POLYPEPTIDE PRODUCTION IN FUNGI
WIPO Patent Application WO/
PAI-2, the main inhibitor of urokinase-type plasminogen activator, is produced with good yield in fungi, especially yeasts such as Saccharomyces cerevisiae, by rDNA techniques. Preferably, the PAI-2 is produced intracellularly as a soluble, correctly-folded product, even though in nature it is secreted.
Inventors:
James, Steven
Application Number:
Publication Date:
02/21/1991
Filing Date:
08/07/1990
Export Citation:
DELTA BIOTECHNOLOGY LIMITED BALLANCE
James, Steven
International Classes:
C07K14/745; C07K14/81; (IPC1-7): C07K15/00; C12N15/00
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Domestic Patent References:
Other References:
CHEMICAL ABSTRACTS, Volume 111, No. 25, 18 December 1989, (Columbus, Ohio, US), G.A. SILVERMAN et al.: "Use of Yeast Artificial Chromosome Clones for Mapping and Walking Within Human Chromosome Segment", see page 205, Abstract 226586t & Proc. Natl. Acad. Sci. U.S.A. ), 7485-9
Science, Volume 244, 16 June 1989, (Washington, DC, US), B.H. BROWNSTEIN et al.: "Isolation of Single-Copy Human Genes from a Library of Yeast Artificial Chromosome Clones", pages
see page 1349, figure 1
The Journal of Biological Chemistry, Volume 263, No. 10, 5 April 1988, The American Society for Biochemistry and Molecular Biology, Inc., (US), R.D. YE et al.: "Mammalian Protein Secretion without Signal Peptide Removal. Biosynthesis of Plasminogen Activator Inhibitor-2 in U-937 Cells", pages
see page 4869, column 1, lines 1-29; page 4873, column 2, lines 41-42 (cited in the application)
1. A process for preparing PAI2 comprising the fermentation o a fungal cell which is transformed with a genetic construct t express PAI2, wherein the genetic construct does not compris an extraneous secretion leader sequence expressible as a fuse polypeptide with the PAI2 and wherein the PAI2 is not secrete from the fungal cell.
2. A process according to Claim 1 wherein the fungal cell is yeast.
3. A process according to Claim 2 wherein the fungal cell i Saccharomyces cerevisiae.
4. A process according to any other of the preceding claim wherein the PAI2 is identical to a naturallyoccurring PAI2 but unglycosylated.
5. A genetic construct for expressing PAI2 in a fungal cell the construct comprising a 5 expression regulatory sequence, coding sequejace for PAI2 and a 3 regulatory sequence, wherei there is no fungallyeffective secretion signal coding sequenc interposed between the 5 regulatory sequence and the PAI coding sequence.
6. A genetic construct according to Claim 5 wherein the codin sequence for PAI2 encodes a naturallyoccurring PAI2.
Description:
POLYPEPTIDE PRODUCTION IN FUNGI The present invention relates to the production of polypeptides, specifically plasiriinogen activator inhibitor 2 (PAI-2), in fungi. PAI-2 is a naturally occurring inhibitor of serin proteases and, more specifically, of plasminogen activators of the urokinase-type (u-PA) and of the tissue-type (t-PA) (se Astedt et al , 1987 for review) . It is a member of a group of structurally and functionally related proteins known as th SERPIN superfamily (Carrell and Travis, 1985), which includes αl-antitrypsin and plasminogen activator inhibitor 1. Suc protease inhibitors act by mimicking the protease's natural substrate and forming a 1:1 covalent inactive complex which i subsequently cleared from the body. The primary determinant o SERPIN specificity is an amino acid at the reactive centre whic is analogous to the amino acid immediately amino' terminal to th peptide bond cleaved in the natural substrate. In the case o PAI-2 this amino acid is arginine, which is also present at th position at which u-PA and t-PA cleave their natural substrat plasminogen. It is clear, however, that other amino acids i the inhibitor are also important for determining specificity. PAI-2 has been isolated from placenta (Kawano et al , 1968j Hol burg et al , 1978; Astedt et al , 1985), human monocyte (Golder and Stephens, 1983) and the human monocyte-lik histiocytic lymphoma cell line U937 (Vassalli et al , 1984 Kruithof et al , 1986). It is found as an unglycosylated 47k
molecule in placental extracts (Astedt et al , 1985) and in a high molecular weight form (58-70kD) in which the protein is glycosylated to a variable degree (Wohlwend et al , 1987; Ye e al , 1988). The latter is secreted from human monocytes and U937 cells (Wohlwend et al , 1987; Genton et al , 1987) and is th predominant form in pregnancy plasma (Lecander and Astedt, 1986). Although the unglycosylated form is detected in culture medium from cultured U937 cells, it is not clear whether this is due to secretion or as a result of cell lysis (Ye et al , 1988). Stephens et al (WO 86/01212) disclosed minactivin, a plasminoge activator inhibitor isolated from human monocyte cultures. This protein is PAI-2 and therefore identical to the molecul previously isolated from placenta (Kawano et al , 1968; Holmber et al , 1978) except that, as the molecular mass was estimated t be 60-70kD, it was probably the glycosylated form of th protein. Antalis et al (EP 238 275) disclose the production of minactivi by recombinant DNA technology. A minactivin (PAI-2) cDNA codin sequence was introduced into an Escherichia coll expressio vector which directed the expression of active minactivin in E. coll .
Webb et al (EP 278 696) used recombinant" DNA technology t produce PAI-2 though they advocated removing 22 amino acids fro the N-terminus of the protein to ensure maximal biologica activity. These amino acids are, however, present in the activ natural molecule and do not constitute a cleavable signa peptide (Ye et al, 1988). The effect of removal of these 2 amino acids from the N-terminus on the activity of PAI-2 has no been examined. However, PAI-2 is secreted in the normal cells which produce i and therefore, although it lacks a cleavable leader sequence, i must have a sequence which directs secretion. This sequenc would have been thought to be effective in fungal cells, a least to the extent of lodging the PAI-2 in the membran fraction. For example, Livi et al (1988) found tha interleukin-lβ, which similarly has an internal secretio signal, is at least partially secreted in yeast. Surprisingly however, it is found that PAI-2 is expressed as a intracellular protein. Moreover, it is obtainable from th soluble fraction when the cells are lysed, unlike when it i expressed in E. coli (EP-A-238 275). Thus, the undesirabl pattern of fungal glycosylation is avoided and the protein ca be recovered relatively easily. We have also found the yield t be surprisingly high.
In essence, therefore, the present invention provides the production of plasminogen activator inhibitor 2 in fungi such as Saccharomyces cerevlslae. A PAI-2 cDNA or other coding sequence is operationally linked to an effective transcription promoter and transcription terminator in a plasmid which can be maintained in the yeast cells. The PAI-2 protein has been found to be expressed as an intracellular, unglycosylated protein which can relatively easily be recovered from cell extracts by simple purification steps. Suitable fungal cells include the genera Plchla , Saccharomyces, Kl uyveromyceε , Candida, Torul opsis , Hansen ul a , Schlzosaccharomyces , Citeromyces, Pachysolen, Debaromyces, Metschunikowia , Rhodosporidium, Leucoεporldlum, Botryoascus, Sporidlobolus , Endomycopsis , and the like. Preferred genera are those selected from the group consisting of Plchla , Saccharomyces,. Kluyveromyces, Yarrowla and Hansenula , because the ability to manipulate the DNA of these yeasts has, at present, been more highly developed than for the other genera mentioned above. Examples of Saccharomyces are Saccharomyces cerevlslae, Saccharomyces Itallcus and Saccharomyces rouxil . Examples of Kluyveromyces are Kluyveromyces fraglllε and Kluyveromyces lactls .
Examples of Hansenula are Hansenula polymorpha , Hansenul anomala and Hansenula capsulata . Yarrowla llpolytica is an example of a suitable Yarrowl species. Saccharomyces cerevlslae and Schlzosaccharomyces pombe ar particularly preferred. Filamentous fungi such as Asperglllu nlger are also suitable. Fungal cells can be transformed by: (a) digestion of the cell walls to
(b) mixing the spheroplasts with transforming DNA (derived fro a variety of sources and containing both native and non native DNA sequences) ; (c) regenerating the transformed cells. The regenerated cells are then screened for the incorporation o the transforming DNA It has been demonstrated that fungal cells of the genera Plchla Saccharomyces , Kluyveromyces , Yarrowla and Hansenula can b transformed by enzymatic digestion of the cells walls to giv
the spheroplasts are then mixed with the transforming DNA and incubated in the presence of calcium ions and polyethylene glycol, then transformed spheroplasts are regenerated in regeneration medium. Methods for the transformation of S. cerevlslae are taught generally in EP 251 744, EP 258 067 and WO 90/01063, all of which are incorporated herein by reference. By "PAI-2" we mean the polypeptide or polypeptides isolated from placentas and the other sources given above and minor variations of such polypeptide(s) which (a) have 80% homology (preferably 85%, 90%, 95% or 99%) with any such polypeptides for the respective corresponding regions of the two molecules, the regions being at least 350 (preferably at least 400) amino acids long, (b) have an arginine residue at the said position of the reactive centre and (c) have a second order rate constant for urokinase-type plasminogen activator inhibition of at least 10~ M~~s~- and generally up to 2 x 107 M-~s~~, for example about 2 x 10- M~~s~~, as measured in the method of Thorsen et al (1988) Eur. J. Blochem. 175, 33-39 (see Table 1 especially). The PAI-2 is unglycosylated.
The PAI-2 may be expressed as a fusion with other polypeptides or may be conjugated to other polypeptides or pharmacologically active compounds by known techniques, except that it is preferred for any fusion polypeptide not to be secreted. Suitable promoters for S. cerevlslae include those associated with the phosphoglycerate kinase (PGK) gene, GALl or GAL10 genes, CYC1 , acid phosphatase { PH05) , TRP1 , ADH1 , ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, glucokinase, -mating factor pheromone, α-mating factor pheromone, the promoter of EP-A-214 638, hybrid promoters involving hybrids of parts of 5 regulatory regions with parts of other 5 regulatory regions or with upstream activation sites (e.g. the promoter of EP-A-258 067) and the PRB1 promoter. Preferably, the promoter is inducible. The preferred promoters are the promoters of EP-A-258 067 and PRBl . The PRBl promoter is described in our co-pending application GB
(our ref. DELF/P7375GB) which is incorporated herein by reference. Example 2 below reproduces the relevant parts thereof for the purposes of this application. The transcription termination signal is preferably the
3' flanking sequence of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation in fungi. Suitable 3' flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter or, in the case of a hybrid promoter, the downstream part thereof. Alternatively, they may be different. Preferably, the termination signal is that of the S. cerevlslae PGK or ADH1 genes. Preferably, the DNA construct according to the present invention is provided at both ends with synthetic oligonucleotide linkers which allow insertion and cloning of the construct in a cloning vector. The fungal expression control sequence (i.e. effective in fungi), the DNA sequence coding for PAI-2 and the fungal transcription termination signals (i.e. effective in fungi) are operably linked to each other, i.e. they are juxtaposed in such a manner that their normal functions are maintained. Thus, the array is such that the expression control sequence effects proper expression of PAI-2 and the transcription termination signals effect proper termination of transcription and polyadenylation. The junction of these sequences is preferably effected by means of synthetic oligonucleotide linkers which may carry the recognition sequence of an endonuclease.
The DNA constructs according to the invention may be prepared b methods known in the art, for example by linking a eukaryotic expression control sequence, a DNA sequence coding for PAI-2 an a DNA sequence containing eukaryotic transcription terminatio signals in such a way that proper expression of the coding sequence is effected in a yeast host. The DNA sequence coding for PAI-2 can be isolated from th sources indicated above, or a complementary double-stranded PAI 2 DNA (PAI-2 ds cDNA) is produced from PAI-2 mRNA, or synthetic gene coding for the amino acid sequence of PAI-2 i produced by means of chemical and enzymatic processes. Genomic PAI-2 DNA and PAI-2 ds cDNA are obtained, for example, according to methods that are known per se. For example, genomic PAI-2 DNA is obtained from a placenta gene bank tha contains the PAI-2 gene by cloning the placenta DNA fragments i a microorganism and identifying clones that contain PAI-2 DNA, for example by colony hybridisation using a radioactivel labelled PAI-2 DNA-specific oligonucleotide that contains a least 15, and preferably from 15 to 30, nucleotides. Th resulting DNA fragments as a rule contain in addition to th PAI-2 gene other undesired DNA constituents that can be remove by treatment with suitable exo- or endo-nucleases.
Double-stranEURed PAI-2 cDNA can be produced by isolating mRN from suitable placenta or monocyte cell lines, or U937 which ar preferably induced to synthesise PAI-2, enriching the PAI-2 mRN in the resulting mRNA mixture in a manner known per se, usin this mRNA as a template for the preparation of single-strande cDNA with RNA-dependent DNA polymerase, synthesising from this with the aid of a DNA-dependent DNA polymerase, ds cDNA, an cloning the latter into a suitable vector. Clones that contai PAI-2 cDNA are identified, for example in the manner describe above, by colony hybridisation using a radioactively labelled, PAI-2 DNA-specific oligonucleotide. The PAI-2 coding sequence can also be produced by chemica synthesis. The process is characterised in that segments of th coding and of the complementary strand of said gene ar chemically synthesised and resulting segments are linke enzymatically into a linear coding sequence for PAI-2. The chemical synthesis of DNA is well-known in the art and make use of conventional techniques. Appropriate techniques hav been compiled by S.A. Narang [ Tetrahedron 39, 3 (1983)]. I particular, the methods described in EP-A-146 785 may be use and are herein incorporated by reference.
According to the present invention there is further provided a hybrid vector having one or multiple DNA inserts each comprising a fungal expression control sequence, a DNA segment consisting of a DNA sequence coding for PAI-2 which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals. The hybrid vectors according to the invention are hybrid plasmids or linear DNA vectors and are selected depending on the host organism envisaged for transformation. The invention relates also especially to hybrid plasmids whic apart from the expression control sequence, the above DN segment and the sequence containing transcription terminatio signals contain additional DNA sequences which are inessential or less important for the function of the promoter, i.e. for th expression of the PAI-2 gene, but which perform importan functions, for example in the propagation of the cell transformed with said hybrid plasmids. The additional DN sequences may be derived from prokaryotic and/or eukaryoti cells and may include chromosomal and/or extra-chromosomal DN sequences. For example, the additional DNA sequences may ste from (or consist of) plasmid DNA, such as bacterial o eukaryotic plasmid DNA, viral DNA and/or chromosomal DNA, suc as bacterial, yeast or higher eukaryotic chromosomal DNA.
Preferred hybrid plasmids contain additional DNA sequences derived from bacterial plasmids, especially Escherlchla coll plasmid pBR322 or related plasmids, bacteriophage, yeast 2μ plasmid, and/or yeast chromosomal DNA. In the preferred hybrid plasmids according to the invention, the additional DNA sequences carry a yeast replication origin and a selective genetic marker for yeast. Hybrid plasmids containing a yeast replication origin, e.g. an autonomously replicating segment (ars), are extrachromosomally maintained within the yeast cells after transformation and are autonomously replicated upon mitosis. Hybrid plasmids containing sequences homologous to yeast 2μ plasmid DNA can be used as well. These hybrid plasmids may be integrated by recombination into 2μ plasmids already present within the cell or may replicate autonomously. The integration vectors of EP-A-251 744 or the "disintegration" vectors of EP-A-286 424 may be used. As to the selective gene marker for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker. Suitable markers for yeast are particularly those expressing antibiotic resistance or, i t the case of auxotrophic yeast mutants, genes which complement host lesions. Corresponding genes confer, for
example, resistance to the antibiotic cycloheximide or provid for prototrophy in an auxotrophic yeast mutant, for example th URA1 , URA3 , ARG4 , LEU2 , HIS 4 , HI S3, TRP5 or TRP1 gene. Advantageously, the additional DNA sequences which are presen in the hybrid plasmids according to the invention also include replication origin and a selective genetic marker for bacterial host, especially Escherlchla coll . There are usefu features which are associated with the presence of an E. col replication origin and an E. coll marker in a yeast hybri plasmid. Firstly, large amounts of hybrid plasmid DNA can b obtained by growth and amplification in E. coll and, secondly the construction of hybrid plasmids is conveniently done in E coll making use of the whole repertoire of cloning technolog based on E. coll . E. coll plasmids, such as pBR322 and th like, contain both E. coll replication origin and E. col genetic markers conferring resistance to antibiotics, fo example tetracycline and ampicillin, and are advantageousl employed as part of the yeast hybrid vectors . The additional DNA sequences which contain, for example replication origin and genetic markers for yeast and a bacteria host (see above) are hereinafter referred to as "vector DNA which, together with the above DNA construct, containing Inte alia the expression control sequence and the PAI-2 gene, i forming a hybrid plasmid according to the invention.
The hybrid vectors according to the invention may contain one or multiple DNA inserts each comprising Inter alia the expression control sequence and the DNA sequence encoding PAI-2. If the hybrid vectors contain multiple DNA inserts, preferably 2 to 4 DNA inserts, these can be present in a tandem array or at different locations of the hybrid vector. Preferred hybrid vectors contain one DNA insert or DNA inserts in a tandem array. The DNA inserts are especially head to tail arranged. The hybrid plasmids according to the invention are prepared b methods known in the art. The process for the preparation o the hybrid vectors comprises introducing one or multiple DN constructs containing a fungal expression control sequence, DNA segment consisting of a DNA sequence coding for PAI-2 whic DNA segment is under transcriptional control of said expressio control sequence, and a DNA sequence containing funga transcription termination signals, as such or introducing th components of said DNA constructs successively in th predetermined order into a vector DNA. The construction of the hybrid plasmids according to th invention is performed applying conventional ligatio techniques. The components of the plasmids are linked throug common restriction sites and/or by means of synthetic linke molecules and/or by blunt end ligation.
Another aspect of the invention involves fungal host organism transformed with a hybrid vector having one or multiple DN inserts each comprising a fungal expression control sequence, DNA segment consisting of a second DNA sequence coding for PAI- which DNA segment is under transcriptional control of sai expression control sequence, and a DNA sequence containin fungal transcription termination signals, and mutants thereof. The host is transformed with a hybrid plasmid having one o multiple DNA inserts each comprising the elements set out above The transformation of the host cells is accomplished by method known in the art. For example, the transformation of yeast wit the hybrid vectors may be accomplished according to the metho described by Hinnen et al [ Proc. Natl . Acad. Sci . USA 75, 192 (1978)]. This method can be divided into three steps: (1) Removal of the yeast cell wall or parts thereof usin various preparations of glucosidases, such as snail gut juice (e.g. GlusulaseR or HelicaseR) or enzyme mixtures obtained fro microorganisms (e.g. ZymolyaseR) in osmotically stabilize solutions (e.g. IM sorbitol) . (2) Treatment of the "naked" yeast cells (spheroplasts) wit the DNA vector in the presence of PEG (polyethylene-glycol) an Ca~+ ions.
16 (3) Regeneration of the cell wall and selection of th transformed cells in a solid layer of agar. This regeneratio is conveniently done by embedding the spheroplasts into agar For example, molten agar (about 50°C) is mixed with th spheroplasts . Upon cooling the solution to yeast growt temperatures (about 30°C) , a solid layer is obtained. This aga layer is to prevent rapid diffusion and loss of essentia macromolecules from the spheroplasts and thereby facilitate regeneration of the cell wall. However, cell wall regeneratio may also be obtained (although at lower efficiency) by platin the spheroplasts onto the surface of preformed agar layers. Preferably, the regeneration agar is prepared in a way to allo regeneration and selection of transformed cells at the sam time. Since yeast genes coding for enzymes of amino aci biosynthetic pathways are generally used as selective markers ( supra ) , the regeneration is preferably performed in yeas minimal medium agar. If very high efficiencies of regeneratio are required the following two step procedure is advantageous (1) regeneration of the cell wall in a rich complex medium, an (2) selection of the transformed cells by replica plating th cell layer onto selective agar plates. When tje DNA vector is a linear DNA vector used for transformin eukaryotic host cells, transformation is preferably done in th presence of a second vector containing a selective marker fo
yeast. This cotransformation allows enrichment for those host cells which have taken up DNA that cannot be directly selected for. Since competent cells take up any type of DNA a high percentage of cells transformed with a selective vector will also harbour any additional DNA (such as the above linear DNA vector). The transformed host cells can be improved in production of PAI-2 by mutation and selection using o methods known in the art. The mutation can be effected, for example, by U.V. irradiation or suitable chemical reagents. Strains which are deficient in protease A and B are pa such strains are generally available. The host cell may be fermented to express PAI-2 in known ways. The PAI-2 may be purified by known techniques, for example separating off the cells, lysing them, collecting the supernatant, concentrating it and chromatographically separating the PAI-2. Separation by copper chelate chromatography is particularly advantageous and forms a further aspect of the invention. The PAI-2 (in some cases labelled with radioactive or other labels ) may be useful for locating and defining the boundaries of tumours in vitro or in vivo and treating inflammation and tumours, for example colorectal, breast, prostatic, pancreatic and renal cell carcinomas or bladder cancer.
Aspects of the invention will now be described by way of example and with reference to the accompanying figures. Figure 1 shows the PAI-2 DNA and amino acid sequences used. Figure 2 shows the construction of plasmid pDBP4; A=A lII, B=Ba_~Hl, Bg=BgrIII, E=EcoRI , E-H±ndIII and P=PstI. Only the Afill sites present.'in the PAI-2 sequence are indicated. Figure 3 shows an SDS reducing polyacrylamide gel of yeast protein extracts, stained with Coo
Lane 1 = S. cerevlslae transformed with pDBP4, Lane 2 = untransformed S. cereviεiae. Figure 4 shows the construction of plasmid pDBPSl. Figure 5 is a plasmid map of pAYE333. Figure 6 describes the two nucleotide substitutions which introduce a .Hindi11 recognition site close to the PRBl translation initiation codon. Figures 7 to 10 are respective plasmid maps of pAYE334, pAYE335, pDBP5 and pDBP6. Figure 11 describes the construction of pDBPl and pDBP2.
Figure 12 is a plasmid map of pDBP7. Only the Bglll and A fill sites present in the PAI-2 sequence are shown. EXAMPLE 1 : PRODUCTION OF PAI-2 WITH A HYBRID PROMOTER Standard recombinant DNA procedures are as described by Maniatis et al (1982) and the second edition thereof (Sambrook et al, 1989) unless otherwise stated. Construction and analysis of M13 recombinant clones was as described by Messing (1983) and Sanger et al (1977) . Strains - E.coli strains TGI [Delta (lac-pro), supE, thi- h βD5/F'traD36, p_roA+B+, lacl**, lacZ Delta M15] and DH5 [F~, φδOdlacZ Delta Ml5, Delta (lacZYA-argF)U169 , recAl , endAl hsdR17 (rj_ ~ mk +), supE44, lambda", thil, gyr , relAl] were used for propagation of M13 phage and plasmids, respectively, and strain Y1090 [Delta lacU169, proA, Delta Ion, araD139, strA, supF, (trpC22: :TnlO) (pMC9)] was used for screening and propagation of the lambda gtll library. S.cerevisiae strains DB1 (a_, leu2) , DS569 (a_, leu2, pral) and DM477 (α, leu2, trpl, ura3, pral, prbl ) were used for expression of PAI-2. A lambda gtll cDNA library constructed from mRNA isolated from phorbol-12-myristate-13-acetate stimulated cells of the human monocyte-like histiocytic lymphoma cell line U937 (obtained from
Clontech Laboratories Inc.) was used as a source of PAI-2 cDNA. The library was screened using radioactively labelle oligonucleotid-e probes corresponding to the DNA sequenc encoding the N-terminus (oligo 1) and complementary to the DN sequence encoding the C-terminal end (amino acids 400-410) o the PAI-2 protein, respectively (Ye et al . , 1987). Oligo 1 5'-ATG GAG GAT CTT TGT GTG GCA AAC ACA CTC TTT-3' Oligo 2 5'-GCC GAA AAA TAA AAT GCA CTT GGT TAT CTT ATG-3' From the^ putative positive clones was selected one clone (lambd gtll-186) which appeared to contain the entire PAI-2 codin region. This was confirmed by sequence analysis of the DN insert in this clone (Fig. 1) following transfer to M13mpl (Norrander et al , 1983) to form pDBPl (Fig. 2). To facilitate insertion into expression vectors, restrictio enzyme recognition sites were created at the 5' and 3' ends o the PAI-2 gerie. A Bglll site was created at the 5' end of th gene using he oligonucleotide primer
5'-TGCCACACAAAGATCTTCCATTGTTTCAATCT-3' to create a mutation in the third position of the second codo as shown below:- M E D L C V A 5' ...AGATTGAAACA ATG GAG GAT CTT TGT GTG GCA...3' 3' ...TCTAACTTTGT TAC CTC CTA GAA ACA CAC CGT...5' changed to:- M E D L C V A 5' ...AGATTGAAACA ATG GAA GAT CTT TGT GTG GCA...3' 3' ...TCTAACTTTGT TAC CTT CTA GAA ACA CAC CGT...5' Bglll An A fill site was created at the 3 end of the gene using th oligonucleotide primer 5'-CAGAAGCAGCACGCTTAGTCTTAAGGTGAGGAAATCTGCC-3' to create mutations in the third position of the last codo (proline) and in the first base after the stop codon as show below:-
G R F S S P STOP 5' ...GGC AGA TTT TCC TCA CCC TAA AACTAAGCGTGCTGCTTCTG..3' 3' ...CCG TCT AAA AGG AGT GGG ATT TTGATTCGCACGACGAAGAC..5' Changed to: G R F S S P STOP 5' ...GGC AGA TTT TCC TCA CCT TAA GACTAAGCGTGCTGCTTCTG..3' 3' ...CCG TCT AAA AGG AGT CCA ATT CTGATTCGCACGACGAAGAC..5' I I A fill These two oligonucle?tides were annealed to single stranded pDBPl and then used in an in vitro mutagenesis procedure (Amersham International pic) carried out according to the manu acturer's recommendations. A clone derived from this procedure and with the correct changes was designated pDBP2 (Fig. 2). Oligonucleotide linkers were then used to position restriction sites at either end of the gene which are suitable for insertion of the gene into an expression vector. The linker positioned at the 5 end of the gene was
Linker 1 5'-AGCTTGTCGACGGATCCAAAAAG ATG GAA ACAGCTGCCTAGGTTTTTC TAC CTT CTAG-5 I I I 1 I I HindiII BamHl Sg ll and the 3 linker was Linker 2 5'-TTAAGTCGACAAGCTTG CAGCTGTTCGAACCTAG-5' I I I I I I A ll ff.__.dIII BamRl These two linkers were ligated with the Bglll-Aflll PAI-2 gene fragment from pDBP2 into Hindlll + BamRl digested M13mpl9 to form pDBP3 (Fig. 2). The BamRl fragment containing the gene was isolated from this plasmid and ligated into pKV50 (GB-A-2 196 635) at Bgrlll to form pDBP4 (Fig. 2). The plasmid ρKV50 contains part of the S. cerevisiae 2 μm plasmid, the
S. cerevisiae LEU2 gene as a selectable marker, a hybrid promoter which is induced in the presence of galactose, and the S. cerevisiae PGK gene transcription terminator. The plasmid pDBP4 was introduced into S. cerevisiae NY4 (leu2~, pral") by transformation (Beggs, 1978) and transformants were selected on a minimal medium lacking leucine. Purified transformants were grown in 10ml YEPGal (1% yeast extract, 2% peptone, 2% galactose) shake flask cultures at 30°C, lysed and assayed for the presence of PAI-2 by Western blotting using polyclonal anti-PAI-2 antibody (American Diagnostica, Inc.). A protein of approximately 47kD, which reacted with the anti-PAI-2 antibody, was detected in the soluble fraction of the cells and represented over 5% of the soluble yeast protein (Figure 3). Small quantities of PAI-2 were also found in the culture supernatant but this was likely to be due to cell lysis since equivalent amounts of the intracellular protein enolase were also present. Purification of recombinant PAI-2 from yeast - Yeast cells were harvested from 6 litres of YEP Gal shake flask culture by centrifugation at 2000xg for 15 min, washed by resuspension in 1 litre of 20mM Na phosphate pH7.0 and repelleted by a second centrifugation. The cells were then resuspended to 50% (w/v) in 200mM Na phosphate pH7.0 and broken with glass beads in a Biospec bead beater (Bartlesville, Oklahoma, USA) in accordance
with the manufacturer's instructions. The beads were removed b filtration through a glass sinter funnel and washed with an equal volume of breakage buffer. The filtrate was centrifuged at 15,000xg for 20 min and then the supernatant was adjusted to ρH7.0 with IM HCl and to IM NaCl with solid NaCl. The slight turbidity generated by these additions was removed by vacuum- assisted filtration through three layers of Whatman No. 1 filte paper. The filtrate was then loaded onto a 200ml (25 x 8cm) Fast Flow Chelate column (Pharmacia), previously charged wit CuS04 and equilibrated with 20mM Na phosphate pH7.0 , IM NaCl. The column was extensively washed with the same high salt buffe followed by two column volumes of 20mM Na phosphate pH7.0. PAI 2 activity was eluted with lOmM imidazole, 20mM Na phosphate. The eluate from the chelate column was adjusted to pH7.6 with I HCl and loaded directly onto a 50ml (10 x 2.6cm) Hiload Sepharose column (Pharmacia) equilibrated with 20mM Tris-HC buffer pH7.6. After washing with the same buffer to restore th A28o baseline, the column was eluted with a 0-250mM gradient o NaCl. The anti-urokinase activity eluted in a broad peak towards th end of the gradient. The front of this peak, pool A, consiste of &95% monomeric PAI-2, as determined by densito etric scannin of a Coomassie stained Phast gel (Pharmacia) whereas pool B ha a high proportion of dimeric PAI-2. This dimer could b
dissociated by reduction. All characterisation work was done on the mono eric material from pool A. Overall, there was a recovery of 33% of the initial uPA inhibitory activity for 2.6% of the initial protein. We have found this purification procedure to be very reproducible. The final material was &95% pure as determined by densitometric scanning of gels and by HPLC analysis. The purified PAI-2 inhibited uPA with similar reaction kinetics to natural PAI-2. EXAMPLE 2 ; PRODUCTION OF PAI-2 WITH THE PRBl PROMOTER The structural gene, PRBl , for the Saccharomyces cerevisiae vacuolar endoprotease protease B has been isolated by Moehle et al . (1987) Genetics 115, 255-263, on two prbl complementing plasmids called MK4 and FP8. When the yeast Saccharomyces cerevisiae is grown on glucose in shake flask culture, very little protease B activity is detected until the cells have catabolised the glucose and are utilising the ethanol accumulated during growth (Saheki, T. and Holzer, H. (1975) Biochem. Biophys. Acta 384, 203-214; Jones et al . (1986) UCLA Symp. Mol. Cell Biol. New Ser. 33, 505-518). This is believed to be a consequence of a transcriptional control mechanism which
represses mRNA accumulation until the glucose has been exhauste and the culture enters the diauxic plateau (Moehle et al . (1987 Genetics 115, 255-263). Studies with protease B (prbl' deficient mutants implicate protease B in the protei degradation that occurs when vegetative cells are starved' o nitrogen and carbon (Wolf, D. and Ehmann, C. (1979) Eur. Biochem 98, 375-384; Zubenko, G. and Jones, E. (1981) Geneti 97, 45-64). The DNA sequence of the PRBl gene has been reported, as ha 150bp of the PRBl promoter (Moehle et al . (1987) Mol. Cell Biol. 7, ). A more extensive DNA sequence of the PR promoter is also available as an entry in the Genbank database release 60, accession number M18097, locus YSCPRBl . The whole of the PRBl promoter may be used, or a smaller portio thereof, as may readily be determined. For example, the roughl lkbp sequence extending upstream from the start codon to t SnaBl site is effective. The transcription termination signal can be the 3 ' flanki sequence of a eukaryotic gene which contains proper signals f transcription termination and polyadenylation. Suitable flanking sequences may, for example, be those of the ge naturally linked to the expression control sequenc
28 Alternatively, they may be different. Preferably, the termination signal is that of the Saccharomyces cerevisiae ADH1 gene. The 1.435kbp mndIII-._.coRI DNA fragment containing the protease B promoter was cloned into by the polylinker of the Ml3 bacteriophage mpl8 (Yanisch-Perron et al . (1985) Gene 33, 103- 119), generating plasmid pAYE333 (Figure 5). Plasmid pAYE333 was linearised by partial digestion with SnaBl and the double standard oligonucleotide linker 3 inserted by ligation. Linker 3 5'-GCGGCCGC-3' 3'-CGCCGGCG-5' Notl This generates a Notl restriction site at the 5' end of the protease B promoter. The promoter element was further modified by site directed mutagenesis (oligonucleotide directed in vitro mutagenesis system-Version 2, Amersham) according to the manufacturer's instructions. Mutagenesis with the oligonucleotide 5'-CGCCAATAAAAAAACAAGCTTAACCTAATTC-3'
introduces a Hindlll restriction site close to the AT translation initiation codon (Figure 6). Plasmid pAAH5 (Goodey et al . 1987: In Yeast Biotechnology, 401- 429, Edited by Berry, D.R. , Russell, I. and Stewart, G.G. Published by Allen and Unwin) was linearised by partiall digesting with BamRl . The 5' protruding ends were blunt-ende with T4 DNA polymerase and ligated with the double-strande oligonucleotide Linker 3. A recombinant plasmid pAYE334 (Figur 7 ) was selected in which a NotI restriction site had replace the BamRl site at the 3' end of the ADH1 terminator. The 0.8kbp Notl-Hindlll modified protease B promoter sequenc was placed upstream of the 0.45kbp Hindlll-Nσtl ADH transcription terminator on a pAT153 based plasmid (Twigg an Sherrat (1980) Nature 283, 216-218) to generate pAYE335 (Figur 8). The large 6.38kbp HindIII-_3&3_-_HI fragment from the yeast E. col shuttle vector pJDB207 (Beggs, J.D. 1981 Molecular Genetics i Yeast, Alfred Benzon Symposium 16, 383-395) was treated with th Klenow fragment of E. coli DNA polymerase to create flush end and ligated with the double stranded oligonucleotide Linker to generate plasmid pDBP5 (Figure 9). The 1.25kbp NotI Proteas
B promoter/ADffl terminator cassette from plasmid pAYE335 (Figur 8) was introduced into the unique NotI site of plasmid pDBP generating pDBP6 (Figure 10). Two double stranded oligonucleotide linkers were used to allo the insertion of a human PAI-2 cDΝA into the expression plasmi pDBP6. Linker 5 5'-AGCTTAACCTAATTCTAACAAGCAAAGATGGAA....-3 ' 3 '- ATTGGATTAAGATTGTTCGTTTCTACCTTCTAG-5' I I I I Hindlll Bgrlll Linker 6 A lll I f 5'-TTAAGTCAGACA CAGTCTGTTCGA Hindlll
Linkers 5 and 6 were ligated with the 1.34kbp Bg ll-Aflll PAI-2 cDNA from pDBP2 (Figure 11) into Hindlll linearised pDBP6 generating plasmid pDBP7 (Figure 12). Stable maintenance of pDBP7 by the S. cerevisiae strains DBl[cir°] and DS569[cir°] can not be accomplished until trans acting functions present on the native 2μ plasmid (Futcher, A.B., (1988) Yeast 4 , 27-40) have been introduced. This was achieved by co-transforming DBl[cir°] and DS569[cir°] with pSAC3 (Chinery, S.A. and Hinchliffe, E. (1989) Current Genetics 16, 21-25, EP286424) and pJDB207 and selecting for transformants on minimal media lacking leucine. Curing of DBl[cir°] pJDB207 and DS569[cir°] pJDB207 of the pJDB207 plasmid results in a cir+ derivatives of the original strains . DBl[cir+] and DS569[cir+] were re-transformed to leucine prototrophy with plasmid pDBP7 and transformants selected on minimal media lacking leucine. DS569[cir+], DS569[cir+] pDBP7; DBl[cir+] and DBl[cir+] pDBP7 were grown for 72 hours in 10ml complex media (1% (w/v) 2% (w/v) bactopeptone and 2% (w/v) sucrose) in shake flask culture at 200rpm, 30°C. Cells were harvested by centrifugation, lysed and the soluble protein extracts analysed by SDS-polyacrylamide gel electrophoresis. An extra protein band of approximately 47Kd is only observed in the soluble protein extracts of those cultures containing the PAI-2 expressing plasmid pDBP7. This extra
protein band is of the predicted molecular weight for full length human PAI-2 and was shown by Western blot analysis to react with anti-PAI-2 antibodies, thereby verifying its identity as PAI-2. Comparative Example In order to direct PAI-2 to the yeast secretory pathway, an expression plasmid was made in which DNA encoding an N-terminal signal sequence was placed upstream of DNA encoding PAI-2. Four oligonucleotides were synthesised and annealed to form Linker 7. Linker 7 oligo 3 M K W V' S F I S L L F L F GATCCAAAAAA ATG AAG TGC GTA AGC TTT ATT TCC CTT CTT TTT CTC TTT GTTTTTT TAC TTC ACC CAT TCG AAA TAA AGG GAA GAA AAA GAG AAA 1 oligo 5
oligo 4 S S A Y S R S L D K R M E AGC TCG GCT TAT TCC AGG AGC TTC GAT AAA AGA ATG GAA TCG AGC CGA ATA AGG TCC TCG AAG CTA TTT TCT TAC CTT CTA G oligo 6 This encodes the HSA/α-factor fusion leader described in WO90/01063 followed by the start of the PAI-2 coding sequence up to the Bglll site. This linker was ligated with the BglII--3a_~HI fragment of pDBP3, encoding the remainder of PAI-2, into the Bglll site of pKV50 to form pDBPSl (Figure 4). This plasmid was introduced into S. cerevisiae NY4 by transformation, selecting for complementation of the leu2 mutation. Transformants were grown for 72 hours in YEPGal and the culture supernatant was analysed by Western blotting using anti-PAI-2 antibody. This revealed the presence of immunoreactive material of high molecular weight in the form of a smear characteristic of a glycosylated protein. Some material of a lower molecular weight was also present. This result indicated that, by provision of an N-terminal signal sequence, PAI-2 is directed to the secretory pathway where it was glycosylated in the manner associated with yeast.
REFERENCES Astedt et al (1985) Thromb. Hae ost. _53_, 122-125. Astedt et al fl987) Fibrinolysis 1 , 203-208. Baldari et al (1987) EMBO J. 6 , 229-234. Barr et al (1988) J. Biol. Chem. ^ 3, . Bunn et al (1989) Fibrinolysis 3 , , Supp 1., 22. Carrell and Travis (1985) Trends Biochem. Sci. 10_, 20-24. Genton et al (1987) -J. Cell. Biol. JL0_4_, 705-712. Golder and Stephens (1983) Eur. J. Biochem. 136, 517-522. Holmberg et al (197'8) Biochem. Biophys. Acta 544, 128-137 Kawano et al (1968) Nature 217, 253-254. Kruithof et al (1986) J. Biol. Chem. 261, . Lecander and Astedt (1986) Br. J. Haematol. ?2, 221-228. Lingappa et al (1979) Nature 281, 117-121. Livi et al (1988) Yeast _4, S446 Maniatis et al (19β2) Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Meek et al (1982) J. Biol. Chem. 251_, . Messing (1983) Methods Enzymol. 101, 20-78. Norrander et al (1983) Gene 2 , 101-106. Sambrook et al (1984 Molecular Cloning : A laboratory manual (2nd Edn. ) Cold Spring Harbor Laboratory, New York. Sanger et al (1977) Proc. Natl. Acad. Sci. USA 7_4, . Tabe et al (1984) J. Mol. Biol. 1 Q_, 645-666. Vassalli et al (1984) J. Exp. Med. 15_9. .
Vlodavsky et al (1987) Proc. Natl. Acad. Sci. USA QA ,
Wohlwend et al (1987) J. Exp. Med. 165, 320-339. Ye et al , (1987) J. Biol. Chem. 2 lt . Ye et al (1988) J. Biol. Chem. 2j53_, .
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