Process for the production of proteins

The invention pertains to the field of recombinant DNA technology and concerns a method for the production of a protein heterologous to yeast in a homogeneous form with the aid of Saccharomyces cerevisiae strain HT393 or a derivative thereof carrying a hybrid vector containing the genes for said protein.

The invention pertains to the field of recombinant DNA technology and 
concerns a method for the production of proteins with the aid of 
genetically engineered yeast cells carrying hybrid vectors comprising the 
genes for said proteins. 
BACKGROUND OF THE INVENTION 
Although in genetic engineering numerous protein expression systems for 
prokaryotic and eukaryotic hosts are already known, there is a continuing 
demand for novel systems which have advantages over the known systems. 
Working on the expression of heterologous proteins in the baker yeast 
Saccharomyces cerevisiae, it has been commonly observed, that a high-level 
expression is dependent on many factors, e.g. plasmid stability, plasmid 
copy number, promoter strength, translation efficiency, low protein 
degradation. 
In this context, one of the very important requisites is the yeast strain 
which is used for the production. 
Recently, quite a number of heterologous proteins have been expressed in 
different yeast strains after transformation of yeast cells with suitable 
expression vectors comprising DNA sequences coding for said proteins, like 
e.g. .alpha.-interferon (Hitzeman et al. Nature (1981), 294, 717-722), 
tissue-type plasminogen activator (EP-A-143081) or certain 
desulfatohirudins (EP-A-225633). In many cases, however, the heterologous 
proteins are not synthesized in pure form, but as a mixture containing 
partially degraded such as C- or N-terminally shortened proteins. For 
instance, the expression of human atrial natriuretic peptide (hANP) in 
yeast resulted in the secretion of two forms of mature hANP differing in 
their C-terminus (Vlasuk et al. J. Biol. Chem. (1986), 261, 4798-4796). 
Similar results have been obtained after the expression of epidermal 
growth factor (EGF) in yeast (George-Nascimento et al. Biochemistry 
(1988), 27, 797-802) where the secreted expression products were 
heterologous in that either the last (Arg 53) or the last two amino acids 
(Leu 52 and Arg 53) were missing and no full-length EGF was produced. 
The separation of mixtures containing full-length proteins such as 
.alpha.-interferon, tissue-type plasminogen activator, inhibitors of 
tissue-type plasminogen activator, or desulfatohirudins as well as 
partially degraded like C- or N-terminally shortened derivatives thereof 
into the individual components and the purification of these components to 
homogeneity, if these derivatives are biologically active at all, is 
laborious and time-consuming. Considering the incidental expenses there is 
a need for improved methods which render possible the economic production 
of homogenous proteins such as desulfatohirudin in yeast. It is an object 
of the present invention to provide methods for the production of proteins 
heterologous to yeast in a homogenous form. 
Surprisingly it has been found, that the use of Saccharomyces cerevisiae 
strain HT393 for the expression of heterologous proteins leads to 
increased yield of biologically active and undegraded form of the 
expressed heterologous protein, compared to other Saccharomyces cerevisiae 
strains that are genetically closely related, e.g., to strain 
cl3-ABYS-86(DSM 9698) that is genetically closest related. 
DESCRIPTION OF THE INVENTION 
The present invention concerns a process for the production of a protein 
heterologous to yeast in a homogenous form characterized in that 
Saccharomyces cerevisiae strain HT393 (DSM 9697) or a derivative thereof 
is used for the expression of said heterologous protein. 
In a preferred embodiment, the present invention relates to an improved 
process for the production of a protein heterologous to yeast in a 
homogenous form comprising culturing Saccharomyces cerevisiae strain 
HT393(DSM 9697) or a derivative thereof that has been transformed with a 
hybrid vector comprising a DNA sequence coding for said heterologous 
protein and isolating said heterologous protein. 
A derivative of HT393 is a strain that is derived from HT393 and shows the 
same properties in respect to the production of heterologous proteins. The 
use of the inventive strains leads, e.g., to an increased yield of a 
biologically active and undegraded form of an expressed heterologous 
protein. 
This heterologous protein can also be processed further, e.g. glycosylated. 
Useful proteins are, for example, enzymes that can be used, for the 
production of nutrients and for performing enzymatic reactions in 
chemistry, or proteins which are useful and valuable as nutrients or for 
the treatment of human or animal diseases or for the prevention thereof, 
for example hormones, polypeptides with immunomodulatory, anti-viral and 
anti-tumor properties, antibodies, viral antigens, vaccines, clotting 
factors, enzyme inhibitors, food-stuffs and the like. 
Such heterologous structural genes are for example those coding for 
hormones such as secretin, thymosin, relaxin, calcitonin, luteinizing 
hormone, parathyroid hormone, adreno adenocorticotropin, 
melanoycte-stimulating hormone, .beta.-lipotropin, urogastrone or insulin, 
growth factors, such as epidermal growth factor, insulin-like growth 
factor (IGF), e.g. IGF-I and IGF-II, mast cell growth factor, nerve growth 
factor, glia derived nerve cell growth factor, or transforming growth 
factor (TGF), such as TGF.alpha. or TGF.beta., e.g. TGF.beta.1, .beta.2 or 
.beta.3, growth hormone, such as human or bovine growth hormones, 
interleukin, such as interleukin-1 or -2, human macrophage migration 
inhibitory factor (MIF), interferons, such as human .alpha.-interferon, 
for example interferon-.alpha.A, .alpha.B, .alpha.D or .alpha.F, 
.beta.-interferon, .gamma.-interferon or a hybrid interferon, for example 
an .alpha.A-.alpha.D- or an .alpha.B-.alpha.D-hybrid interferon, 
especially the hybrid interferon BDBB, inhibitors such as proteinase 
inhibitors such as .alpha..sub.1 -antitrypsin, SLPI, an inhibitor of the 
plasminogen activator (PAI-2) 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, tumor necrosis factor, somatostatin, 
renin, .beta.-endorphin, 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, 
e.g. sCD23 and the like, calcitonin, human calcitonin-related peptide, 
blood clotting factors, such as factor IX or VIIIc, erythropoietin, eglin, 
such as eglin C, desulfatohirudin, such as desulfatohirudin variant HV1, 
HV2 or PA, human superoxide dismutase, viral thymidin kinase, 
.beta.-lactamase, glucose isomerase. 
Preferred genes are those coding for a human .alpha.-interferon or hybrid 
interferon, particularly hybrid interferon BDBB and an inhibitor of the 
plasminogen activator (PAI-2). 
In a preferred embodiment of the invention, the expressed heterologous 
protein is not secreted. 
S. cerevisiae strain HT 393 (E95-1-2A) is obtained from strain cl3-ABYS-86 
(DSM 9698) as described in Heinemeyer et al., EMBO J. (1991), 10, 555-562. 
The wording derivatives of S. cerevisiae strain HT 393 embraces strains 
that are derived by genetic engineering from HT 393 and are, e.g., strains 
that are additionally cleared from two-micron (2.mu.) DNA (cir.sup.0), 
have a different mating type (MAT.alpha.) and/or different selection 
marker. Preferred is HT 393 and derivatives thereof that are also 
deficient for protease A, protease B, carboxypeptidase Y, carboxypeptidase 
S and proteinase yscE. 
Essentially preferred are HT 393 and derivatives thereof that show at least 
the following genetic characterization MATa, leu2-3, leu2-112, 
ura3.DELTA.5, prb1-1, cps1-3, prc1-1, pra1-1 and pre1-1. 
EXPRESSION CASSETTES 
A suitable expression cassette comprises a promoter operably linked to a 
DNA sequence coding for the protein and to a DNA sequence containing 
transcription termination signals. 
The expression cassette may additionally comprise a DNA sequence encoding a 
signal peptide linked in the proper reading frame to the DNA sequence 
coding for the inventive protein. 
In a preferred embodiment, the promoter, the signal sequence, if present, 
and the terminator are recognized by the yeast expression system. 
Promoter suitable for expression in a certain host are well known. Examples 
are the promoter of the TRP1 gene, the ADC1 gene (coding for the alcohol 
dehydrogenase I) or ADR2 gene (coding for the alcohol dehydrogenase II), 
acid phosphatase (PHO5) gene, CUP1 gene, isocytochrome c gene, or a 
promoter of the genes coding for glycolytic enzymes, such as TDH3, 
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a shortened version of 
GAPDH (GAPFL), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate 
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, 
phosphoglucose isomerase, invertase and glucokinase genes, a promoter of 
the yeast mating pheromone genes coding for the a- or .alpha.-factor, or 
the GAL/CYC1 hybrid promoter (intergenic region of the GAL1-GAL10 
gene/Cytochrome1 gene; Guarente et al. Proc. Natl. Acad. Sci. (1982), 79, 
7410-7414). Preferred vectors of the present invention contain, e.g., 
promoters with transcriptional control that can be turned on or off by 
variation of the growth conditions, e.g. the PHO5, the ADR2, or the 
GAL/CYC1 promoter. For example, the PHO5 promoter can be repressed or 
derepressed at will, solely by increasing or decreasing the concentration 
of inorganic phosphate in the medium. 
The DNA sequence encoding a signal peptide ("signal sequence"), e.g. a 
yeast signal peptide, is derived, e.g., from a yeast gene, coding for a 
protein which is ordinarily secreted. Yeast signal sequences are, for 
example, the signal sequences of the yeast invertase (SUC2), 
.alpha.-factor, pheromone peptidase (KEX1), "killer toxin" and repressible 
acid phosphatase (PHO5) genes and the glucoamylase signal sequence from 
Aspergillus awamori. Additional sequences, such as pro- or 
spacer-sequences which may carry specific processing signals can also be 
included in the constructions to facilitate accurate processing of 
precursor molecules. For example, the processing signals contain a Lys-Arg 
residue, which is recognized by a yeast endopeptidase located in the Golgi 
membranes. 
A DNA sequence containing transcription termination signals, e.g. yeast 
transcription termination signals, is preferably the 3' flanking sequence 
of a gene, e.g. a yeast gene, which contains proper signals for 
transcription termination and polyadenylation. The preferred flanking 
sequence is that of the yeast PHO5, the FLP and the .alpha.-factor gene. 
The DNA coding for the protein according to the invention may be isolated 
from a gene bank of the natural host by methods known in the art like 
excision of the desired fragment using suitable restriction enzymes, PCR 
or may be synthesized chemically, using, e.g., the preferred codon usage 
of the host. 
The promoter, the DNA sequence coding for the protein and the DNA sequence 
containing 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 protein or, if a signal sequence is present, the 
signal sequence-protein complex; the transcription termination signals 
effect proper termination of transcription and polyadenylation. In case a 
signal sequence is used, the signal sequence is linked in the proper 
reading frame to the protein gene in such a manner that the last codon of 
the signal sequence is directly linked to the first codon of the gene for 
the protein. The signal sequence, if present, has its own ATG for 
translation initiation. The junction of these sequences may, for example, 
be effected by means of synthetic oligodeoxynucleotide linkers carrying 
the recognition sequence of an endonuclease. Examples for related 
expression cassettes are described e.g. in EP-A-341215. 
Preferred expression cassettes comprise the PHO5, the ADR2, or the GAL/CYC1 
promoter, the DNA coding for a protein as defined above and the PHO5, 
.alpha.-factor or FLP terminator. 
Especially preferred are expression cassettes as comprised, for example in 
pPAI-2A-10, pPAI-2A-20, pPAI-2B-10, pPAI-2B-20, or in SEQ ID NO:1 or a 
functional fragment or derivative thereof. 
A functional fragment or derivative of said recombinant DNA molecule is, 
for example, a fragment coding for a shortened or elongated version of a 
above mentioned protein or an expression cassette containing a recombinant 
DNA molecule coding for said protein. 
Recombinant Plasmids 
The expression cassette as described above are normally inserted in a 
plasmid, suitable for expression of heterologous proteins in yeast. These 
plasmids are based, i.e., on the two-micron, pMB354 or the pEMBLyex 
plasmids (Cesareni and Murray, Genetic Engineering (1987), 4, 135-154). 
Suitable recombinant plasmids contain, for example, apart from the protein 
expression cassette, a DNA segment originating from two-micron DNA 
containing the origin of replication or total two-micron DNA. For example, 
plasmids according to 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 linearized 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 IR2 sites of two-micron DNA as well as small "FLP 
recognition target" (FRT) sites, located near the center of each inverted 
repeat (IR) at which the FLP recombinase acts, are maintained. Optionally, 
the restriction site is chosen such that the D gene of two-micron DNA is 
kept intact too. Suitable restriction sites are, for example, the unique 
Hpal and SnaBl sites located outside of all of said genes and sites. 
However, it is likewise possible to insert the expression cassette and 
further components (cf. below) at different (such as two) restriction 
sites, especially those mentioned above, within two-micron DNA. 
Such a plasmid derivative may comprise two invertedly repeated FRT sites or 
an additional, third FRT site. The former kind of plasmid is hereinafter 
called a "symmetric two-micron-like hybrid vector". The latter kind of 
plasmid is hereinafter called "symmetric two-micron-like disintegration 
vector" despite it is not a real symmetric plasmid but gives rise to a 
symmetric two-micron-like hybrid vector in the yeast cell transformed 
therewith. 
A symmetric two-micron-like hybrid vector of the invention does 
preferentially not contain bacterial or viral DNA sequences, i.e. DNA 
derived from a bacterial genome, plasmid or virus. However, a 
two-micron-like disintegration vector of the invention may comprise DNA 
sequences of prokaryotic origin between the two directly repeated FRT 
sites which are excised from the vector in the transformed yeast cell in 
which the symmetric two-micron-like hybrid vector is generated from the 
disintegration vector. These DNA sequences are bacterial sequences as 
described below and can provide to the vector essential structural or 
functional features or can also only have the function of filling up the 
two regions between the two invertedly repeated FRT sites of an 
unsymmetric two-micron-like plasmid derivative or of an "unsymmetric" 
disintegration vector in order to construct a symmetric two-micron-like 
hybrid vector or a symmetric disintegration vector. 
Preferably, the expression plasmids according to the invention include one 
or more, especially one or two, selective genetic markers, e.g. a marker 
for yeast and a marker and (except for symmetric two-micron like hybrid 
vectors) an origin of replication for a bacterial host, especially 
Escherichia coli. 
As to the selective gene markers, any marker gene can be used which 
facilitates the selection for transformants due to the phenotypic 
expression of the marker gene. Suitable markers 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, LEU2, LYS2, HIS3, or TRP1 gene. 
As the amplification of the expression plasmids is conveniently done in a 
prokaryote, such as E. coli, a prokaryotic (e.g. E. coli), genetic marker 
and a prokaryotic (e.g. E. coli), replication origin are included 
advantageously. These can be obtained from corresponding prokaryotic 
plasmids, for example E. coli plasmids, such as pBR322 or a pUC plasmid, 
for example pUC18 or pUC19, which contain both prokaryotic, e.g. E. coli, 
replication origin and genetic marker conferring resistance to 
antibiotics, such as ampicillin. 
Apart from the protein expression cassette, replication origin(s) and 
genetic marker(s) the expression plasmids according to the invention can 
contain optionally additional expression cassettes, such as 1 to 3 
additional protein expression cassettes. The additional protein expression 
cassette(s) are identical to or different from each other and are 
identical to or different from the protein expression cassette already 
present on the vector. 
Isolation of Proteins 
The protein can be isolated by conventional means. For example, the first 
step consists usually in lysing or mechanically breaking the cell wall and 
removing the cell debris by centrifugation or, in the case of secretory 
proteins, in separating the cells from the culture fluid by means of 
centrifugation. The supernatant can be enriched for 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 sulfate or by extraction with a suitable solvent. Host 
proteins, if present, can also be precipitated, for example, by means of 
acidification with acetic acid (for example 0.1%, pH 4-5). 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 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. 
A further embodiment of the invention concerns the use of Saccharomyces 
cerevisiae strain HT393(DSM 9697) or a derivative thereof for the 
production of a protein heterologous to yeast in a homogenous form.