WO1991002057A1 - Production de polypeptides a l'interieur de cellules fongiques - Google Patents

Production de polypeptides a l'interieur de cellules fongiques Download PDF

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Publication number
WO1991002057A1
WO1991002057A1 PCT/GB1990/001229 GB9001229W WO9102057A1 WO 1991002057 A1 WO1991002057 A1 WO 1991002057A1 GB 9001229 W GB9001229 W GB 9001229W WO 9102057 A1 WO9102057 A1 WO 9102057A1
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Prior art keywords
pai
dna
sequence
yeast
plasmid
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PCT/GB1990/001229
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English (en)
Inventor
David James Ballance
John Steven
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Delta Biotechnology Limited
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Priority claimed from GB898918191A external-priority patent/GB8918191D0/en
Priority claimed from GB898927480A external-priority patent/GB8927480D0/en
Application filed by Delta Biotechnology Limited filed Critical Delta Biotechnology Limited
Publication of WO1991002057A1 publication Critical patent/WO1991002057A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8132—Plasminogen activator inhibitors

Definitions

  • the present invention relates to the production of polypeptides, specifically plasudinogen activator inhibitor 2 (PAI-2), in fungi.
  • PAI-2 is a naturally occurring inhibitor of serine proteases and, more specifically, of plasminogen activators of the urokinase-type (u-PA) and of the tissue-type (t-PA) (see
  • SERPIN superfamily (Carrell and Travis, 1985), which includes ⁇ l-antitrypsin and plasminogen activator inhibitor 1.
  • protease inhibitors act by mimicking the protease's natural substrate and forming a 1:1 covalent inactive complex which is subsequently cleared from the body.
  • the primary determinant of SERPIN specificity is an amino acid at the reactive centre which is analogous to the amino acid immediately amino terminal to the peptide bond cleaved in the natural substrate. In the case of PAI-2 this amino acid is arginine, which is also present at the position at which u-PA and t-PA cleave their natural substrate plasminogen. It is clear , however , that other amino acids in the inhibitor are also important for determining specificity.
  • PAI-2 has been isolated from placenta (Kawano et al, 1968; Holmburg et al, 1978; Astedt et al, 1985), human monocytes (Golder and Stephens, 1983) and the human monocyte-like histiocytic lymphoma cell line U937 (Vassalli et al, 1984; Kruithof et al , 1986).
  • minactivin a plasminoge activator inhibitor isolated from human monocyte cultures. This protein is PAI-2 and therefore identical to the molecule previously isolated from placenta (Kawano et al , 1968; Holmberg et al , 1978) except that, as the molecular mass was estimated to be 60-70kD, it was probably the glycosylated form of the protein.
  • Antalis et al disclose the production of minactivin by recombinant DNA technology.
  • a minactivin (PAI-2) cDNA coding sequence was introduced into an Escherichia coli expression vector which directed the expression of active minactivin in - E. coll .
  • Webb et al used recombinant" DNA technology to produce PAI-2 though they advocated removing 22 amino acids from the N-terminus of the protein to ensure maximal biological activity. These amino acids are, however, present in the active natural molecule and do not constitute a cleavable signal peptide (Ye et al, 1988). The effect of removal of these 22 amino acids from the N-terminus on the activity of PAI-2 has not been examined.
  • PAI-2 is secreted in the normal cells which produce it and therefore, although it lacks a cleavable leader sequence, it must have a sequence which directs secretion. This sequence would have been thought to be effective in fungal cells, at least to the extent of lodging the PAI-2 in the membrane fraction.
  • interleukin-1 ⁇ which similarly has an internal secretion signal, is at least partially secreted in yeast.
  • PAI-2 is expressed as an intracellular protein.
  • it is obtainable from the soluble fraction when the cells are lysed, unlike when it is expressed in E. coli ( EP-A-238 275 ) .
  • 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 uyveromyces , Candida, Torul opsis , Hansen ul a ,
  • Preferred genera are those selected from the group consisting of Plchla , Saccharomyces, Kluyveromyces, Yarrowia 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.
  • Saccharomyces are Saccharomyces cerevlslae, Saccharomyces itallcus and Saccharomyces rouxii .
  • Kluyveromyces are Kluyveromyces fragilis and Kluyveromyces lactis .
  • Hansenula are Hansenula polymorpha , Hansenula anomala and Hansenula capsulata .
  • Yarrowia llpolytica is an example of a suitable Yarrowia species.
  • Saccharomyces cerevlslae and Schlzosaccharomyces pombe are particularly preferred.
  • Filamentous fungi such as Asperglllus nlger are also suitable.
  • Fungal cells can be transformed by:
  • the regenerated cells are then screened for the incorporation of the transforming DNA
  • 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 5 M -1 s -1 and generally up to 2 ⁇ 10 7 M -1 s -1 , for example about 2 ⁇ 10 6 M -1 s -1 , as measured in the method of Thorsen et al (1988) Eur.
  • 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, GAL1 or GAL10 genes, CYC1 , acid phosphatase ( PHO5), 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, ⁇ -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 (
  • PRBl promoter is described in our co-pending application GB 8927480.7 (our ref. DELF/P7375GB) which is incorporated herein by reference.
  • Example 2 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.
  • the termination signal is that of the S. cerevlslae PGK or ADH1 genes.
  • 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.
  • the DNA sequence coding for PAI-2 and the fungal 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 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 by methods known in the art, for example by linking a eukaryotic expression control sequence, a DNA sequence coding for PAI-2 and a DNA sequence containing eukaryotic transcription termination signals in such a way that proper expression of the coding sequence is effected in a yeast host.
  • PAI-2 ds cDNA complementary double-stranded PAI-2 DNA
  • PAI-2 ds cDNA synthetic gene coding for the amino acid sequence of PAI-2 is 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.
  • genomic PAI-2 DNA is obtained from a placenta gene bank that contains the PAI-2 gene by cloning the placenta DNA fragments in a microorganism and identifying clones that contain PAI-2 DNA, for example by colony hybridisation using a radioactively labelled PAI-2 DNA-specific oligonucleotide that contains at least 15, and preferably from 15 to 30, nucleotides.
  • the resulting DNA fragments as a rule contain in addition to the PAI-2 gene other undesired DNA constituents that can be removed by treatment with suitable exo- or endo-nucleases.
  • Double-stranded PAI-2 cDNA can be produced by isolating mRNA from suitable placenta or monocyte cell lines, or U937 which are preferably induced to synthesise PAI-2, enriching the PAI-2 mRNA in the resulting mRNA mixture in a manner known per se, using this mRNA as a template for the preparation of single-stranded cDNA with RNA-dependent DNA polymerase, synthesising from this, with the aid of a DNA-dependent DNA polymerase, ds cDNA, and cloning the latter into a suitable vector.
  • Clones that contain PAI-2 cDNA are identified, for example in the manner described above, by colony hybridisation using a radioactively labelled, PAI-2 DNA-specific oligonucleotide.
  • the PAI-2 coding sequence can also be produced by chemical synthesis.
  • the process is characterised in that segments of the coding and of the complementary strand of said gene are chemically synthesised and resulting segments are linked enzymatically into a linear coding sequence for PAI-2.
  • 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.
  • 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 which apart from the expression control sequence, the above DNA segment and the sequence containing transcription termination signals contain additional DNA sequences which are inessential or less important for the function of the promoter, i.e. for the expression of the PAI-2 gene, but which perform important functions, for example in the propagation of the cells transformed with said hybrid plasmids.
  • the additional DNA sequences may be derived from prokaryotic and/or eukaryotic cells and may include chromosomal and/or extra-chromosomal DNA sequences.
  • the additional DNA sequences may stem from (or consist of) plasmid DNA, such as bacterial or eukaryotic plasmid DNA, viral DNA and/or chromosomal DNA, such as bacterial, yeast or higher eukaryotic chromosomal DNA.
  • Preferred hybrid plasmids contain additional DNA sequences derived from bacterial plasmids, especially Escherichia coli plasmid pBR322 or related plasmids, bacteriophage, yeast 2 ⁇ plasmid, and/or yeast chromosomal DNA.
  • 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)
  • a yeast replication origin e.g. an autonomously replicating segment (ars)
  • ars autonomously replicating segment
  • Hybrid plasmids containing sequences homologous to yeast 2 ⁇ plasmid DNA can be used as well.
  • These hybrid plasmids may be integrated by recombination into 2 ⁇ 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.
  • 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, in the case of auxotrophic yeast mutants, genes which complement host lesions.
  • Corresponding genes confer, for example, resistance to the antibiotic cycloheximide or provide for prototrophy in an auxotrophic yeast mutant, for example the URA1 , URA3 , ARG4 , LEU2 , HIS 4 , HIS3, TRP5 or TRP1 gene.
  • the additional DNA sequences which are present in the hybrid plasmids according to the invention also include a replication origin and a selective genetic marker for a bacterial host, especially Escherlchla coli .
  • a replication origin for a bacterial host
  • Escherlchla coli there are useful features which are associated with the presence of an E. coli replication origin and an E. coli marker in a yeast hybrid plasmid.
  • large amounts of hybrid plasmid DNA can be obtained by growth and amplification in E. coli and, secondly, the construction of hybrid plasmids is conveniently done in E. coli making use of the whole repertoire of cloning technology based on E. coli .
  • E. coli plasmids, such as pBR322 and the like contain both E. coli replication origin and E. coli genetic markers conferring resistance to antibiotics, for example tetracycline and ampicillin, and are advantageously 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 bacterial host (see above) are hereinafter referred to as "vector DNA" which, together with the above DNA construct, containing inter alia the expression control sequence and the PAI-2 gene, is 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 by methods known in the art.
  • the process for the preparation of the hybrid vectors comprises introducing one or multiple DNA constructs containing 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 fungal transcription termination signals, as such or introducing the components of said DNA constructs successively in the predetermined order into a vector DNA.
  • hybrid plasmids The construction of the hybrid plasmids according to the invention is performed applying conventional ligation techniques.
  • the components of the plasmids are linked through common restriction sites and/or by means of synthetic linker molecules and/or by blunt end ligation.
  • Another aspect of the invention involves fungal host organisms transformed with a hybrid vector having one or multiple DNA inserts each comprising a fungal expression control sequence, a DNA segment consisting of a second DNA sequence coding for PAI-2 which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing fungal transcription termination signals, and mutants thereof.
  • the host is transformed with a hybrid plasmid having one or multiple DNA inserts each comprising the elements set out above.
  • the transformation of the host cells is accomplished by methods known in the art.
  • the transformation of yeast with the hybrid vectors 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:
  • glucosidases such as snail gut juices (e.g. Glusulase R or Helicase R ) or enzyme mixtures obtained from microorganisms (e.g. Zymolyase R ) in osmotically stabilized solutions (e.g. 1M sorbitol).
  • snail gut juices e.g. Glusulase R or Helicase R
  • enzyme mixtures obtained from microorganisms e.g. Zymolyase R
  • osmotically stabilized solutions e.g. 1M sorbitol
  • 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 biosynthetic pathways are generally used as selective markers (-supra ) , the regeneration is preferably performed in yeast minimal medium agar. If very high efficiencies of regeneration are required the following two step procedure is advantageous:
  • tjie DNA vector is a linear DNA vector used for transforming eukaryotic host cells
  • transformation is preferably done in the presence of a second vector containing a selective marker for 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 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 particularly preferred; 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.
  • FIG 1 shows the PAI-2 DNA and amino acid sequences used.
  • Figure 4 shows the construction of plasmid pDBPS1.
  • Figure 5 is a plasmid map of pAYE333.
  • Figure 6 describes the two nucleotide substitutions which introduce a HindIII recognition site close to the PRB1 translation initiation codon.
  • Figures 7 to 10 are respective plasmid maps of pAYE334, pAYE335, pDBP5 and pDBP6.
  • Figure 11 describes the construction of pDBP1 and pDBP2.
  • Figure 12 is a plasmid map of pDBP7. Only the BglII and AflII sites present in the PAI-2 sequence are shown.
  • Strains - E.coli strains TGI [Delta (lac-pro), supE, thi, hsdD5/F'traD36, proA + B + , lacI q , lacZ Delta M15] and DH5 ⁇ [F-, ⁇ 80dlacZ Delta Ml5, Delta (lacZYA-argF)U169, recA1, endA1 hsdR17 (r k - m k + ), supE44, lambda-, thil, gyrA, relA1] were used for propagation of M13 phage and plasmids, respectively, and strain Y1090 [Delta lacU169, proA, Delta Ion, araD139, strA, supF, (trpC22::Tn10) (pMC9)] was used for screening and propagation of the lambda
  • S.cerevisiae strains DB1 (a, leu2), DS569 (a, leu2, pra1) and DM477 ( ⁇ , leu2, trp1, ura3, pra1, prb1) were used for expression of PAI-2.
  • the library was screened using radioactively labelled oligonucleotide probes corresponding to the DNA sequence encoding the N-terminus (oligo 1) and complementary to the DNA sequence encoding the C-terminal end (amino acids 400-410) of the PAI-2 protein, respectively (Ye et al . , 1987).
  • restriction enzyme recognition sites were created at the 5' and 3' ends of the PAI-2 gerie.
  • a BglII site was created at the 5' end of the gene using the oligonucleotide primer 5 ' -TGCCACACAAAGATCTTCCATTGTTTCAATCT-3 '
  • 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
  • the two linkers were ligated with the BglII-AflII PAI-2 gene fragment from pDBP2 into HindIII + BamHI digested M13mp19 to form pDBP3 (Fig. 2).
  • the BamHl fragment containing the gene was isolated from this plasmid and ligated into pKV50 (GB-A-2 196 635) at BglII to form pDBP4 (Fig. 2).
  • the plasmid ⁇ KV50 contains part of the S. cerevisiae 2 ⁇ m plasmid, the S. cerevisiae LEU2 gene as a selectable marker, a hybrid promoter which is injduced 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.
  • the filtrate was centrifuged at 15,000xg for 20 min and then the supernatant was adjusted to ⁇ H7.0 with IM HCl and to IM NaCl with solid NaCl. The slight turbidity generated by these additions was removed by vacuumassisted filtration through three layers of Whatman No. 1 filte paper. The filtrate was then loaded onto a 200ml (25 ⁇ 8cm) Fast Flow Chelate column (Pharmacia), previously charged with CuSO 4 and equilibrated with 20mM Na phosphate pH7.0, 1M NaCl. The column was extensively washed with the same high salt buffer 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 1M HCl and loaded directly onto a 50ml (10 ⁇ 2.6cm) Hiload Q Sepharose column (Pharmacia) equilibrated with 20mM Tris-HCl buffer pH7.6. After washing with the same buffer to restore the A 280 baseline, the column was eluted with a 0-250mM gradient of NaCl.
  • pool A The anti-urokinase activity eluted in a broad peak towards the end of the gradient.
  • pool A consisted of >95% monomeric PAI-2, as determined by densitometric scanning of a Coomassie stained Phast gel (Pharmacia) whereas pool B had a high proportion of dimeric PAI-2. This dimer could be dissociated by reduction. All characterisation work was done on the monomeric 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.
  • the structural gene, PRB1 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.
  • MK4 and FP8 two prbl complementing plasmids.
  • the DNA sequence of the PRB1 gene has been reported, as has 150bp of the PRB1 promoter (Moehle et al . (1987) Mol. Cell. Biol. 7, 4390-4399). A more extensive DNA sequence of the PRB1 promoter is also available as an entry in the Genbank database, release 60, accession number M18097, locus YSCPRB1.
  • the whole of the PRB1 promoter may be used, or a smaller portion thereof, as may readily be determined.
  • the roughly lkbp sequence extending upstream from the start codon to the SnaB1 site is effective.
  • the transcription termination signal can be the 3' flanking sequence of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence. Alternatively, they may be different. Preferably, the termination signal is that of the Saccharomyces cerevisiae ADH1 gene.
  • Plasmid pAYE333 was linearised by partial digestion with SnaB1 and the double standard oligonucleotide linker 3 inserted by ligation.
  • 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
  • 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 partially digesting with BamHl. The 5' protruding ends were blunt-ended with T4 DNA polymerase and ligated with the double-stranded oligonucleotide Linker 3. A recombinant plasmid pAYE334 ( Figure 7) was selected in which a NotI restriction site had replaced the BamHl site at the 3' end of the ADH1 terminator.
  • Two double stranded oligonucleotide linkers were used to allow the insertion of a human PAI-2 cD ⁇ A into the expression plasmid pDBP6.
  • Linkers 5 and 6 were ligated with the 1.34kbp BglII-AflII PAI-2 cDNA from pDBP2 ( Figure 11) into HindIII linearised pDBP6 generating plasmid pDBP7 ( Figure 12).
  • DB1[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; DB1[cir + ] and DB1[cir + ] pDBP7 were grown for 72 hours in 10ml complex media (1% (w/v) yeast extract; 2% (w/v) bactopeptone and 2% (w/v) sucrose) in shake flask culture at 200rpm, 30°C.
  • an expression plasmid was made in which DNA encoding an N-terminal signal sequence was placed upstream of DNA encoding PAI-2.
  • Linker 7 Four oligonucleotides were synthesised and annealed to form Linker 7.
  • This linker was ligated with the BglI -BamHl fragment of pDBP3, encoding the remainder of PAI-2, into the BglII site of pKV50 to form pDBPS1 ( 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.

Abstract

Le PAI-II qui est le principal inhibiteur de l'activateur de plasminogène de type uroquinase, est produit avec un bon rendement à l'intérieur de cellules fongiques, en particulier à l'intérieur de levures telles que la Saccharomyses cerevisiae, par des techniques d'ADNr. Le PAI-II est de préférence produit par voie intracellulaire sous la forme d'un produit soluble correctement enveloppé, même si à l'état naturel il est sécrété.
PCT/GB1990/001229 1989-08-09 1990-08-07 Production de polypeptides a l'interieur de cellules fongiques WO1991002057A1 (fr)

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GB8918191.1 1989-08-09
GB898918191A GB8918191D0 (en) 1989-08-09 1989-08-09 Polypeptide production in fungi
GB8927480.7 1989-12-05
GB898927480A GB8927480D0 (en) 1989-12-05 1989-12-05 Mutant fungal strain detection and new promoter

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Cited By (3)

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WO1992010206A1 (fr) * 1990-12-08 1992-06-25 Delta Biotechnology Limited Utilisation de l'inhibiteur de type 2 des activateurs du plasminogene pour le traitement du cancer
US7807621B2 (en) 1997-07-25 2010-10-05 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions
US9873746B2 (en) 2003-10-22 2018-01-23 Keck Graduate Institute Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy

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WO1988001273A1 (fr) * 1986-08-19 1988-02-25 Scripps Clinic And Research Foundation Essai de depistage d'inhibiteurs d'activateurs de plasminogene de type tissulaire et de type urokinase, genes de codage de l'inhibiteur

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WO1992010206A1 (fr) * 1990-12-08 1992-06-25 Delta Biotechnology Limited Utilisation de l'inhibiteur de type 2 des activateurs du plasminogene pour le traitement du cancer
US7807621B2 (en) 1997-07-25 2010-10-05 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions
US8110543B2 (en) 1997-07-25 2012-02-07 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions
US9873746B2 (en) 2003-10-22 2018-01-23 Keck Graduate Institute Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy
US10155819B2 (en) 2003-10-22 2018-12-18 Alderbio Holdings Llc Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy
US10259883B2 (en) 2003-10-22 2019-04-16 Keck Graduate Institute Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy
US11447560B2 (en) 2003-10-22 2022-09-20 Keck Graduate Institute Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy

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