IE66006B1 - Derivative of tissue-type plasminogen activator - Google Patents
Derivative of tissue-type plasminogen activatorInfo
- Publication number
- IE66006B1 IE66006B1 IE184890A IE184890A IE66006B1 IE 66006 B1 IE66006 B1 IE 66006B1 IE 184890 A IE184890 A IE 184890A IE 184890 A IE184890 A IE 184890A IE 66006 B1 IE66006 B1 IE 66006B1
- Authority
- IE
- Ireland
- Prior art keywords
- ser
- gly
- leu
- arg
- ala
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21069—Protein C activated (3.4.21.69)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Diabetes (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Luminescent Compositions (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The tissue plasminogen activator (t-PA) derivative is not glycosylated and has the following amino-acid sequence: <IMAGE>
Description
Description The invention concerns a new t-PA derivative, a DNA sequence which codes for the new t-PA derivative, expression plasmids which contain a DNA sequence which codes for the t-PA derivative as well as a process for the preparation of such plasmids, a process for the production of the t-PA derivative and an agent for dissolving blood clots which contains the t-PA derivative.
Coagulated blood contains polymeric fibrin which is the main component of the protein matrix. Fibrin is dissolved under physiological conditions by a fibrinolytic system in a reaction cascade which is similar to that of blood coagulation. The central reaction in this is the activation of plasminogen to plasmin which is for example mediated by the tissue-type plasminogen activator t-PA. Plasmin, in turn, dissolves fibrin which is the main component of the protein matrix of coagulated blood. The enzymatic activity of natural t-PA or t-PA obtained from eukaryotes by genetic engineering, i.e. the catalytic activation of plasminogen to plasmin, is very low in the absence of fibrin or fibrinogen cleavage products, but it can be substantially increased in the presence of these proteins, namely by more than ten-fold.
T-PA is cleaved by proteases present in the blood into an A-chain and a B-chain. Both parts of the chain remain bound via a cysteine-bridge. The ability to stimulate the activity of t-PA is a significant advantage in comparison with other known plasminogen activators such as, for example urokinase or streptokinase (cf. for example M. Hoylaerts et al., J. Biol. Chem. 257 (1982), 2912-2919; W. Nieuwenhuizen et al., Biochem. Biophys. Acta, 755 (1983), 531-533).
The mechanism of action of t-PA in vivo is described for example in Korniger and Collen, Thromb. Hamostasis 46 (1981), 561-565. The focus of enzymatic activity on the fibrin surface would seem to make it a suitable agent for the treatment of pathological vascular occlusions (for example myocardial infarction) which has been confirmed to a large extent by clinical trials (Collen et al., Circulation 70 (1984), 1012; Circulation 73 (1986), 511).
A disadvantage of t-PA is however the rapid decrease in its plasma concentration (clearance rate). As a result, a relatively large amount of t-PA is necessary to achieve an effective lysis of thrombi. On the other hand, high therapeutic doses result in side effects such as for example bleeding.
A natural degradation product of t-PA is described in EP 0 196 920 which only contains the kringle 2 and the protease domains of the t-PA, and whose N-terminal amino acid is alanine 160 of the amino acid sequence described by Pennies et al. in Nature 301 (1983), 214-221. The clearance rate of this product of t-PA degradation does not, however, differ significantly from that of the natural t-PA. An improvement of this can only be achieved by a chemical modification of the catalytic domain via attachment of a blocking group.
It is therefore the object of the invention to modify t-PA such that the resultant derivative has a much reduced clearance rate and thus a longer half-life in blood plasma. In this process the ability to lyse thrombi as well as the ability to be stimulated by fibrin should be preserved.
The object of the invention is therefore a tissue—type plasminogen activator (t-PA derivative, also denoted K1K2P in the Examples) which is characterized in that it is not glycosylated and consists of the following amino acid sequence: (M) Ser Tyr Gin Val lie 5 Asp Thr Arg Ala Thr 10 Cys Tyr Glu Asp Gin 15 Gly lie Ser Tyr Arg 20 Gly Thr Trp Ser Thr 25 Ala Glu Ser Gly Ala 30 Glu Cys Thr Asn Trp 35 Asn Ser Ser Ala Leu 40 Ala Gin Lys Pro Tyr 45 Ser Gly Arg Arg Pro 50 Asp Ala lie Arg Leu 55 Gly Leu Gly Asn His 60 Asn Tyr Cys Arg Asn 65 Pro Asp Arg Asp Ser 70 Lys Pro Trp Cys Tyr 75 Val Phe Lys Ala Gly 80 Lys Tyr Ser Ser Glu 85 Phe Cys Ser Thr Pro 90 Ala Cys Ser Glu Gly 95 Asn Ser Asp Cys Tyr 100 Phe Gly Asn Gly Ser 105 Ala Tyr Arg Gly Thr 110 His Ser Leu Thr Glu 115 Ser Gly Ala Ser Cys 120 Leu Pro Trp Asn Ser 125 Met He Leu He Gly 130 Lys Val Tyr Thr Ala 135 Gin Asn Pro Ser Ala 140 Gin Ala Leu Gly Leu 145 Gly Lys His Asn Tyr 150 Cys Arg Asn Pro Asp 155 Gly Asp Ala Lys Pro 160 Trp Cys His Val Leu ί65 Lys Asn Arg Arg Leu 170 Thr Trp Glu Tyr Cys 175 Asp Val Pro Ser Cys 180 Ser Thr Cys Gly Leu 185 Arg Gin Tyr Ser Gin 190 Pro Gin Phe Arg lie Lys 195 Gly Gly Leu Phe 200 Ala Asp lie Ala Ser 205 His Pro Trp Gin Ala Ala lie Phe Ala Lys His Arg Arg Ser Pro Gly Glu Arg Phe 210 215 220 Leu Cys Gly Gly lie Leu lie Ser Ser Cys Trp lie Leu Ser Ala Ala 225 230 235 240 His Cys Phe Gin Glu Arg Phe Pro Pro His His Leu Thr Val He Leu 245 250 255 Gly Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu Gin Lys Phe Glu 260 265 270 Val Glu Lys Tyr He Val His Lys Glu Phe Asp Asp Asp Thr Tyr Asp 275 280 285 Asn Asp lie Ala Leu Leu Gin Leu Lys Ser Asp Ser Ser Arg Cys Ala 290 295 300 Gin Glu Ser Ser Val Val Arg Thr Val Cys Leu Pro Pro Ala Asp Leu 305 310 315 320 Gin Leu Pro Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His 325 330 335 Glu Ala Leu Ser Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His Val 340 345 350 Arg Leu Tyr Pro Ser Ser Arg Cys Thr Ser Gin His Leu Leu Asn Arg 355 360 365 Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly Gly 370 375 380 Pro Gin Ala Asn Leu His Asp Ala Cys Gin Gly Asp Ser Gly Gly Pro 385 390 395 400 Leu Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly He He Ser 405 410 415 Trp Gly Leu Gly Cys Gly Gin Lys Asp Val Pro Gly Val Tyr Thr Lys 420 425 430 Val Thr Asn Tyr Leu Asp Trp He Arg Asp Asn Met Arg Pro 435 440 445 It was established that, surprisingly, deletion of the other domains which are present in native t-PA had no effect on the thrombolytic efficacy of the protein and that the fibrin-dependent stimulatability of the mutein was the same as that of native t-PA.
A further object of the present invention is a DNA sequence which codes for the t-PA derivative according to the present invention and contains the following sequence: ATGTCTTACCAAGTGATCGATACCAGGGCCACGTGCTACGAGGACCAGGGCATCAGCTAC AGGGGCACGTGGAGCACAGCGGAGAGTGGCGCCGAGTGCACCAACTGGAACAGCAGCGCG TTGGCCCAGAAGCCCTACAGCGGGCGGAGGCCAGACGCCATCAGGCTGGGCCTGGGGAAC CACAACTACTGCAGAAACCCAGATCGAGACTCAAAGCCCTGGTGCTACGTCTTTAAGGCG GGGAAGTACAGCTCAGAGTTCTGCAGCACCCCTGCCTGCTCTGAGGGAAACAGTGACTGC TACTTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCC TGCCTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGT GCCCAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAG CCCTGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCC TGCTCCACCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTC 0 TTCGCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCG CCCGGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCC GCCCACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACA TACCGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCAT AAGGAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGAT TCGTCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGAC CTGCAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTG TCTCCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGC TGCACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGAC 7 ACTCGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGC 0 CCCCTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTG GGCTGTGGACAGAAGGATGTCCCGGGTGTGTACACAAAGGTTACCAACTACCTAGACTGG ATTCGTGACAACATGCGACCG 1341 The DNA sequence according to the present invention serves to express the t-PA derivative according to the present invention when it is present on an expression plasmid. An expression plasmid of this kind is a further object of the invention as well as an expression plasmid with a different DNA sequence which, however, also codes for the t-PA derivative according to the present invention. Due to the degeneracy of the genetic code sequences which differ from the DNA sequence shown are suitable for this purpose.
Besides the sequence coding for the t-PA derivative the expression plasmid preferably also contains, apart from an origin of replication, a promotor structure which can be regulated (e.g tac promotor), an efficient terminator (e.g. fd), and a selection marker (e.g. £-lactamasegene).
A further object of the present invention is the plasmid pA33. The preparation of this plasmid is described in Example 1; it contains a DNA sequence which codes for the t-PA derivative according to the present invention.
Yet a further object of the invention is a process for the construction of one of the expression plasmids according to the present invention, wherein a DNA sequence which codes for the t-PA protein according to the present invention or a derivative thereof which contains further regions of the t-PA protein in addition to the kringle I, kringle II and the protease domains is incorporated into a plasmid and those domains which code for amino acids which are not present in the t-PA derivative according to the present invention are deleted by site-directed mutagenesis.
The choice of plasmid, into which the DNA sequence coding for the t-PA derivative according to the present invention is to be incorporated, is dependent on the host cells which are later to be used to express the derivative. Suitable plasmids, as well as the minimum requirements for such a plasmid (e.g. origin of replication, restriction site) are known to the expert. Within the scope of the invention a cosmid, the replicative double-stranded form of phages Gt, M13), and other vectors known to the expert can be used instead of a plasmid. The method of site-directed mutagenesis is described by Morinaga et al., Biotechnolgy 21, (1984), 634, and is carried out essentially as described.
Yet a further object of the invention is a process for the production of a t-PA derivative according to the present invention, which is characterized in that one of the plasmids according to the present invention is expressed in suitable host cells and the product is isolated from the culture medium or, after lysis of the host cells, from the fluid in which the lysis was carried out (e.g. buffer or also the culture medium). Prokaryotic cells are preferably used as the host cells to produce the t-PA derivative according to the present invention. In this connection, it is particularly preferable to first separate the so-called inclusion bodies (insoluble protein aggregates) which form during this process from the soluble cell particles, to solubilize the inclusion bodies containing t-PA by treatment with guanidine hydrochloride, subsequently to derivatise them with oxidized glutathione and finally to renature the t-PA derivative by addition of L-arginine and guanidine hydrochloride. Exact instructions for the t activation of t-PA from inclusion bodies are for example disclosed in the patent applications EP-A Ο 219 874 and EP-A 0 241 022. According to the present invention any other method for the isolation of the active protein from inclusion bodies can, however, be employed as well.
The process according to the present invention is preferably carried out in the presence of L-arginine, in particular in a concentration of 25 to 1000 mmol/1. 1.0 The removal of foreign proteins according to the present invention by affinity chromatography is carried out in a preferred embodiment of the invention over an ETI (Erythrina Trypsin Inhibitor) adsorber column. In this connection, ETI is fixed on a carrier material (adsorber) such as e.g. BrCN-Sepharose. The purification over an ETI adsorber column has the advantage that the ETI adsorber column material can be loaded directly from the concentrated reoxidation preparation even in the presence of such high concentrations of arginine as 0.8 mol/1 arginine. In this way, an aggregation of p-t-PA, which can occur at low arginine concentrations under 25 mmol/1, is avoided. Thus, it is especially preferred to carry out the purification of the p-t-PA preparation over an ETI adsorber column in the presence of 0.2 to 1.0 M, preferably 0.5 to 1.0 M arginine. In this process the solution containing the K1K2P/Pro has preferably a pH of more than 6.5, particularly preferably of more than 7.
The elution from the ETI column is effected by lowering the pH in the presence of arginine under conditions which allow a good solubility of t-PA which was expressed in prokaryotes. Preferably the pH is in the acid range during the elution, particularly preferably in the range of 4 to 6. ο A preparation of K1K2P/Pro produced according to the present invention has a specific t-PA activity of >0.4x10® IU/mg, preferably >0.6x10® IU/mg whose stimulatability by fibrin cleavage products (activity in the presence of fibrinogen peptides/activity without fibrinogen peptides) is larger than ten-fold and preferably larger than twenty-fold. The purity of the preparation according to the present invention is more than 90 % and preferably more than 95 %, especially more than 98 %.
The t-PA derivative according to the present invention is therefore particularly suitable for use in a pharmaceutical agent for dissolving blood clots which again is a further object of the invention.
The invention is elucidated by the following Example in conjunction with the Figures.
Fig. 1 shows schematically the construction of plasmid pA33.
Fig. 2 shows the time course of the plasma 20 concentration of the t-PA activity after intravenous bolus injection of commercially available t-PA (ActilyseR) in comparison with the t-PA derivative according to the present invention in a linear graph and Fig. 3 shows the time course of the concentration in a logarithmic graph. 1 Example 1 Construction of the plasmid pA33 The plasmid pePa 133 served as the starting plasmid which is described in the application EPA 0 242 836. All experimental steps for the specific mutagenesis i.e. for the deletion of the domains F and E from pePa 133 (see Fig. 1) were carried out essentially using the method of Morinaga et al., Biotechnologie 21 (1984), 634. For this, two cleavage preparations were prepared from pePa 133. Preparation A was cleaved with EcoRl and the largest fragment was isolated. Preparation B was cleaved with Xhol and in this way the product was linearized.
The following oligonucleotide was used for the heteroduplex formation: ' TAC CAA GTG ATC GAT ACC AGG GCC 3’ Those clones which contained the sought-after plasmid pA33 were determined by colony hybridization with the above-mentioned mutagenesis oligonucleotide. This plasmid was characterized by restriction analysis and checked for the absence of the Sspl restriction cleavage site which is located in the part of the t-PA expression cassette coding for the finger domain.
Example 2 Preparation of active K1K2P/Pro from E. coli a) Cell lysis and preparation of the inclusion bodies (IB's) 1.6 kg cell wet-weight (E. coli, DSM 3689), transformed with the plasmid pA33 was suspended in 10 1 0.1 mol/1 Tris-HCl, 20 mmol/1 EDTA, pH 6.5, 4*C. 2.5 g lysozyme was added to this and incubated for 30 minutes at 4C; afterwards complete cell lysis was carried out by high S' pressure dispersion. 5 1 0.1 mol/1 Tris-HCl, 20 mmol/1 EDTA, 6 % Triton X100 and 1.5 mol/1 NaCl, pH 6.5 was added and mixed with the lysate solution and incubated for a further 30 minutes at 4’C. Following this the inclusion bodies (IB's) were separated by centri fugat ion.
The pellet was suspended in 10 1 0.1 mol/1 Tris-HCl, mmol/1 EDTA, pH 6.5, incubated for 30 minutes at 4’C and the IB-preparation was isolated by subsequent centrifugation. b) Solubilization of the IB's 8.7 g IB's (wet-weight) were suspended in 100 ml 0.1 M Tris, 6 M guanidine, 0.1 M DTE, pH 7.5 and stirred for 30 minutes at 25’C.
After adjustment of the pH to pH 3 with HCl (25 %), the solution was dialysed against 4 mol/1 guanidine-HCl, pH 2.5 (3x31, 24 h, 4’C). 3 c) Derivatization The above-mentioned dialysate was adjusted to 50 mmol/1 with oxidized glutathione (GSSG) and to 100 mmol/1 with Tris and the pH value was titrated to 7.5 with 5 mol/1 NaOH. The preparation was incubated for 90 min at 25*C. After adjusting the pH value to pH 3 with HC1 (25 %), a dialysis against 10 mmol/1 HC1 (3 x 100 1, 48 h, 4*C) was carried out. d) Renaturation A 10 1 reaction vessel was filled with 0.8 mol/1 Arg/HCl, pH 8.5, 1 mmol/1 EDTA, 1 mmol/1 GSH (glutathione). The renaturation was carried out at 20C by a three-fold addition at 24 hour intervals of 100 ml of the derivative which had previously been dialysed against 6 mol/1 guanidine-HCl, pH 2.5 .
After the renaturation a preparation is obtained with a specific activity of 50 - 75 KU/mg (test according to H. Lill, Z. gesamte inn. Med. ihre Grenzgeb. (1987), 42. 478-486) e) Concentration of the renaturation preparation The renatured preparation can, if required, be ' concentrated on a haemodialyzer. 4 Example 3 Purification of K1K2P/Pro from E. coli The purification of K1K2P/Pro from E. coli is carried out by affinity chromatography on Erythrina-Trypsin5 Inhibitor (ETI)-Sepharose.
The renaturation preparation was concentrated 1 : 5 on a haemodialyzer (Asahi AM 300) and dialysed overnight against 0.8 mol/1 Arg/HCl, pH 7.5 and centrifuged. 1.8 1 dialysate was applied (4 column volumes (CV)/h) to an ETI-Sepharose column (120 ml) which had been equilibrated with 0.8 mol/1 Arg/HCl, pH 7.5 and was washed with buffer (0.8 mol/1 Arg/HCl, pH 7.5, 0.5 mol/1 NaCl) until the absorbance of the eluate at 280 nm reached the blank value for the buffer. After washing for a second time with 5 CV 0.3 mol/1 Arg/HCl, pH 7.0 the elution was carried out with 0.3 mol/1 Arg/HCl, pH 4.5.
Volume Activity cprot. (ml) KU/ml mg/ml SA F* * KU/mg dialysate 1800 74 1.05 70 15 Κ1Κ2Ρ/ΡΓΟ 200 610 0.82 744 28 *F = stimulation by fibrin = activity in the presence of fibrin/activity in the absence of fibrin.
Examp le_4 Characterization of purified K1K2P/Pro from E. coli a) Characterization of the protein - SDS-PAGE and Reversed-Phase HPLC 5 The homogeneity of the preparation purified by affinity chromatography on ETI-Sepharose was demonstrated by SDS-PAGE and reversed-phase HPLC (RP-HPLC). An apparent molecular weight for K1K2P from E. coli of 50800 Da + 2000 Da was calculated from the relative distance of migration in an electrophoretic analysis of the purified material. The densitometric analysis showed a purity of > 90 %.
RP-HPLC is based on the different interactions of proteins with hydrophobic matrices. This property was used as an analytical method to quantify the degree of purity.
The analysis of the purified K1K2P/Pro from E.coli was carried out on a Nucleosil 300 separation column (Knauer) using a trifluoroacetic acid/acetonitrile gradient (buffer A: 1.2 ml trifluoroacetic acid in 1000 ml H2O; buffer B: 300 ml H2O, 700 ml acetonitrile, 1 ml trifluoroacetic acid; - 100 %). Integration of the chromatographic analysis yielded a purity of >95 %. Ιβ - N-terminal sequence The N-terminal amino acid sequence was determined using an ABI 470 sequencer with a standard programme and on-line PTH detection.
The determined sequence S1-Y2-Q3-V4-I5-D6T7-R8-A9—T10-C11—Y12—E13—D14 agreed with the expected sequence deduced from the DNA-sequence.
Activity determination The in vitro activity of K1K2P/Pro from E. coli was determined according to H. Lill, Z. gesamte inn. Med. ihre Grenzgeb. (1987), 42, 478-486. The specific activity (SA) was 650000 IU/mg + 200000 IU/mg. The stimulatability of K1K2P/Pro from E.coli in this test system by BrCN-fibrinogen fragments (activity in the presence of fibrinogen fragments divided by activity in the absence of fibrinogen fragments) was 25-30.
In vitro binding to fibrin The in vitro binding of K1K2P/Pro to fibrin was determined according to the method described by Higgins and Vehar (Higgins, D.L. and Vehar, G.A. (1987), Biochem. 26, 7786-7791).
It shows that K1K2P/Pro compared to t-PA from CHO cells has no significant binding to fibrin.
Example 5 Pharmacokinetics of K1K2P/Pro in the rabbit The pharmacokinetic properties of K1K2P/Pro were compared to those of ActilyseR (Alteplase, Thomae GmbH, Biberach, FRG) in New-Zealand white rabbits. Both fibrinolytic agents were injected intravenously for 1 min at a dose of 200000 IU/kg body weight '(bw) . Plasma samples were taken before and at defined times after the injection. The t-ΡΆ activity in the plasma was measured with a spectrophotometric test according to J. H. Verheijen et al., (Thromb. Haemostas. 48. 266, 1982), modified according to H. Lill (Z. ges. Inn. Med. 42. 478, 1987).
A computer programme for non-linear regression modified according to H.Y. Huang (Aero-Astronautics-Report 64. Rice University, 1-30, 1969) was used to calculate the pharmacokinetic parameters. When fitting the curves, a 1 or 2 compartment model was assumed which is different from individual to individual. In each case, before the calculation, the value for the endogenous basal concentration was subtracted from the subsequent values.
K1K2P/Pro is eliminated in only 2 of 6 animals with a fast a and a slow β phase. In these 2 animals the alpha phase portion .was 53 % Of the total elimination. In contrast, all animals in the Actilyse* group show a fast alpha phase whose.portion amounts to more than 70 % of the total elimination. K1K2P/Pro is eliminated mainly with only one half-time which is 15.1 + 3.1 min. In contrast, ActilyseR has a fast half-time of 1.63 min and a slow half-time of 10.1 min (Tab. 1). The total plasma clearance of K1K2P/Pro is 6.3 + 1.5 ml/kg/min and is thus considerably lower than that of ActilyseR (Cltot = 22.9 + 9.1 ml/kg/min).
All in all, K1K2P/Pro represents a t-PA mutant which, in comparison with ActilyseR as the state of the art, has a clearly improved pharmokinetic profile (Fig. 2 and 3).
Example 6 Phatmacodynamics of K1K2P/Pro in the rabbit The rabbit model for jugular vein thrombosis established by D. Collen et al. (J. Clin. Invest. 71. 368, 1983) was used to examine the thrombolytic efficacy. The fibrinolytic agents or the solvent were injected intravenously over 1 min as a bolus. Afterwards the rate of thrombolysis was determined and selected parameters of the coagulation system as well as the number of platelets were determined (Table 2).
At the same dose (200000 IU/kg body weight) K1K2P/Pro achieved a statistically significantly higher rate of thrombolysis of 50.7 + 6.5 % after intravenous bolus injection than ActilyseR (24.1 + 3.7 %). The dose of ActilyseR with a comparable thrombolysis efficacy to K1K2P/Pro is 800000 IU/kg body weight (Table 2).
\ With an equipotent dosage of K1K2P/Pro in comparison with ActilyseR there were less effects on the coagulation parameters fibrinogen, plasminogen and alpha2-antiplasmin compared with the solvent group, which, however, do not differ from the effects of an equipotent dose of ActilyseR.
K1K2P/Pro is thus a t-PA mutant which has a thrombolytic efficacy after bolus injection in the rabbit model of jugular vein thrombosis which is greatly increased in comparison with ActilyseR. In this K1K2P/Pro has maintained its fibrin specificity to an extent which also applies for ActilyseR.
Table 1 Pharmaeokinetio parameter· derived from computer calculations of plasma concentration-time data based on the t-PA activity in anaesthetised rabbits after i.v. bolus injection of 200000 ZU/kg body weight KlX2P/Pro or Aotilyse Fibrinolytictl/2 afcl/2 0cinj .Vc*cltot*AUCextrapol. agent (min) (min) (IU/ml) (ml/kg) (mlxkg"1xmin"1) (IUxml-1xh) ActilyseR 1.63 10.1 3051.9 63.2 22.9 161.1 (n=6) (n=5) ±1318.8 ±29.3 ±9.1 ±49.8 1 K1K2P/Pro 8.9 15.1 1695.9 119.1 6.3 556.4 (n=6) (n=2) ±3.1 ±345.4 ±26.6 ±1.5 ±118.8 Mean + SEM *vc = Volume of distribution of the central compartment Cltots Total plasma clearance AUC = area under the curve; the area under the curve (AUC") is of particular interest 15 for the application of a fibrinolytic agent as a bolus injection, since it enables a comparison over time of the prevailing plasma concentrations.
Table Rate of thrombolysis, number of platelets and selected parameters of the coagulation system after i.v. bolua injection (over X min) of Aotilyse*, K1K2P/Pro or solvent in the rabbit model of jugular vein thrombosis Solvent ActilyseR 200000 IU/kg bw K1K2P/Pro 200000 IU/kg bw ActilyseR 800000 IU/kg bw Thrombolysis rate (%) 12.9 + 0.9 24.1 + 3.7 50.7 + 6.5 44.6 + 4.8 Fibrinogen (%) 89.5 + 2.1 90.5 ± 2.6 81.1 + 5.1 81.4 + 3.4 Plasminogen (%) 90.5 + 2.7 83.8 + 4.4 60.1 + 4.3 69.6 + 3.3 a2-Antiplasmin (%) 90.9 ± 4.4 100.8 ±5.3 67.6 + 6.7 74.2 + 5.9 PTT (%) 113.8 ± 4.9 112.9 + 4.3 113.1 + 3.0 115.1 + 5.5 Thrombocytes (%) 86.1 +12.1 91.4 + 4.9 84.4 + 8.3 86.3 + 2.8 Coagulation parameters and number of thrombocytes in % of the initial value before injection PTT = partial thromboplastin time Mean + SEM each group n - 6 animals bw.» body weight Example 7 Optimized expression in E. coli To increase the yield of expression product, the sequence encoding the KlK2P-gene was subcloned in a plasmid with a high copy number. Plasmid pePa 126.1 described in the patent application P 39 31 933.4 was used for this. This plasmid is composed mainly of the vector pKK223-3 and the sequence coding for t-PA as described in the application EP-A 0 242 835.
An fd-terminator sequence was first integrated into this plasmid. For this, the plasmid pePa 126.1 was linearized with the restriction enzyme Hind III. The plasmid cleaved in this manner was separated by gel electrophoresis and isolated preparatively. The plasmid pLBUl (Gentz et al., (1981) PNAS 78 (8):4963) was cleaved with Hind III and a Hind III fragment of about 360 bp which contained the fd-terminator was isolated preparatively by gel electrophoresis and gel elution.
The linearized plasmid pePa 126.1 and the 360 bp Hind III fragment from pLBUl were ligated. The ligation preparation was cotransformed with the plasmid pUBS 500, described in the application P 39 31 933.4 in E. coli, DSM 2102. From the clones, those were selected that contained the desired plasmid pePa 126 fd which differs from the starting plasmid pePa 126.1 in that it contains a second Hind III cleavage site.
Two fragments were isolated from the plasmid pePa 126 fd: a BamHI/PvuI-fragment of 3.4 kb size and a Pvul/Xmal fragment of 1.3 kb size. Both these fragments were ligated with a BamHI/Xmal fragment of about 1.6 kb ο *1 *> from the plasmid pA33 and transformed with the plasmid pUBS 500 into £. coli. The resultant plasmid was named pA33 fd and can be distinguished from pePa 126 fd in that in a restriction digest with EcoRl the second smallest EcoRl fragment from pePa 126 fd of about 610 bp length is about 250 bp shorter in pA33 fd.
Claims (3)
1. Derivative of tissue-type plasminogen activator (t-ΡΆ), wherein it is not glycosylated and has the following amino acid sequence: (M) Ser Tyr Gin Val lie 5 Asp Thr Arg Ala Thr 10 Cys Tyr Glu Asp Gin 15 Gly He Ser Tyr Arg 20 Gly Thr Trp Ser Thr 25 Ala Glu Ser Gly Ala 30 Glu Cys Thr Asn Trp 35 Asn Ser Ser Ala Leu 40 Ala Gin Lys Pro Tyr 45 Ser Gly Arg Arg Pro 50 Asp Ala He Arg Leu 55 Gly Leu Gly Asn His 60 Asn Tyr Cys Arg Asn 65 Pro Asp Arg Asp Ser 70 Lys Pro Trp Cys Tyr 75 Val Phe Lys Ala Gly 80 Lys Tyr Ser Ser Glu 85 Phe Cys Ser Thr Pro 90 Ala Cys Ser Glu Gly 95 Asn Ser Asp Cys Tyr 100 Phe Gly Asn Gly Ser 105 Ala Tyr Arg Gly Thr 110 His Ser Leu Thr Glu 115 Ser Gly Ala Ser Cys 120 Leu Pro Trp Asn Ser 125 Met lie Leu lie Gly 130 Lys Val Tyr Thr Ala 135 Gin Asn Pro Ser Ala 140 Gin Ala Leu Gly Leu 145 Gly Lys His Asn Tyr 150 Cys Arg Asn Pro Asp 155 Gly Asp Ala Lys Pro 160 Trp Cys His Val Leu 165 Lys Asn Arg Arg Leu 170 Thr Trp Glu Tyr Cys 175 Asp Val Pro Ser Cys 180 Ser Thr Cys Gly Leu 185 Arg Gin Tyr Ser Gin 190 Pro Gin Phe Arg lie 195 Lys Gly Gly Leu Phe 200 Ala Asp lie Ala Ser 205 His Pro Trp Gin Ala 210 Ala lie Phe Ala Lys 215 His Arg Arg Ser Pro 220 Gly Glu Arg Phe Leu 225 Cys Gly Gly He Leu 230 lie Ser Ser Cys Trp 235 lie Leu Ser Ala Ala 240 His Cys Phe Gin Glu 245 Arg Phe Pro Pro His 250 His Leu Thr Val He 255 Leu Gly Arg Thr Tyr 260 Arg Val Val Pro Gly 265 Glu Glu Glu Gin Lys 270 Phe Glu Val Glu Lys 275 Tyr He Val His Lys 280 Glu Phe Asp Asp Asp 285 Thr Tyr Asp Asn Asp 290 lie Ala Leu Leu Gin 295 Leu Lys Ser Asp Ser 300 Ser Arg Cys Ala Gin 305 Glu Ser Ser Val Val 310 Arg Thr Val Cys Leu 315 Pro Pro Ala Asp Leu 320 Gin Leu Pro Asp Trp 325 Thr Glu Cys Glu Leu 330 Ser Gly Tyr Gly Lys 335 His Glu Ala Leu Ser 340 Pro Phe Tyr Ser Glu 345 Arg Leu Lys Glu Ala 350 His Val Arg Leu Tyr 355 Pro Ser Ser Arg Cys 360 Thr Ser Gin His Leu 365 Leu Asn Arg Thr Val 370 Thr Asp Asn Met Leu 375 Cys Ala Gly Asp Thr 380 Arg Ser Gly Gly Pro 385 Gin Ala Asn Leu His 390 Asp Ala Cys Gin Gly 395 Asp Ser Gly Gly Pro 400 Leu Val Cys Leu Asn 405 Asp Gly Arg Met Thr 410 Leu Val Gly He He 415 Ser Trp Gly Leu Gly 420 Cys Gly Gin Lys Asp 425 Val Pro Gly Val Tyr 430 Thr Lys Val Thr Asn Tyr Leu Asp Trp He Arg Asp Asn Met Arg Pro 435 440 445
2. DNA sequence, wherein it codes for a t-PA derivative as claimed in claim 1 and contains the following sequence: ATGTCTTACCAAGTGATCGATACCAGGGCCACGTGCTACGAGGACGAGGGCATCAGCTAC AGGGGCACGTGGAGCACAGCGGAGAGTGGCGCCGAGTGCACCAACTGGAACAGCAGCGCG TTGGCCCAGAAGCCCTACAGCGGGCGGAGGCCAGACGCCATCAGGCTGGGCCTGGGGAAC CACAACTACTGCAGAAACCCAGATCGAGACTCAAAGCCCTGGTGCTACGTCTTTAAGGCG : GGGAAGTACAGCTCAGAGTTCTGCAGCACCCCTGCCTGCTCTGAGGGAAACAGTGACTGC; TACTTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCC: TGCCTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGT GCCCAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAG CCCTGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCC TGCTCCACCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTC TTCGCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCG CCCGGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCC GCCCACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACA TACCGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCAT AAGGAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGAT TCGTCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGAC CTGCAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTG TCTCCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGC TGCACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGAC ACTCGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGC CCCCTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTG GGCTGTGGACAGAAGGATGTCCCGGGTGTGTACACAAAGGTTACCAACTACCTAGACTGG ATTCGTGACAACATGCGACCG 1341 3. Expression plasmid, wherein it contains the DNA sequence as claimed in claim 2 or a different DNA . sequence within the scope of the degeneracy of the genetic code which codes for a protein as claimed in claim 1. Plasmid pA33 according to Fig. 1 5. Process for the construction of an expression plasmid as claimed in one of the claims 3 or 4, wherein a DNA sequence which codes for the whole t-PA protein or a derivative thereof containing further regions of the t-PA protein in addition to the kringle I, kringle II and the protease domains is incorporated into a plasmid and those domains which code for amino acids which are not present in the t-PA derivative as claimed in claim 1 are removed by site-directed mutagenesis. 6. Process as claimed in claim 5, wherein the corresponding cDNA is used as the DNA sequence for the t-PA protein or a derivative thereof. 7. Process for the production of a t-PA derivative as claimed in claim 1, wherein a plasmid according to one of the claims 3 or 4 is introduced into suitable host cells and the expression product is isolated from the culture medium or after lysis of the host cells. 8. Process as claimed in claim 7, wherein prokaryotic cells and in particular E. coli are used as host cells. 9. Process as claimed in claim 8, wherein the yield of active protein is increased by isolating the inclusion bodies” that form and solubilizing them by treatment with guanidine hydrochloride, followed by derivatization with oxidized glutathione and finally renaturation of the t-PA derivative by addition of L-arginine and GSH. 28. 10. Process as claimed in one of the claims 7 to 9, wherein one works in the presence of 25 to 1000 mmol/1 L-arginine. 11. Process as claimed in one of the claims 7 to 10, wherein after the renaturation, the t-ΡΆ in the renaturation mixture is concentrated and subsequently a chromatographic purification by means of affinity chromatography on an ETI adsorber column is carried out. , 12. Process as claimed in claim ll, wherein the chromatographic purification is carried out with the concentrate of the renaturation mixture which contains 25 to 1000 mmol/1, preferably 200 to 1000 mmol/1 L-arginine. 13. Process as claimed in claim 12, wherein the concentrate used for the chromatographic purification is adjusted to a pH value of more than 7.0. 14. Process as claimed in one of the claims 11 to 13, wherein the elution is carried out at a pH of 4 to 6. 15. Agent for dissolving blood clots containing a t-PA derivative as claimed in claim 1. 16. A derivative of tissue-type plasminogen activator (t-PA) according to claim 1, substantially as hereinbefore described and exemplified. 17. A DNA sequence according to claim 2, substantially as hereinbefore described and exemplified. 18. An expression plasmid according to claim 3, substantially as hereinbefore described and exemplified. 19. 20. 21. 10 22. 23. -29A process for the construction of an expression plasmid according to claim 3, substantially as hereinbefore described and exemplified. An expression plasmid according to claim 3, whenever obtained by a process claimed in any one of claims 5, 6 or 19. A process for the production of a t-PA derivative according to claim 1, substantially as hereinbefore described and exemplified. A t-PA derivative according to claim 1, whenever produced by a process claimed in any one of claims 7 - 14 or 21. An agent according to claim 15, substantially as hereinbefore described. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS. BOhri”I N GER MANNHEIM GmbH
3. Sheets
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE3917781 | 1989-05-31 | ||
DE3923339A DE3923339A1 (en) | 1989-05-31 | 1989-07-14 | FABRIC PLASMINOGEN ACTIVATOR DERIVATIVE |
Publications (2)
Publication Number | Publication Date |
---|---|
IE901848L IE901848L (en) | 1990-11-30 |
IE66006B1 true IE66006B1 (en) | 1995-11-29 |
Family
ID=25881452
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
IE184890A IE66006B1 (en) | 1989-05-31 | 1990-05-22 | Derivative of tissue-type plasminogen activator |
Country Status (17)
Country | Link |
---|---|
EP (1) | EP0400545B1 (en) |
JP (1) | JPH0327286A (en) |
AT (1) | ATE119940T1 (en) |
AU (1) | AU623781B2 (en) |
CA (1) | CA2017636C (en) |
CZ (1) | CZ282035B6 (en) |
DD (1) | DD300109A5 (en) |
DE (2) | DE3923339A1 (en) |
ES (1) | ES2070210T3 (en) |
FI (1) | FI101475B (en) |
HU (1) | HU216870B (en) |
IE (1) | IE66006B1 (en) |
IL (1) | IL94539A (en) |
NO (1) | NO902405L (en) |
NZ (1) | NZ233796A (en) |
PT (1) | PT94227B (en) |
ZA (1) | ZA904089B (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3942144A1 (en) * | 1989-12-20 | 1991-06-27 | Boehringer Mannheim Gmbh | STABILIZATION OF K1K2P PRO |
AU2660397A (en) * | 1996-04-05 | 1997-10-29 | Board Of Regents, The University Of Texas System | Methods for producing soluble, biologically-active disulfide bond-containing eukaryotic proteins in bacterial cells |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MX197183A (en) * | 1982-05-05 | 1994-02-28 | Genentech Inc | HUMAN TISSUE PLASMINOGEN ACTIVATOR |
FR2594845B1 (en) * | 1986-02-21 | 1989-12-01 | Genetica | MICROBIOLOGICAL PREPARATION OF THE HUMAN PLASMINOGEN TISSUE ACTIVATOR (T-PA) AND CONVERSION OF THE ENZYME SO OBTAINED IN ITS ACTIVE FORM |
DE3643158A1 (en) * | 1986-04-21 | 1987-11-19 | Boehringer Mannheim Gmbh | TISSUE PLASMINOGEN ACTIVATOR (TPA) DERIVATIVE AND ITS PRODUCTION |
FI100106B (en) * | 1986-12-05 | 1997-09-30 | Novartis Ag | A process for the preparation of a plasminogen single-stranded recombinant activator |
IL87276A (en) * | 1987-08-03 | 1995-07-31 | Fujisawa Pharmaceutical Co | Analog of tissue plasminogen activator comprising only the kringle 2 and protease domain dna encoding the same processes for the preparation thereof and pharmaceutical compositions containing the same |
US5094953A (en) * | 1988-03-21 | 1992-03-10 | Genentech, Inc. | Human tissue plasminogen activator variants |
DE3903581A1 (en) * | 1989-02-07 | 1990-08-16 | Boehringer Mannheim Gmbh | FABRIC PLASMINOGEN ACTIVATOR DERIVATIVE |
-
1989
- 1989-07-14 DE DE3923339A patent/DE3923339A1/en not_active Withdrawn
-
1990
- 1990-05-22 IE IE184890A patent/IE66006B1/en not_active IP Right Cessation
- 1990-05-24 NZ NZ233796A patent/NZ233796A/en unknown
- 1990-05-28 AU AU56009/90A patent/AU623781B2/en not_active Ceased
- 1990-05-28 ES ES90110096T patent/ES2070210T3/en not_active Expired - Lifetime
- 1990-05-28 EP EP90110096A patent/EP0400545B1/en not_active Expired - Lifetime
- 1990-05-28 AT AT90110096T patent/ATE119940T1/en not_active IP Right Cessation
- 1990-05-28 IL IL9453990A patent/IL94539A/en not_active IP Right Cessation
- 1990-05-28 DE DE59008693T patent/DE59008693D1/en not_active Expired - Fee Related
- 1990-05-28 CA CA002017636A patent/CA2017636C/en not_active Expired - Fee Related
- 1990-05-29 JP JP2139539A patent/JPH0327286A/en active Pending
- 1990-05-29 ZA ZA904089A patent/ZA904089B/en unknown
- 1990-05-30 CZ CS902676A patent/CZ282035B6/en not_active IP Right Cessation
- 1990-05-30 FI FI902700A patent/FI101475B/en not_active IP Right Cessation
- 1990-05-30 DD DD341150A patent/DD300109A5/en unknown
- 1990-05-30 NO NO90902405A patent/NO902405L/en unknown
- 1990-05-30 HU HU903267A patent/HU216870B/en not_active IP Right Cessation
- 1990-05-31 PT PT94227A patent/PT94227B/en active IP Right Grant
Also Published As
Publication number | Publication date |
---|---|
NZ233796A (en) | 1992-07-28 |
PT94227B (en) | 1997-01-31 |
DE59008693D1 (en) | 1995-04-20 |
ES2070210T3 (en) | 1995-06-01 |
CZ267690A3 (en) | 1997-04-16 |
HU216870B (en) | 1999-09-28 |
EP0400545B1 (en) | 1995-03-15 |
HUT54734A (en) | 1991-03-28 |
PT94227A (en) | 1991-02-08 |
FI902700A0 (en) | 1990-05-30 |
CA2017636C (en) | 2000-05-02 |
AU5600990A (en) | 1990-12-06 |
FI101475B1 (en) | 1998-06-30 |
IL94539A (en) | 1996-08-04 |
ATE119940T1 (en) | 1995-04-15 |
HU903267D0 (en) | 1990-10-28 |
JPH0327286A (en) | 1991-02-05 |
NO902405L (en) | 1990-12-03 |
EP0400545A1 (en) | 1990-12-05 |
CA2017636A1 (en) | 1990-11-30 |
DE3923339A1 (en) | 1990-12-06 |
CZ282035B6 (en) | 1997-04-16 |
NO902405D0 (en) | 1990-05-30 |
FI101475B (en) | 1998-06-30 |
IE901848L (en) | 1990-11-30 |
DD300109A5 (en) | 1992-05-21 |
IL94539A0 (en) | 1991-03-10 |
AU623781B2 (en) | 1992-05-21 |
ZA904089B (en) | 1991-03-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5223256A (en) | Thrombolytically active non-glycosylated protein | |
EP0191606B1 (en) | Vectors and methods for expression of human protein c activity | |
JP2631645B2 (en) | Novel compound, production method thereof and pharmaceutical composition containing the same | |
Winkler et al. | Purification and characterization of recombinant single-chain urokinase produced in Escherichia coli | |
Orsini et al. | Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies | |
AU620927B2 (en) | A preparation of plasminogen activator expressed in prokaryotes | |
US5866358A (en) | Production of human prourokinase | |
CA2017636C (en) | Derivative of tissue-type plasminogen activator | |
EP0666920B1 (en) | Thrombin activatable plasminogen derivatives | |
US5723122A (en) | Use of the protease domain of human plasminogen activator for the treatment of thromboembolic diseases | |
CA2215531C (en) | Mutant of the erythrina caffra type inhibitor and the use of the said mutant for purifying serin proteases | |
CA2262751A1 (en) | Plasminogen activator capable of being activated by thrombin | |
AU624869B2 (en) | Production of human prourokinase | |
EP0796323A1 (en) | Use of the protease domain of human plasminogen activator for the treatment of thromboembolic diseases | |
CA2017177A1 (en) | Human prourokinase variants, methods of producing same, dna sequences, plasmids and hosts therefor | |
MXPA99000966A (en) | Plasminogen activator capable of being activated by thrombin |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MM4A | Patent lapsed |