CA2043953C - Method for the isolation and expression of a gene which codes for streptokinase, nucleotide sequence obtained, recombinant dna and transformed microorganisms - Google Patents

Abstract

The present invention relates to the field of biotechnology and genetic engineering and in particular a novel nucleotide sequence which codes for a streptokinase, as well as the recombinant DNA obtained therefrom which is used for the transformation of various host organisms. The technical object which is pursued with the proposed solution is to produce the protein in commercially useful quantities, by the recombinant method, with the possibility of alternatively using different hosts to obtain a high-quality streptokinase capable of being used as a thrombolytic agent in the treatment of different disorders. The present invention is based on the isolation of a new gene which codes for streptokinase from Streptococcus equisimilis of type C (strain ATCC-9542) and the cloning and expression thereof in prokaryotic (E. coli) and eukaryotic (Pichia pastoris) hosts, for which is includes the vehicles of expression which contain the genetic sequences of said gene, as well as the microorganisms transformed with these vectors capable of producing streptokinase. The protein obtained thereby can be used in clinical medicine as a therapeutic agent, in the treatment of disorders such as thromboembolic obstructions including coronary thrombosis.

Description

~.-:. 2043953 Title: Method for the isolation and expression of a gene which codes for streptokinase, nucleotide sequence obtained, recombinant DNA and transformed microorganisms.
The present invention relates to the field of biotechnology and genetic engineering techniques and in particular a method for the isolation and cloning of a novel nucleotide sequence which codes for a streptokinase, as well as the recombinant DNA obtained therefrom which is used for the transformation of different host organisms.
Streptokinases are plasminogen activators from prokaryotic. cells, which are usually secreted into the culture medium by a large number of haemolytic Strepto oc ~ of different serotypes. Being proteins of bacterial origin, some antigenic responses to them have been detected (Dykewies, M.S. et al., 1985 and McGroth, K.G. et al., 1985).
Their molecular weight is approximately 97,000 dalton.
The function in the pathogenicity of tr p~~oCGi is not known exactly, although potentially it must contribute to elimination or avoidance of the formation of fibrin barriers around the infection.
During interaction of streptokinase with the plasminogen which is in human plasma, it was found that the protein is capable of converting the latter to plasmin, which displays proteolytic activity and can degrade fibrin clots into soluble products.
Streptokinase, urokinase and tissue-type plasminogen activator are at present used~as thrombolytic agents in the treatment of disorders which collectively represent one of the greatest causes of death in the world, such as myocardial infarct, pulmonary, arterial or venous thromboembolism, surgical complications and other cases of thrombosis.
There are physicochemical and immunological differences anal differences in rESpect. of .~~ubstrate specificity which beat witness to molecular heterogeneity of streptokinases of A

. . . _ . .. .. . .. .. ~04~~~~ ..

different origins, although they are all closely related in respect of function.
Combined with the low yields obtained in production of the protein and the pathogenicity of its natural host, the ,tre~tQcoccus strains used for the industrial production of streptokinase secrete other products into the culture medium, such as deoxyribonucleases, streptolysin br hyaluronidase and proteases, which makes the process of purifying the desired protein difficult. On the other hand, it has not yet been possible to obtain genetically improved strains from these hosts due to the lack of a developed methodology for the gene transfer.
It is due to these drawbacks that the cloning of different isolated genes which code for these proteins has been attempted, using prokaryotic and eukaryotic hosts.
The German Democratic Republic patent no. 249493, IPC: C12 N 15/00, describes the cloning and expression of a gene which codes for streptokinase from strain H46A of Streptococcus ~cuisimilis, belonging to Streptococcus type C, using ~. coli bacteria as the host, wherein levels of excretion into the medium from 0.1 to 1.8 mg/1 are obtained, depending on the age of the culture.
The coding sequence for this gene, which was called SKC, was subsequently determined, including its signal peptide, as well as the identification of the primary structure of adjacent regions involved in the control of transcription and translation of said gene (Malke, H. et al., 1985).
Other nucleotide sequences of genes which code for streptokinases from Streptococcus types G and A have been characterised (Water, F. et al., 1989), In particular, the gene which determines streptokinase type A (gene SKA) from strain 49 type M of Streptococcus pio,genes was cloned and expressed in strain JM109 of ~, coli and in Streptococcus sanauis Challis 57E (Huan , T.T, et al., 1989). In both cases,",;
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protein levels of 0.64 mg/1 and 40 ~.g/1 respectively were produced. In the case of ~. coii, 94$ of the'protein recovered ,,, . .

.204393 _ was in the periplasmic space and 6~ in the cytosol, whereas in pan uis all the enzyme was found extracellularly. Moreover, many clones producing streptokinase in ~. coli were very unstable, as in some cases the SKA gene was deleted or the host cells died owing to some lethal activity of the gene product in question.
In the case of ~. s~"uis, the protein molecule obtained was approximately 3,000 dalton less than native streptokinase and the absence of 32 amino acids from the C-terminal end was detected; however, biological activity was not affected (Huang, T.T. et al., 1989).
Subsequently, conclusive results were obtained with respect to the difficulty of cloning and expressing the isolated SKC gene in ~. coli, it being found that the gene product interferes with the normal physiology of the host, which is shown by the mucosity of the cells which carry this gene, by the incomplete export of streptokinase into the periplasmic space, by the structural instability of the .plasmids which carry the SKC gene and which are designated for the expression of high levels of the protein, as well as by the drawbacks encountered in cloning streptokinase genes from additional serotypes of Streptococcus in plasmids of ~. _roli and unsuccessful attempts to express heterologous genes under the control of expression-excretion signals of the SKC gene itself (Muller, J. et al., 1989).
More recently the company Phillips Petroleum (patent DD257646 IPC: C 12 N 15/00, and Ha , ggenson, M.J. et al., 1989) has obtained the expression of~~streptokinase in the methylotrophic yeast Pichia pastoris, under the control of gene regulatory sequences of alcohol oxidase, wherein yields of the desired product using continuous fermentation of the order of 77-250 mg/1 culture medium were obtained, with an . intermediary cell density of 46 g/1. This system uses the ~KG~ge~ne,- contained i~n the vpla'si~~ti ~~MFS "Which is'''lice~rised' to this company by Dr. J.J Ferretti of Oklahoma University, USA, in the expression vectors.

The controllability of the system makes it attractive, taking into account that it can easily be repressed by using glucose or glycerol and induced with methanol; nevertheless, its application is limited to this host.
It is the object of the present invention to obtain high levels of streptokinase yield in different host systems, by the use of expression vectors which carry a novel nucleotide sequence which represents a genetic variant not described before and which codes for a bioactive streptokinase, which contains the active portion corresponding to streptokinase from ~t~rP.~gt-ococcus ~gllssims>>s of type C, strain ATCC-9592.
The novel isolated gene is called SKC-2, and codes for a protein of the same molecular weight as the one encoded by SKC; it has the fundamental characteristic of stability in vectors of E_. soli and yeasts, adverse effects on cells growth or viability not being found in a single case, which makes it possible to obtain yields greater than those reported up to now in both hosts, in respect of the product obtained, which indicates a streptokinase different to those known up to nova, with the desired biological activity.
A

s , 4a Drawings: The invention will now be described with reference to the following drawings:
Figure 1. Plasmid pEKG-3, which carries the sequence of the SKC-2 gene under the tryptophan promoter of E. coli and displays at the 3~ end of the gene the signal for termination of bacteriophage T4 to give greater stability to expression.
Figure 2. Comparison between the amino acid sequences derived from the base sequences of the genes SKC-2, SKC, SKG and SKA.
Figure 3. Plasmids pPESKC-4 and pPISKC-6, for extracellular and intracellular expression respectively in P.
pastoris.
Figure 4. Relative positions of DNA fragments SK1, SK2 and SK3.
JJ:lcd The present invention relates to a method for the isolation and expression of a novel nucleotide sequence corresponding to the SKC-2 gene, the product of which is a protein of approximately 97,000 dalton. Said protein belongs to the streptokinases, which are distinguished by their fibrinolytic activity.
The present invention also relates to this gene, l.he sequence of which corresponds to seq. id. No. 1 in the Sequence T~isting.
It was obtained from the genome of the ~ re~tocsccus eauis~.rnilis type C strain (ATCC-9592) by gene amplificat.ian using the polymerase chain reaction (PCR) from three synthetic oligonucleot.ides called SK1, SK2 and SK3 having the seyuer~c S..K1 5' . ... TGG1AATTCAT.G11AA1~~'~'1~~',TTj~T,CT . . . 3_' sect . id . Ne .

SK2 5'...'TGGATCCTTATTTGTCGTTAGGGTTATC...3' seq. id. Nm. i SK3 5'...GGnATTCATGATTGCTGGACCTGAGTGGCTG...3' seq. id. No. 4 JJ:lcd a and constitute a novel aspect for this method. The relative positions of the DNA fragments SK1, SK2 and SK3 are depicted in Fig. 4. These primers further carry restriction sites which are not found in the gene and which allow direct cloning 5 in an expression vector. On the other hand, the SK3 which hybridises at the 5~ end carries an ATG which acts as a site for initiating translation and removes the signal peptide of SKC-2. The three oligonucleotides were synthesised from the SKC sequences published by Malke et al., 1985; and with them were marked the boundaries of the exact fragment of the gene which codes for the mature protein or the complete gene including the signal peptide.
Another novel aspect of this method is the possibility of expressing the isolated gene in both bacteria and yeasts, high levels of expression being achieved in both cases.
The present invention also relates to recombinant DNA which includes the SKC-2 gene, such as vectors for the expression of this gene, in bacteria pEKG3 (Fig. 1), and in yeasts pPESKC-4 and pPISKC-6 (Fig. 3). In particular for expression in ~. coli, the SKC-2 gene with or without its signal peptide is cloned under the tryptophan promoter and with the transcription termination signal of phage T4. For yeasts there was used an expression vector in which the SKC-2 gene is located behind the signal peptide of sucrose invertase (SUC2) controlled by the alcohol oxidase gene promoter (AOX1) of ich' a pastoris and which carries the termination signal of the glyceraldehyde-3-phosphate dehydrogenase (GAPt) gene of Saccharomyces cerevisiae for the extracellular expression variant, this vector being called pPESKC-4. For the intracellular expression of SKC-2, the vector pPISKC-6 is used which does not contain the signal peptide of SUC-2 behind the JJ:lcd a 2043953 _ 5a AOX1 promoter, and this is obtained by inserting the SKC-2 gene in the expression vector pNAO (kindly provided by Muzio, OIGB, Havana, Cuba) (Fig. 3). The HISS gene of S. cerevisiae is used as a selection marker in both vectors, and the expression cassette referred to above JJ:lcd Bt is flanked by 5' and 3' sequences of the AOX gene of ~ hia gast-oris for integration.
The present invention also relates to the microorganisms resulting from transformation of ~.. ,~oli strain 4V3110 with vector pEKG3, and to mutant MP-36 of P.ichia ~asroris with expression vectors pPESKC-4 and pPISKC-6, which axe characterised as expressing high levels of streptokinase, and having good viability (the product has no adverse effects on cell growth) and high stability of the strains transformed.
The transformed ~.. coli clone was called HSK-M and exhibits levels of expression of the product of the SKC-2 gene greater than 350 mg/1 culture medium..
The transformed Pichia has oris strains MSK-M9 and MSK-M6 produce streptokinase levels intracellularly and extracellularly respectively which vary between 1.0 and 1.2 g/1 culture medium.
The method described in the present invention, given the levels of expression never before reported for this product, makes it possible to reach optimum purity thereof for its administration to human beings and animals, without the need to develop a complex and costly process for purification.
Examples The following examples are intended to illustrate but not limit the invention. ~. ~oli and ~i~hia ~~storis are used as host systems in these examples; nevertheless, other eukaryot.i.c and prokaryotic cells can be~used for the method described in the present invention.
Fxamg,~e 1 For the isolation of genomic DNA of ~trPp~~occus ~,L~imilis of type C, strain ATCC-9542 was used as a source thereof for cloning the SK gene. The cells of ~~rPnrococcus aauis.;mills were =grown -in Br..aHin Heart .Infusion .Medi..um .(GLBCO) at 240 r.p.m. for 12 hours at 37°C in 5-ml pre-cultures acting as an inoculum which was grown for 12 hours in a 300-ml *Trade-mark ,~ 7 203953 Erlenmeyer flask. The cells were collected by centrifugation at 3,000 r.p.m. and resuspended in 8 ml lyse (4.5 g glucose, 1.$6 g EDTn, 1.51 g tris-HC1, in 500 ml sterile water, pH=8), 80 ~1 of lysozyme were added at a concentration of 10 mg/ml, and the suspension was incubated for 30 minutes at 37°C. Next, to obtain efficient cell rupture, 500 ~1 pronase (Boehringer), 1 ml SDS at 10~ and 200 ail EDTA, 0,5 M, pH=8, were added and the suspension was incubated for 2 hours at 50°C with smooth agitation. Successive treatments with phenol, phenol-chloroform and chloroform were carried out, and the genomic DNA was precipitated with absolute ethanol and NHqAc" 7.5 M.
The yield obtained was 100 Hg per 300-ml Erlenmeyer culture flask. The presence of the gene which codes for streptokinase was verified by the Southern blot technique (Maniatis et al., 1982).
For subcloning in bacteria, 1 ~g genomic DNA of the ~trPntococcus Pau~s~m>>;s type C strain (ATCC-9592) was taken and the gene which codes for SKC-2 was amplified by a PCR
(Randall et al., 1988) using the oligonucleotides SK1 and SK2 for cloning the gene with its signal peptide and SK2-SK3 for cloning without it.
In each reaction 100 pmol of each oligonucleotide, 2 units of Taq polymerase (Perkin Elmer, USA) and 200 )unol of each dNTP were used and the reactions were performed in 10 mM MgCl~, 100 mM dTT, 10 mM NaCl and 100 ~g/ml gelatine.
Thirty amplification cyc~.es were performed, wherein in each one the reaction was incubated at 95°C for 1 minute for denaturisation, at 52°C for 95 seconds for hybridisation of the oligonucleotides and at 70°C for 80 seconds for extension.
An efficiency greater than 5~ of amplification was obtained.
For cloning in bacteria (F. co i), a new genetic construct was used, wherein the tryptophan promoter of E_. coli and the termination signal of bacteriophage T4 are used.
A

The fragments amplified in the PCR were digested with BamHI
and ligated with the vector ptrip-N_coI-S1-CHI
(Estrada et al., 1988). This construct was transformed into a preparation of competent cells prepared according to Dagert et al. (1979) and Hanahan et al. (1983), of ~. coli strain HB101 ((rg-mg-), sunEq4, ara-19, aalK-2, cYl, proA2, rnsL20, (StrR) , ~cyl-5, ~1-5, ~1-1, recAl3 } , which had a frequency greater than 10~ transformants per g DNA.
The colonies obtained were applied to plates of LB medium (10 g/1 trypton, 5 g/1 yeast extract, 10 g/1 sodium chloride) and 50 ~tg/ml ampicillin, and hybridised according to Maniatis et al. (1982), using as a probe the fragment resulting from the amplification in the PCR, which was marked using dATP32 (Amersham, UK) and the Klenow fragment of DNA-polymerase I of ~. ~oli (Maniatis et al., 1982) in whatman 541 filters for 30 minutes at 37°C, the reaction being terminated by EDTA and heat. 4~ of the colonies were positive clones, which were examined by restriction analysis and had the same pattern of digestion with more than 10 restriction enzymes;
moreover the positive clones were checked by double chain DNA
sequencing (Sanger et al., 1977), using therefor an oligo-nucleotide of 17 bases (5'...ATCATCGAACTAGTTAA...3', seq. id.
No. 5) which hybridises at the 3' end of the promoter, with which it was corroborated that joining of the latter to the SKC-2 gene was as desired.
The selected clone was called pEKG-3 (E'ig. 1), which was subjected to fermentation to realise the characterisation of the product.
The plasmid of clone pEKG3 was purified using a CsCl gradient and the sequences of the SKC-2 gene were established, each time using 2 g of plasmid, and using the oligonucleotides which appear below as primers:
SSK-O1 5'...GAATCAAGACATTAGTC...3' seq. id. No. 6 SSK-02 5'...GTGGCGCGATGCCAC..,3' seq. ld. NO. 7 SSK-03 5'...GCAACCATTACTGATCG...3' seq. id. No. 8 SSK-04 5'...CCAGTACAAAATCAAGC...3' seq> id. No. 9 *Trade-mark a ,~~ 204395 SSK-05 5'...CTAGCTATCGGTGACAC...3' seq. id. No. 10 SSK-06 5'...CAGAGATCAGGTCAG...3' seq.~id. No. ll SSK-07 5'...GTTAAGAGCTGCTCGC...3' seq. id. No. 12 SSK-08 5'...CCAGTTAAGGTATAGTC...3' seq. id. No. 13 SSK-09 5'...TCTCGTTCTTCTTCGG...3' seq. id. No. 14 The protocol followed was basically according to Sanger et al. (1977), and dATP32 and S35dATP (Amersham, UK) were used.
Fig. 2 shows a comparison between the amino acid sequence derived from the base sequence of SKC-2, and those of the genes SKC (Malke et al., 1986), SKA and SKG (Water, F. et al., 1989) .
The plasmid pEKG3 was transformed in several E,, coli strains such as W-3110, JM-101, LE392 and MC-1061 and the expression of streptokinase was compared between them. The best results were obtained with strain W3110 (F- supF suoE
~R g~",],K ~gR metB ~Y tonA), owing to which it was selected to be subjected to fermentation, wherein stable expression levels greater than 20~ of the total protein content of the cells were obtained, and 350-400 mg streptokinase per litre of culture medium were obtained.
ExamBle 3 For subcloning of the SKC-2 gene in yeast, strain MP36 of Piehia nastori~ was used as the host, and variants were made for intracellular and extracellular expression from the plasmids pNAO and pPS7 (Fig. 3), using the signal peptide of sucrose invertase for the extY~acellular construct, and in both cases subcloning the gene under the control of the alcohol oxidase (AOX) promoter, using as the terminator at the 3' end the termination signal of the glyceraldehyde-3-phosphate '. dehydrogenase gene of ~. e-erevisiae and a non-coding 3' region of the AOX gene for integration by homology in the genome of the ~'e.ast,.... fuxt,~e~;, ,~e.~'~,n,~ ~ ~ ;k~~", ~e ~nco, i ... 35~....,..which w 1~ ' .."~, ..k~ ,9.~ . .." ,.. ~
,~.9.:~~;~,~~~~~.~?~,"~~... ..,....",... , .
as used for selection in strain MP36 his'.

204395"3 The vector pPESKC4 (plasmid for extracellular expression of the protein) was obtained from the vector construct pPS7-NcoI-S1 nuclease-phosphatase ligated with the SKC-2 gene amplified by PCR from pEKG3 with the 5 oligonucleotide SK2 and a new primer which hybridises with the 5' end of the gene and eliminates the ATG which had been inserted for expression in bacteria. This new primer is a fragment of SK-3 which does not contain the ATG. In the case of intracellular expression, pPISKC6 was obtained (plasmid for 10 intracellular expression of the protein) from the vector pNAO-NcoI-EcoRI-S1 nuclease-phosphatase ligated with the band of SKC-2 amplified by PCR, with the primers SK2 and SK3 with which is obtained the exact gene which codes for streptokinase with an ATG at its 5' end (Fig. 3). Both plasmids were transformed in strain MP36 his-, using the protocol according to Cregg, J. et al. (1985).
The positive clones were studies by the Southern blot method (Maniatis et al., 1982), and out of those which had the correct integration were selected the most productive in each case for characterisation of the product.
The expression of recombinant streptokinase obtained in P_. pastoris extracellularly was 1-1.2 g/1 culture supernatant, and in case of the intracellular construct more than 1.0 g/1 culture.
In the construct for extracellular expression, the glycosylated protein was obtained with a molecular weight greater than 67,000 dalton, it being corroborated by Western blotting that it decreases to the molecular weight of native streptokinase when it is digested with endoglycosidase H.
This was carried out by taking a portion having a concentration equal to 1 mg/ml in a sodium citrate solution, 0.05 molar, pH=5.5, and denaturing it by the addition of SDS
JJ:1 cd 10a (final concentration 0.02%) and heating at 100°C for 10 minutes, then leaving it to cool to ambient temperature and adding 20 milli-units (mU) of endoglycosidase H (endo H) and leaving it for 16 hours at 37°C, at the end of which it is subjected to subsequent heating at 100°C for 5 minutes and 10 mU of endo H are added, followed by 12 hours incubation at 37°C, and it is JJ:Icd °', ~,'"'""""
. . . . . . . _ . . . 204~:9..~~ ...
11 ' applied to a 12.5% polyacrylamide ge,l and compared with an undeglycosylated sample.
The streptokinase produced in .fichia gastoris in both constructs maintains biological activity, not varying its affinity for plasminogen and being in fact another variant for the use of this protein in clinical medicine.
Example 4 To verify the biological activity of the product of the SKC-2 gene, the pure recombinant streptokinase obtained from both bacteria and yeast was used for acute and subacute toxicology tests on rats, wherein satisfactory and acceptable results were obtained to allow its use in human and animal therapeutics. Its in vivo fibrinolytic activity was verified in clinical tests on animals, wherein there was success in dissolving clots in the coronary and femoral arteries of dogs, blood parameters being maintained similar to those reported in the literature with this type of product.
The product of the SKC-2 gene showed a specific activity of 50,000-100,000 IU/mg, which was measured on plates of agarose-fibrin (Astrup et al.., 1952), chromogenic substrate (Friberger et al., 1982) and in vitro clot lysis according to Westlund et al. (1985).
Exam 1~ a 5 To verify the amino acid sequence derived from the base sequence of the SKC-2 gene, an analysis was made of the pure product by high-performance Liquid chromatography in reverse phase (HPLC-RP), using therefor a C8 4.6 x 250 mm column (Baker, USA), wherein there was used the gradient 5 minutes at 0% buffer B and up to 90% B in 55 minutes, with buffer A
(trifluoroacetic acid (TFA, Pierce, USA) at 0.1% in distilled water) and buffer 8 (TFA at 0,.,5% ~.n acetonitrile .
,, , ~ ,(~L.ichrosolv, ,Merck,T, FRG) ),,..,. ~a~"~,~air)in~ :a, flow , r,ate",off ,, M , "" , ,. , ""
0.8 ml/min.
."

. _ . _ _ .. 12 ~0 ~3~ j~ .
With the protein with a high degree of purity, the amino acid sequence derived from the base sequence obtained from the SKC-2 gene was verified by sequencing it by mass spectrometry.
For this the protein was digested with different enzymes and with combinations of them. The enzymes used were chymotrypsin, endoproteinase Glu-C, endoproteinase Lys-C and trypsin .
From the analysis of the mass spectra of the°peptides obtained in each of the digestions with the different enzymes, the map of the amino acid sequence of the protein was constructed by superposition, which made it possible to verify that there is in this case 100 correspondence between the sequence of the SKC-2 gene and the amino acid sequence of the protein obtained.
Strain deposits The ~. coli HSK-M [pEKG3] strain, based on the ~. coli strain W3110 and containing the plasmid pEKG3, was deposited on June 11, 1990, with the Centraalbureau voor Schimmelcultures (CBS), Baarn, The Netherlands, and obtained deposit number CBS 243.90.
Likewise, the Pichia pas oris MSK-M4 [pPESKC-4] strain, based on the Pir,~.zia gastoris strain MP-36 and containing the plasmid pPESKC-4, was deposited on June 11, 1990, with the Centraalbureau voor Schimmelcultures (CBS), Baarn, The , Netherlands, and obtained deposit number CBS 244.90.
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',: ' R1~ FERENCES
Astrup, 'T., and Mullertz, S., 1952, Arch. Biochem. Biophys.
40: 346-351 Burnett, N.N., 1981, nnal. Biochemistry 112: 195-203 Cregg, J.M., Barringer, K.J., Iiessler, A.Y., and Madden, K.R., 1985, Mol. Cell. Biol. 5: 3376-3385 Dagert, M., and Ehrlich, S.D., 1974, Gene 6: 23-28 Dykewicz, M.S., McGrath, K.G., Harris, K.E., and Patterson, R., 1985, Int. Arch. Allergy Appl. Immun. 78: 386-390 Estrada, M.P., Hernandez, O., and de la Fuente, J., Interferon y Biotecnolog~.a 5: 152-156 Fiberger, P., 1982, J. Clin. Lab. Invest. 92, Suppl. 162:

Hanaha~~, D., 1983, J. Mol. Biol. 166: 557-580 Huang, T.T., Malke, Ii., and Ferrett_i, J.J., 1989, Molec.
Microb . 3 (2 ) : 197-205 Malke, ti., and Ferretti, J.J., 1984, Proc. Natl. Acad. Sci.
USA, 81: 3557-3561 Malke, H., Roe, B., and Ferretti, J.J., 1985, Gene 34: 357-362 Maniatis, T., Frisch, E.F., and Sambrook, J., 1982, Cold Spring Harbor Laboratory, USA
McGrath, K.G., Zeffren, B., Alexander, J., Kaplan, K., and Patterson, R.J., 1985, Allergy Clin. Immunol. 76: 453-457 Muller, J . , Reinert, fi . , and Malke, H . , 1989, Journal of Bacteriology, Apr.: 2202-2208 Randall, K., Gelfond, D.H., Stoffel, S., Scharf, S., t~iguchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A., 1988, Science 239: 987-491 Sanger, F., Nickler, S., and Coulson, A.K., 1977, Proc. Natl.
Acad. Sci. USA 74: 5463-5467 Tombin, H., Stahelin, T., and Gordon, J., 1979, Proc. Natl.
Acad. Sci. USA 76: 4350-4354 Walter, F., Siegel, M., and Malke, H., 1989, Nucl. Acids Res.
17 (3) : 1262 Westtund, L.E., and Anderson, L.O., 1985, Thrombosis Research 37: 213-223 Haggenson M.J. et al., Enzyme Microb. and Technol. 11, 650-656 (1989) ~4 20,4,3953 SEQUENCE LISTING
SEQ ID NO:1 SEQUENCE TYPE: Nucleotide with corresponding protein SEQUENCE LENGTH: 1245 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: genomic DNA
ORIGINAL SOURCE ORGANISM: Streptococcus equisimilis from group C
of Lanfield definition IMMEDIATE EXPERIMENTAL SOURCE: ATCC-9542 strain FEATURES: from 1 to 1245 by mature peptide PROPERTIES: Streptokinase gene The gene product binds to human plasminogen The gene product is an activator of human plasminogen GCT CTA

CCT CGT
GAG CCA
TGG TCT
Ile GTC
Ala AAC
Gly AAC
Pro AGC
Glu Leu Trp Leu 1 Asp Arg CAA Pro TTA Ser GTT Val GTT Asn AGC Asn GTT Ser Gln 10 Leu 15 Val GCT
Val GGT
Ser ACT
Val GTT

GGG
ACG
AAT
CAA
GAC
Ala Gly Thr Val Glu Gly Thr Asn Gln Asp IleSer Leu Lys PhePhe GluIle Asp LeuThr Ser Arg ProAla His GlyGly Lys Thr GluGln GlyLeu Ser ProLys Ser Lys ProPhe Ala ThrAsp,Ser Gly AlaMet ProHis Lys LeuGlu Lys Ala AspLeu Leu LysAla Ile Gln GluGln LeuIle Ala AsnVal His Ser AsnAsp Asp TyrPhe Glu Val IleAsp PheAla Ser AspAla Thr Ile ThrAsp Arg GGC Lys Val TTT Asp Lys AspGly Ser Val ThrLeu Pro Asn 115 GCT
Gly Tyr 120 125 Phe Ala t~.

~5 2043953 ThrGlnPro Val Gln GluPhe LeuLeu Ser Gly HisVal ArgVal Arg 130 135 ~ 140 ProTyrLys Glu Lys ProIle GlnAsn Gln Ala LysSer ValAsp Val GluTyrThr Val Gln PheThr ProLeu Asn Pro AspAsp AspPhe Arg ProGlyLeu Lys Asp ThrLys LeuLeu Lys Thr LeuAla IleGly Asp ThrIleThr Ser Gln GluLeu LeuAla Gln Ala GlnSer IleLeu Asn AAAACC,CAC CCA GGC TATACG ATTTAT GAA CGT GACTCC TCAATC GTC 672 LysThrIsisPro Gly TyrThr IleTyr Glu Arg AspSer SerIle Val 210-~ 215 220 ThrHisAsp Asn Asp IlePhe ArgThr Ile Leu ProMet AspGln Glu PheThrTyr His Val LysAsn ArgGlu Gln Ala TyrGlu IleAsn Lys LysSerGly Leu Asn GluGlu IleAsn Asn Thr AspLeu IleSer Glu AAATAT,TAC GTC CTT AAAAAA GGGGAA AAG CCG TATGAT CCCTTT GAT 864 LysTyrTyr Val Leu LysLys'GlyGlu Lys Pro TyrAsp ProPhe Asp ArgSerHis Leu Lys LeuPhe ThrIle Lys Tyr ValAsp ValAsn Thr AsnGluLeu Leu Lys SerGlu GlnLeu Leu Thr AlaSer GluArg Asn TTAGACTTC AGA GAT TTATAC GAT,~ CGT GAT AAGGCT AAACTA CTC 1008 CT

LeuAspPhe Arg Asp "LeuTyr Asp'1 Arg Asp LysAla LysLeu Leu ~~ro TyrAsnAsn Leu Asp AlaPhe GlyIle Met Asp TyrThr LeuThr Gly a 2p~3953 LysVal GluAsp Asn HisAsp AspThr Asn ArgIle Ile Thr ValTyr MetGly LysArg Pro GluGly GluAsn Ala SerTyr.His Leu AlaTyr AspLys AspArg Tyr ThrGlu GluGlu Arg GluVal Tyr Ser TyrLeu ArgTyr ThrGly Thr ProIle ProAsp Asn ProAsn Asp .Lys A

2043953 _.
SEQ ID N0:2 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 26 bases TGGAATTCAT GAAAAATTAC TTATCT
SEQ ID N0:3 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 28 bases TGGATCCTTA TTTGTCGTTA GGGTTATC
SEQ ID N0:9 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 31 bases GGAATTCATG ATTGCTGGAC CTGAGTGGCT G
SEQ ID N0:5 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 17 bases ATCATCGAAC TAGTTAA
SEQ ID N0:6 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 17 bases GAATCAAGAC ATTAGTC
SEQ ID N0:7 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 15 bases GTGGCGCGAT GCCAC
SEQ ID N0:8 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 17 bases GCAACCATTA CTGATCG

.,, 18 204r395~3 SEQ ID N0:9 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 17 bases SEQ ID NO:10 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 17 bases CTAGCTATCG GTGACAC
SEQ ID NO:11 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 15 bases CAGAGATCAG GTCAG
SEQ ID N0:12 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 16 bases GTTAAGAGCT GCTCGC
SEQ ID N0:13 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 17 bases CCAGTTAAGG TATAGTC
SEQ ID N0:14 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 16 bases TCTCGTTCTT CTTCGG
A

Claims (30)

1. A method for producing streptokinase by expression of a gene encoding said streptokinase which comprises the steps of transforming a host cell with an expression vector containing a streptokinase gene (SKC-2) comprising nucleotide sequence:

operably linked to an effective promoter and transcription terminator, growing said transformed host cell and isolating the streptokinase so produced.

2. The method of Claim 1 wherein said SKC-2 gene is isolated from Streptococcus equisimilis type C strain ATCC-9542.

3. The method of Claim 1 wherein said SKC-2 gene is used without its signal peptide-encoding region.

4. The method of Claim 1 wherein said SKC-2 gene is used with its signal peptide-encoding region.

5. The method of Claim 1 wherein said host cell is a bacterium.

6. The method of Claim 5 wherein said bacterium is Escherichia coli.

7. The method of Claim 5 wherein said bacterium is Escherichia cold strain W3110.

8. The method of Claim 5 wherein said expression vector is a plasmid containing the SKC-2 gene operably linked to the Escherichia coli tryptophan promoter and the T4 transcription terminator.

9. The method of Claim 8 wherein said expression vector is the plasmid pEKG3 contained in Escherichia coli strain CBS 243.90.

10. The method of Claim 1 wherein said host cell is a yeast.

11. The method of Claim 10 wherein said yeast is Pichia pastoris.

12. The method of Claim 10 wherein said yeast is Pichia pastors HIS- strain MP-36 (CBS 244.90).

13. The method of Claim 10 wherein said expression vector is a plasmid containing the SKC-2 gene operably linked to the Pichia pastoris alcohol oxidase promoter (AOX1) and the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase termination sequence (GAPt).

14. The method of Claim 13 wherein said expression vector contains the Saccharamyces cerevisiae HIS3 gene as a selection marker.

15. The method of Claim 14 wherein said expression vector is the plasmid pPESKC-4 contained in Pichia pastoris strain CBS
244.90.

16. The method of Claim 14 wherein said expression vector contains the SKC-2 gene, without a signal peptide-encoding region.

17. An isolated and purified nucleic acid consisting essentially of the nucleotide sequence shown in claim 1.

18. An expression vector for the expression of streptokinase in a host cell, said vector containing a streptokinase gene (SKC-2) consisting essentially of the nucleotide sequence shown in Claim 1 operably linked to an effective promoter and transcription terminator.

19. The expression vector of Claim 18 containing the SKC-2 gene operably linked to the Escherichia coli tryptophan promoter and the T4 transcription terminator for expression of said SKC-2 gene in bacteria.

20. The expression vector of Claim 19 which is the plasmid pEKG3 contained in Escherichia coli strain CBS 243.90.

21. The expression vector of Claim 18 containing the SKC-2 gene operably linked to the Pichia pastoris alcohol oxidase promoter (AOX1) and the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase termination sequence (GAPt) for expression of said SKC-2 gene in yeast.

22. The expression vector of Claim 21 containing the Saccharomyces cerevisiae HIS3 gene as a selection marker.

23. The expression vector of Claim 22 which is the plasmid pPESKC-4.

24. A host cell for producing streptokinase by expression of a gene encoding said streptokinase, said host cell being transformed with an expression vector containing a streptokinase gene (SKC-2)comprising the nucleotide sequence shown in claim 1 operably linked to an effective promoter and transcription terminator.

25. The host cell of Claim 24 which is a bacterium.

26. The host cell of Claim 24 which is Escherichia coli.

27. The host cell of Claim 24 which is Escherichia coli strain W3110.

28. The host cell of Claim 24 which is a yeast.

29. The host cell of Claim 24 which is Pichia pastoris.

30. The host cell of Claim 24 which is Pichia Qastoris HIS-strain MP-36 (CBS 244.90).