide with immunomodulatory, anti-viral and anti-tumor properties, an antibody, viral antigen, blood clotting factor, a fibrinolytic agent, a growth regulation factor, a protein, especially an enzyme useful in the foodstuff and beverage industries and the like.

Examples of such proteins are e.g. hormones such as secretin, thymosin, relaxin, calcitonin, luteinizing hormone, parathyroid hormone, adrenocorticotropin, melanocytestimulating hormone, ss-lipotropin, urogastrone, insulin, growth factors, such as epidermal growth factor (EGF), insulin-like growth factor (if), e.g.IGF-I and IGF-II, mast cell growth factor, nerve growth factor, glia derived nerve cell growth factor, platelet derived growth factor (PDGF), or transforming growth factor (TGF), such as TGFss, growth hormones, such as human or bovine growth hormones, interleukin, such as interleukin-1 or -2, human macrophage migration inhibitory factor (mew), interferons, such as human a-interferon, for example interferon-aA, aB, aD or aF, p-interferon, r-interferon or a hybrid interferon, for example an aA-aD- or an aB-aD-hybrid interferon, especially the hybrid interferon BDBB, proteinase inhibitors such as al-antitrypsin, SLPI and the like, hepatitis virus antigens, such as hepatitis B virus surface or core antigen or hepatitis A virus antigen, or hepatitis nonA-nonB antigen, plasminogen activators, such as tissue plasminogen activator or urokinase, hybrid plasminogen activators, such as K2tuPA, tick anticoagulant peptide (TAP), human atrial natriuretic peptide (hANP), connective tissue activating peptide-III (CTAPE), epidermal growth factor tumour necrosis factor, somatostatin, renin, immunoglobulins, such as the light and/or heavy chains of immunoglobulin D, E or G, or human-mouse hybrid immunoglobulins, immunoglobulin binding factors, such as immunoglobulin E binding factor, human calcitonin-related peptide, blood clotting factors, such as factor IX or vmc, platelet factor 4, erythropoietin, eglin, such as eglin C, hirudin, desulfatohirudin including variants and mutants thereof, corticostatin, echistatin, cystatins, human superoxide dismutase, viral thymidin kinase, p-lactamase or glucose isomerase. Preferred proteins comprise human a-interferon e.g.

interferon aB, or hybrid interferon, particularly hybrid interferon BDBB (see EP 205,404), transforming growth factor ss, human calcitonin, human atrial natriuretic peptide (hANP), connective tissue activating peptide-III (CTAPIII), epidermal growth factor, insulin-like growth factors I and II and desulfatohirudin including mutants and variants thereof.

In the most preferred aspect the invention relates to an improved method for the production of desulphatohirudin comprising culturing a yeast strain lacking carboxypeptidase yscY activity, disposing of endopeptidase yscA and carboxypeptidase ysca activities, lacking endogenous two-micron DNA and which has been transformed with a hybrid vector comprising a desulphatohirudin expression cassette consisting of a yeast promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence coding for desulphatohirudin, a DNA sequence containing yeast transcription termination signals and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites, and isolating desulphatohirudin.

The term "desulphatohirudin" is intended to embrace the desulphatohirudin compounds described in literature or obtainable from a transformed microorganism strain containing DNA which codes for a desulphatohirudin. Such desulphatohirudins are, for example, desulphatohirudin variant HV1, HV1 (modified a, b), HV2, HV2 (modified a, b c), PA, variants of PA and des(Val2)-desulphatohirudin.

Preferred desulphatohirudins are those having the formula X1 Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Gln Gly Asn X2 Cys lie Leu Gly Ser Asp Gly Glu X3 Asn Gln Cys Val Thr Gly Glu Gly Thr Pro X4 Pro Gln Ser Xs Asn Asp Gly Asp Phe Glu Glu lie Pro Glu X6 (D, in which a) X1 represents the dipeptide residue Val-Val and X2, X3 and X4 are each Lys, X5 is His and X6 is the peptide residue Glu-Tyr-Leu-Gln (HV1), or b) X2 is lie or Glu and X1 and X3 - X6 are as defined in a) (HV1 modified a), or c) X3 is Ile or Glu and X1, X2 and X4 - X6 are as defined in a) (HV1 modified a), or d) X4 is Ile or Glu and X1 - X3 and Xs and X6 are as defined in a) (HV1 modified a), or e) Xs is Leu or Asp and X1 - X4 and X6 are as defined in a) (FiVi modified a), or f) X6 is selected from the group consisting of Glu-Tyr, Glu-Tyr-Leu, Glu-Asp-Leu-Gln, Glu-Glu-Leu-Gln, Glu-Tyr-Lys-Arg, Glu-Asp-Lys-Arg, Glu-Lys-Leu-Gln, Ser-Phe-Arg-Tyr, Trp-Glu-Leu-Arg, Glu-Tyr-Leu-Gln-Pro and Glu-Tyr-Leu-Gln-Arg and X1 - Xs are as defined in a) (HV1 modified b), or g) in which X1 represents Leu-Thr and X2 - X6 are as defined in a), or having the formula Y1 Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys lle Leu Gly Ser Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Giu Gly Thr Pro Y2 Pro Glu Ser His Asn Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Y3 Leu Gln in which a) Y1 represents the N-terminal dipeptide residue Ile-Thr, Y2 is Asn and Y3 is Tyr (HV2), or b) Y2 is Lys, Arg or His and Y1 and Y3 are as defined in a) (HV2 modified a), or c) Y3 is Glu or Asp and Y1 and Y2 are as defined in a) (HV2 modified b), or d) in which Y1 represents the N-terminal dipeptide residue Val-Val and Y2 and Y3 are as defined in a) (HV2 modified c), or having the formula Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu Gly Ser Gln Gly Lys Asp Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp Phe Glu Pro Ile Pro Glu Asp Ala Tyr Asp Glu (Ill) HV3 and variants of said HV3 which are characterized by a shortening of the primary structure by 1 or 2 amino acids at the N-terminus or by 18, 10,9, 6,4 or 2 amino acids at the C-terminus.

The preferred desulphatohirudin compounds are those of formula I in which X1 represents the dipeptide residue Val-Val and X2, X3 and X4 are each Lys, Xs is His and X6 is the peptide residue Glu-Tyr-Leu-Gln (Ia), or X1 represents Leu-Thr and X2, X3 and X4 are each Lys, X5 is His and X6 is the peptide residue Glu-Tyr-Leu-Gln (Ig) or the desulfatohirudin compound of the formula II in which Y1 represents the N-terminal dipeptide residue Ile-Thr, Y2 is Lys and Y3 is Tyr.

The most preferred protein is desulfatohirudin HV1 having the formula I in which X1 represents the dipeptide residue Val-Val and X2, X3 and X4 are each Lys, Xs is His and X6 is the peptide residue Glu-Tyr-Leu-Gln.

The homologous or heterologous proteins expressed in yeast can be accumulated inside the cells or can be secreted into the culture medium. In the case of desulphatohirudin irrespective of the yeast strain, promoter and signal peptide used, most of the produced protein is secreted into the culture medium whereas only a minor part remains cell associated. The precise ratio (secreted compoundslcell associated compounds) depends on the fermentation conditions and the recovery process applied. In general it amounts to about or more than 8:1. Accordingly, secreted desulphatohirudin is always strongly dominating.

The homologous or heterologous protein can be isolated from the culture medium by conventional means. For example, the first step consists usually in separating the cells from the culture fluid by means of centrifugation. The resulting supernatant can be enriched for the desired protein by treatment with polyethyleneimine so as to remove most of the non-proteinaceous material, and precipitation of the proteins by saturating the solution with ammonium sulphate. A protein homologous to yeast can also be precipitated by means of acidification with acetic acid (for example 0.1 %, pH 45). A further enrichment of a heterologous protein can be achieved by extracting the acetic acid supernatant with n-butanol.Other purification steps include, for example, desalination, chromatographic processes, such as ion exchange chromatography, gel filtration chromatography, partition chromatography, HPLC, reversed phase HPLC and the like.

The separation of the constituents of the protein mixture is also effected by dialysis, according to charge by means of gel electrophoresis or carrier-free electrophoresis, according to molecular size by means of a suitable Sephadex column, by affinity chromatography, for example with antibodies, especially monoclonal antibodies, or with thrombin coupled to a suitable carrier for affinity chromatography, or by other processes, especially those known from the literature.

If the protein is not secreted or if it is desired to isolate any additional protein which is cell associated, i.e. which has accumulated intracellularly or in the periplasmic space, some supplementary purification steps are required. Thus, in case the product has accumulated within the cells, the first step for the recovery thereof consists in liberating it from the cell interior. In most procedures the cell wall is first removed by enzymatic digestion with glucosidases (infra). Subsequently, the resulting spheroplasts are treated with detergents, such as Triton. Alternatively, mechanical forces, such as shearing forces (for example X-press, French-press) or shaking with glass beads, are suitable for breaking cells.In the case where the protein is secreted by the host cells into the periplasmic space, a simplified protocol can be used: The protein is recovered without cell lysis by enzymatic removal of the cell wall or by treatment with chemical agents, e.g. thiol reagents or EDTA, which give rise to cell wall damages permitting the product to be released.

The known proteins obtainable by the process according to the invention can be used in a manner known per se, e.g. in the therapy and prophylaxis of human and animal diseases.

The invention concerns especially the transformed yeast strains, the methods for the production thereof and the method for the production of a protein homologous or heterologous to yeast, as described in the Examples.

Brief description of the drawings In the following experimental part various embodiments of the present invention are described with reference to the following drawings in which Fig. 1 depicts the production of hirudin compounds 1-65 (full-length hirudin), 1-64 and 1-63 by yeast strains H449/pDP34/GAPFL-mIR(A), Tr 1357/pDP34/GAPFL-YHIR(B) and Tr 1 195/pDP34/GAPFL-YHIR(C) during 168 h of fermentation in YPD-broth.

Fig. 2 shows the production of hirudin compounds 1-65, 1-64 and 1-63 by yeast strains H449/pDP34/GAPFLtYHIR(A), Tr 1357/pDP34/GAPFL-YHIR(B) and Tr 1195/pDP34/GAPFL-YHIR(C) during 168 h of fermentation in HE 42 medium.

The following examples serve to illustrate the present invention but should not be construed as a limitation thereof.

Reference Example: Preparation of S. cerevisiae strain Tr 1195 by disruption of the gene PRAl encoding endoprotease vscA in S. cerevisiae strain H449 In order to eliminate carboxypeptidase yscY mediated C-terminal degradation processes one can either eliminate its maturating enzyme yscA which results in accumulation of biologically inactive precursor protein or, alternatively, eliminate the yscY encoding gene PRC1 directly.

Proteinase yscA activity is eliminated from S. cerevisiae strain H449 [MATa, prbl, cpsl, ura3A5, leu 2-3, 2-112, cir ] through disruption of the genomic PRA1 gene [Ammerer, G.

et al. (1986) Mol. Cell. Biol. 6,2490]. For this purpose the PRA1 gene is identified in a yeast genomic library and cloned into a suitable vector. The URA3 gene, which serves as a selective marker, is inserted in the structural gene of PRAl to disrupt its reading frame.

The hybrid plasmid DNA comprising the URA3 gene flanked on either side by PRA1 sequences is introduced into the ura yeast strain H449. The sequence homology of the PRA1 gene on the plasmid and on the chromosome allows in vivo recombination, transforming yeast cells from Urar to Ura+ and concomitantly from PRA1 to pral strains which due to a disrupted PRA1 gene do not synthesize a functional yscA protein.

PRA1 is isolated from total genomic yeast DNA of wildtype S. cerevisiae S288C, which is obtained from the yeast Genetic Stock Center, Berkeley, USA, digested with SacI and PstI. 2 kb fragments are isolated from a preparative 0.6 % agarose gel, electroeluted and ligated into the PstI-SacI sites of the polylinker region of pUC19 [J. Norrander et al.

(1983), Gene 26, 101]. The vector is transformed into E. coli JM 109 [Yanisch-Perron, C.

et al. (1985) Gene 33 103-119] and 280 individual colonies are picked. The colonies are grown individually in wells of microtiter plates containing LB +amp medium (10 g/l tryptone, 5 gil NaCl, 5 gil bacto yeast extract, 0.1 gn ampicillin, pH 7.5). Colony hybridization is carried out essentially as described [Woods, D.E. et al. (1982) Proc. Nail.

Acad. Sci. USA 79 5651] with the following 32P-labelled oligonucleotide probe 5'- AAGCCTAGTGACCTAGT -3' which is derived from the published PRA1 sequence [Ammerer et al., supra]. 3 positive clones are picked, DNA of one - pUC19/PRA1 - is cut with SacI and XhoI and the 1.9 kb fragment containing the entire PRA1 gene subcloned into the KS polylinker region of the Bluescript vector M13+ (Stratagene Cloning Systems, San Diego, USA). A 1.2 kb Hindi fragment containing the entire URA3 gene [Rose, M. et al. (1984) Gene 29, 113]) is inserted into the unique Hindi site within the coding region of the PRAl-insert. The resulting plasmid is designated M13+/pral::URA3. M13+/pral::URA3 is digested with SacI/XhoI and the 3.1 kb fragment without separation from the vector used to transform S.

cerevisiae H 449 as described by Hinnen et al. EBroc. Natl. Acad. Sci. USA (1978) 75, 1929].

S. cerevisiae strain H449 is harvested from the early logarithmic stage in 100 ml YPD (20 gil bacto peptone, 10 gil bacto yeast extract, 20 gil glucose) medium (OD600 =0.2), washed with 25 ml 0.8 M sorbitol and resuspended in 5 ml of the same sorbitol solution.

To this 5 ml cell suspension, 30 pl zymolyase (ZYMOLYASE-100 T from Arthrobacter luteus, Seikagaku Kogyo Co., Tokyo, Japan; 10 mg/ml in 0.8 M sorbitol) are added. The mixture is gently agitated in a 100 ml shake flask on the shaker (110 revs/min) at 300C for about 30 min. At 5 min intervals a 100 ttl aliquot is taken, diluted in 10 ml distilled water and the absorbances of the dilutes at 600 nm are measured to control the progressive course of spheroplasting. To get a good spheroplast formation, the difference in absorbance before and after the treatment with zymolyase must be greater than a factor of 10.The spheroplasted cells are washed with 0.8 M sorbitol twice, resuspended in 25 ml medium HE 30 (1 M sorbitol, 20 gil bacto peptone, 10 gil bacto yeast extract, 20 gil glucose) and incubated in a 100 ml shake flask with gentle agitation on the shaker (110 revs/min) at 300C for one hour. The cells are centrifuged (3000 rpm, 5 min), and resuspended in 1 ml HE 31 solution (10 mM Tris-HCl, pH 7.5, 10 mM Cacti2, 0.9 M sorbitol) with care. To 100 Fl cell suspensions 4 Rg plasmid DNA is added.The mixture is incubated for 15 min at room temperature. 1 ml of 20 % polyethyleneglycole (PEG) 4000 is added and incubated for additional 30 min at room temperature, centrifuged (3000 rpm, 3 min) and resuspended in 500 A 0.8 M sorbitol. The spheroplasts with DNA are mixed with 10 ml regeneration agar (1 M mannitol, 6.8 gil yeast nitrogen base w/o aa, 10 gil L-asparagine, 1.0 gil L-histidine, 1.0 gil adenine, 1.0 gil threonine, 1.0 gil lysine and 3 % agar) and poured as an overlay onto plates containing a basic agar layer of the same composition. The plates are incubated at 300C for 96 hours until the transformed colonies appear.

Uracil independent transformants are picked, DNA prepared and SacI/XhoI digested and checked for correct PRA1 gene disruption by Southern blotting. One transformant with the correct shift of the SacVXhoI fragment from 1.9 kb to 3.1 kb hybridizing with PRA1 is designated Tr 1186.

In the next step Tr 1186 - with the disruption in its PRA1 gene by pral::URA3 - is again made uracil dependent by introducing a deletion in the pral::URA3 gene insert. Tr 1186 is transformed with 1 g of plasmid YEpl3 [Broach, J.R. et al. (1979) Gene 8, 121) together with 10 ug of plasmid pUC12ura3delta5 containing a 200 bp deletion in the URA3 gene [Sengstag, C. et al. (1978) Nucleic Acids Research 15, 233]). 3000 leucine prototrophic yeast transformants are resuspended in 5 ml minimal medium (0.84 % yeast nitrogen base w/o aa, to which 2 % glucose, 0.1 % leucine, 0.1 % uracil and 0.25 % fluoroorotic acid are added) in a small shake flask and incubated for 60 hours at 300C and 180 rpm.

Transformants which grow are resistant to the toxic analogue fluoroorotic acid and therefore carry a replacement in the pral::URA3 region by ura3delta5. The grown cells are plated out on full medium composed of (gull): peptone 20, yeast extract 10, glucose 20 and after growth for 48 hours at 30"C replica-plated onto minimal medium (0.84 % yeast nitrogen base w/o aa, supplemented with 2 % glucose and 0.1 % leucine) to detect uracil auxotrophs. Several auxotrophs are picked and tested for plasmid YEpl3 loss conferring leucine auxotrophy.One individual colony requiring leucine and uracil is picket Said strain is designated Tr 1195 (MATa, prbl, cups1, pral:: ura 3A5, ura 3A5, leu 2-3, 2-112, [cir ]) and used for further experimentation.

Example 1: Preparation of S. cerevisiae strain Tr 1357 bv disruption of the gene PRC1 encoding carboxyyentidase vscY in S. cerevisiae strain H449.

Alternatively, carboxypeptidase yscY activity in H449 is eliminated directly. PRC1 coding for yscY is isolated from the yeast genomic library in the centromer vector pCS 19 [Sengstag, C. et al. (1978) Nucleic Acids Research 15,233] by colony hybridization with 2 synthetic radiolabelled oligonucleotides 5'-GAAAGCATTCACCAGTTTACTATGTGG-3' and 5' -CGAATGGATCCCAACGGGTITCTCC -3, corresponding to the 5' and 3' end of the PRC1 coding sequence . One positive clone is designated as pCS 19/cpy8. pCS 19/cpy8 DNA is digested with ClaI/PvuII, loaded on a 0.6 % preparative agarose gel and a 2.6 kb fragment is isolated and electroeluted. This ClaI/PvuII fragment, containing the entire PRC1 gene, is further subcloned into the NarI/SmaI sites of pUC19.The resulting plasmid pUC19/PRC1 is cut at the unique StuI site, into which the 1.2 kb URA3 fragment (see Example 1) is located. For this purpose, the sticky HindIII ends of the URA3 containing fragment are filled in a reaction with Klenow DNA polymerase to fit into the blunt-ended Stul-site of pUC/PRC1.

The resulting plasmid pUC19/prcl::URA3 is digested with AatlI and the AatII fragment without separation from the vector used to transform S. cerevisiae H449 as described.

Uracil independent transformants are tested for correct PRC1 gene disruption by Southern blotting. One strain with the correct PRC1 disruption is designated Tr 1357 (MATa, prbl, cpsl, prcl:: URA3, ura 3A5, leu 2-3, 2-112, [cir ]) .

Example 2: Transformation of Saccharomyces cerevisiae strains H449, Tr 1195 and Tr 1357 with plasmid pDP34/GAPFL-YHIR Yeast strains H449, Tr 1195 and Tr 1357 are transformed with plasmid pDP34/GAPFLYHIR (European Patent Application No. 340170) using the transformation protocol described by Hinnen et al. (Proc. Natl. Acad. Sci. USA 75, 1929 (1978)).

Transformed yeast cells derived from H449 or Tr 1195 are selected on yeast minimal medium plates supplemented with leucine and deficient in uracil. Transformed yeast cells derived from Tr 1357 are selected on yeast minimal medium plates deficient in leucine.

Single transformed yeast cells are isolated and referred to as S accharomyces cerevisiae H449/pDP34/GAPFLYHIR, Saccharomyces cerevisiae Tr 1 195/pDP34/GAPFL-YHIR, S accharomyces cerevisiae Tr 1 3 57/pDP34/G APFL-YHIR.

Example 3: Fermentation of S. cerevisiae strains H449/pDP34/GAPFL-YHlR, Tr 1195/pDP34/GAPFL-YHIR and Tr I 357/pDP34/GAPFL-YHIR in rich medium (YPD-broth) Cells of Saccharomyces cerevisiae strains H449/pDP34/GAPFL-YHIR and Tr 1 195/pDP34/GAPPL-YHIR are each grown in a preculture of 10 ml minimal medium composed of (go): yeast nitrogen base w/o amino acids (Difco) 8.4 L-asparagine (Fluka) 10.0 L-histidine (Fluka) 1.0 L-leucine (Fluka) 0.1 glucose (Amylum) 20.0 Cells of Saccharomyces cerevisiae strain Tr 1357/pDP34/GAPFL-YHIR are grown in a 10 ml preculture of the identical medium, but without the addition of leucine.

The precultures are grown for 72 h at 28"C and 250 rpm. The main culture medium (YPD) is composed of (g/l): bacto peptone (Difco) 20.0 bacto yeast extract (Difco) 10.0 glucose (Amylum) 20.0 The main cultures (50 ml) are inoculated from the precultures to a concentration of 106 cells/ml and incubated up to 168 h at 280C and 250 rpm. Approximately 3x108 cells/ml are obtained at the end of the fermentation. At several time points during the fermentation, aliquots of the cultures are taken and subsequently analyzed for desulfatohirudin by HPLC (infra).

Example 4: HPLC-analysis of hirudin production bv S. cerevisiae strains H449/pDP34/GAPFLYHIR. Tr 1195/pDP34/GAPSYHIRe and Tr 1357/pDP34/GAPFL-YH1R fermented in rich medium (YPD-broth) Samples are withdrawn from the yeast cultures (see Example 3) after various periods of time (Fig. 1). The cells are removed by centrifugation and the supernatant is diluted 1:5 (v/v) with 1M acetic acid. The samples are subjected to reversed phase HPLC analysis under the following conditions.

A LiChroCart (Merck) column (4 mm x 125 mm) is filled with LiChrospher 100-5 C18 material (Merck), a spherical stationary phase with a particle diameter of 5 llm and a porosity of 100 A.

Mobile phase A is made from water (NANOPUREB, Barnstead) containing 0.1 % (v/v) trifluoroacetic acid.

Mobile phase B is made from 80 % (v/v) of acetonitrile (HPLC grade, Merck) and 20 % water (NANOPURE, Barnstead), containing 0.8 % (v/v) trifluoroacetic acid.

The detection wavelength for the eluent is 216 nm.

Chromatographic separations are performed at a constant flow rate of 1.5 mI/min using the following gradient: time (min) % A % B 0 88 12 1 79 21 5 79 21 18 65 35 19 0 100 22 0 100 24 88 12 32/0 88 12 The sample volume is 50 A at a protein concentration of 50-100 Fg/ml.

A standard solution for calibration is made by dissolving 100 mg of pure recombinant yeast desulfatohirudin variant HV1 in 1 liter of 0.1M ammonium formiate, pH 3.3. 50 A of this standard solution are applied to the column and chromatographed as described above to calibrate the system.

In addition a control run is performed to install the retention times for hirudin derivatives desulfatohirudin 1-64 and desulfatohirudin 1-63, C-terminal degradation products of recombinant hirudin that usually occur upon expression of the protein in wild type strains of Saccharomvces cerevisiae (European Patent Application No. 225 633). Therefore, 50 A supernatant ofa culture of strain S. cerevisiae GRF18/pJDB207/PH05-HIR grown for 42 h as described in European Patent Application No. 225 633 are applied to the column.

Under the conditions described above, recombinant hirudin 1-65 shows a retention time of 17.45, whereas hirudin 1-64 and hirudin 1-63 have a retention time of 18.90 and 14.64, respectively. As the method outlined cannot unequivocally detect other possible hirudin derivatives, "total hirudin" is defined as the sum of hirudin 1-65, 1-64 and 1-63.

Fig. 1 shows the production of hirudin compounds 1-65 (full-length intact hirudin), 1-64 and 1-63 by yeast strains H449/pDP34/GAPFL-YHIR(A), Tr 1357/pDP34/GAPFL YHIR(B) and Tr 1 195/pDP34/GAPFL-YHIR(C) during 168 h of fermentation in YPD-broth. Strain H449/pDP34/GAPFL-YHIR shows an extensive degradation of hirudin 1-65 to 1-64 and 1-63, which increases dramatically during prolonged periods of fermentation. After 168 h of fermentation intact hirudin 1-65 constitutes approximately 30 % of the total hirudin detected by the method described. In contrast, proteolysis of hirudin 1-65 to 1-64 and 1-63 is less pronounced in strains Tr 1357/pDP34/GAPFL-YHIR and Tr 1195/pDP34/GAPFL-YHIR.Full-length hirudin 1-65 makes up approximately 85 % of total hirudin detected in the culture supernatants of both strains, this proportion remaining constant over prolonged fermentation, i.e. 72 h and 168 h. However, the absolute amount of detected hirudin produced by strain Tr 1 195/pDP34/GAPFL-YHIR is about 30 % lower than the amount produced by strain Tr 1357/pDP34/GAPFL-YHIR.

Example 5: Fermentation of S. cerevisiae strains H449/DDP34/GAPFGYHIR, Tr 1195/pDP34/GAPFL-YHJR and Tr 1357/pDP34/GAPSYEIIR in minimal medium (Me 42-broth) Cells of Saccharomyces cerevisiae strains H449/pDP34/GAPFL-YHIR and Tr 1 195/pDP34/GAPFL-YHIR are each grown in a preculture of 10 ml minimal medium composed of (girl): yeast nitrogen base w/o aa (Difco) 8.4 L-asparagine (Fluka) 10.0 L-histidine (Fluka) 1.0 L-leucine (Fluka) 0.1 glucose (Amylum) 20.0 Cells of Saccharomvces cerevisiae Tr 1357/pDP34/GAPFL-YHIR are grown in a 10 ml preculture of the identical medium, but without the addition of leucine.

The precultures are grown for 72 h at 280C and 250 rpm. The main culture medium (He 42) is composed of (g/l): yeast nitrogen base w/o aa (Difco) 5.0 L-asparagine (Fluka) 7.5 casamino acids (BBL/Becton-Dickinson) 8.5 2-morpholino ethane sulfonic acid (Fluka) 10.0 adenine (Fluka) 0.05 L-histidine (Fluka) 0.04 L-leucine (Fluka) 0.1 L-tryptophane (Fluka) 1.0 calcium-D-panthotenate (Fluka) 0.03 glucose (Amylum) 30.0 The main cultures (50 ml) are inoculated from the preculture to a concentration of 106 cells/ml and incubated up to 168 h at 280C and 250 rpm. Appproximately 3x108 cells/ml are obtained at the end of the fermentation. At several time points during the fermentation, aliquots of the cultures are taken and subsequently analyzed for desulfatohirudin by HPLC (infra).

Example 6: HPLC-analvsis of hirudin production by S. cerevisiae strains H449/pDP34/GAPFL-YHIR. Tr 1195/pDP34/GAPFL-YHIR and Tr 1357/DP34/GAPPL-YHIR fermented in minimal medium (He 42-broth) Samples are withdrawn and subsequently analyzed for hirudin content by reversed phase HPLC analysis as described in example 4 (supra).

As depicted in Fig. 2, only a neglectable amount of full-length 1-65 can be found in culture supernatants of strain H449/pDP34/GAPFL-YHIR(A) when fermented in He 42 medium. The predominant hirudin compound is hirudin 1-63, which after long fermentation times (72 to 168 h) constitutes almost all of the hirudin detected by HPLC.

In contrast, hirudin 1-65 can be recovered from culture supernatants of Tr 1357/pDP34/GAPFL-YHIR(B), representing approximately 80 % of the total hirudin detected at any given time point. Conversion of hirudin 1-65 to 1-64 and 1-63 is also decreased in Tr 1 195/pDP34/GAPFL-YHIR(C) when compared to H449/pDP34/GAPFL YHIR. However, in contrast to the results obtained with Tr 1357/pDP34/GAPFL-YHlR hirudin 1-65 degradation increases during long fermentation times and leads to a relatively low proportion of full-length hirudin 1-65 (about 30 % of total hirudin detected after 168 h of fermentation).Moreover, as can be observed during fermentation in rich medium (see example 4), the overall hirudin production by Tr 1 195/pDP34/GAPPL-YHIR is about 30 % lower when compared to the hirudin production by strain Tr 1357/pDP34/GAPFL YHIR.

Summarizing the data presented in example 4 and 6, it can be concluded that strains deficient in either protease yscA (Tr 1 195/pDP34/GAPPL-YHIR) or carboxypeptidase yscY (Tr 1357/pDP34/GAPFL-YHIR) show significantly greater production of full-length hirudin 1-65 than a strain exhibiting both protease activities (H449/pDP34/GAPFL YHIR). The reduced C-terminal degradation of hirudin 1-65 to hirudin 1-64 and 1-63 is most likely due to the absence of active carboxypeptidase yscY in both strains. The degree of residual hirudin proteolysis varies depending on the growth media.

However, concerning the total hirudin titer exhibited a strain with a deficiency in carboxypeptidase yscY (Tr 1357/pDP34/GAPFL-YHIR) is by far superior to a strain defective in protease yscA (Tr 1 195/pDP34/GAPFL-YHIR) (appr. 30 %). This superiority is independent of the medium used and of the degree of C-terminal hirudin degradation.

Deposition of microorganisms Saccharomyces cerevisiae strain H449 was deposited at the Deutsche Sammlung von Mikroorganismen (DSM), Mascheroder Weg lb, D-3300 Braunschweig on February 18, 1988, accession number DSM 4413.

Claims (27)

Claims:

1. A yeast strain lacking carboxypeptidase yscY activity, disposing of protease yscA and carboxypeptidase ysca activities, lacking endogenous two-micron DNA and which has been transformed with a hybrid vector comprising a yeast promoter operably linked to a DNA sequence coding for a protein homologous or heterologous to yeast, a DNA sequence containing yeast transcription termination signals and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites.

2. A yeast strain according to claim 1 wherein the hybrid vector comprises a yeast promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding a homologous or heterologous protein, a DNA sequence containing yeast transcription termination signals and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites.

3. A yeast strain according to claim 1 wherein the DNA sequence codes for a heterologous protein or a mutant thereof.

4. A yeast strain according to claim 2 wherein the heterologous protein is desulfatohirudin or a variant or mutant thereof.

5. A yeast strain according to claim 1 which additionally lacks peptidase activity selected from the group consisting of yscB, yscS and vacuolar aminopeptidases.

6. A yeast strain according to claim 6 which additionally lacks yscB activity.

7. A yeast strain according to claim 6 which additionally lacks yscB and yscS activities.

8. A yeast strain according to claim 1 which has been transformed with a hybrid vector comprising the complete two-micron DNA additionally including an intact D gene.

9. A yeast strain according to claim 1 wherein the hybrid vector comprises a yeast promoter selected from the group consisting of the MFal promoter, GAL1 promoter, a promoter of a gene encoding a glycolytic enzyme, ADHI promoter, TRPI promoter and the PHOS promoter which has optionally been deprived of its upstream activation sites.

10. A yeast strain according to claim 2 wherein the hybrid vector comprises a first DNA sequence selected from the group consisting of the signal and prepro sequences of the yeast invertase, a-factor, pheromone peptidase (KEX1), "killer toxin" and repressible acid phosphatase (PHO5) genes and the glucoamylase signal sequence from Aspergillus awamon.

11. A yeast strain according to claim 2 wherein the hybrid vector comprises a second DNA sequence which codes for a desulphatohirudin compound.

12. A yeast strain according to claim 1 wherein the hybrid vector comprises one or more selective genetic markers for yeast.

13. A yeast strain according to claim 1 wherein the hybrid vector comprises a selective genetic marker and an origin of replication for a bacterial host

14. A Saccharomvces cerevisiae strain according to claim 1.

15. A transformed yeast strain according to claim 1 which is S. cerevisiae Tr l357/pDP34/GAPFL-YHIR as herein described.

16. Method for the production of a transformed yeast strain according to claim 1 comprising transforming a yeast strain which lacks carboxypeptidase yscY activity, disposes of protease yscA and carboxypeptidase ysca activities and is free of endogenous two-micron DNA with said hybrid vector.

17. Method for the production of a protein homologous or heterologous to yeast comprising culturing a yeast strain lacking carboxypeptidase yscY activity, disposing of protease yscA and carboxypeptidase ysca activities and lacking endogenous two-micron DNA which has been transformed with a hybrid vector comprising a yeast promoter operably linked to a DNA sequence coding for said protein, a DNA sequence containing yeast transcription termination signals and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites, and isolating said protein.

18. Method for the production of a protein homologous or heterologous to yeast according to claim 17 wherein the hybrid vector comprises a yeast promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence coding for said protein, a DNA sequence containing yeast transcription termination signals and the complete two-micron DNA including intact REP1, REP2 and FLP genes, as well as intact ORI, STB, IR1 and IR2 sites.

19. Method according to claim 17 wherein the homologous or heterologous protein is susceptible to posttranslational C-terminal degradation by carboxypeptidase yscY.

20. Method for the production of a protein which is heterologous to yeast according to claim 17.

21. Method for the production of a protein heterologous to yeast according to claim 20 wherein said heterologous protein originates from a virus, a prokaryotic or a eukaryotic organism.

22. Method for the production of a protein heterologous to yeast according to claim 20 wherein said heterologous protein originates from a eukaryotic organism.

23. Method for the production of a protein heterologous to yeast according to claim 20 wherein said protein is selected from the group consisting of human a-interferon, hybrid interferon, transforming growth factor P, human calcitonin, human atrial natriuretic peptide, connective tissue activating peptide-III, epidermal growth factor, insulin-like growth factors I and II and desulphatohirudin including variants and mutants thereof.

24. Method for the production of a protein heterologous to yeast according to claim 18 wherein said heterologous protein is desulphatohirudin.

25. Method according to claim 24 for the preparation of a desulphatohirudin compound selected from the group consisting of desulphatohirudin variant HV1, HVl modified (a, b), HV2, HV2 modified (a, b, c), HV3, variants of HV3 and des(Val2)-desulphatohirudin.

26. Method according to claim 24 for the preparation of desulphatohirudin variant HV1.

27. Method according to claim 17 comprising culturing a yeast strain according to any one of claims 1 to 15.