Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1990-07-11
1999-02-02
Railey, II, Johnny F.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 711, 435215, 4353201, 536 241, C12N 121, C12N 972, C12P 2102
Patent
active
058663584
DESCRIPTION:
BRIEF SUMMARY
This application is the national stage application of PCT/EP89/01168, filed Oct. 6, 1989.
The present invention relates to a recombinant DNA method of producing non-glycosylated single-chain prourokinase (hereinafter referred to as proUK). More particularly, it relates to a method of producing non-glycosylated proUK which comprises recovering mRNA from an established cell line, preparing cDNA based on said mRNA, inserting the cDNA into a vector, introducing the resulting plasmid into a bacterial cell to thereby produce a transformant and recovering non-glycosylated proUK from said bacterial cell. The invention concerns also certain expression plasmids employed in the above method.
INTRODUCTION
The increasing knowledge of the molecular interactions that regulate physiological fibrinolysis has lead to important implications in the understanding of the mechanisms that dissolve blood clots, and in the development of new thrombolytic agents.
In the human fibrinolytic system a proenzyme, plasminogen, can be activated to the active enzyme, plasmin, by several types of plasminogen activators (Collen, D. and Lijnen, H. R., CRC Critical Reviews in oncology/hematology, 4, n. 3,p. 249, 1986; Verstraete, M. and Collen, D.: Blood, 67, n. 6,p. 1529, 1986). Plasmin is the major protease responsible for the degradation of the fibrin component of a blood clot (Rakoczi, I., Wiman, B. and Collen, D.: Biochim. Biophys. Acta, 540, p. 295, 1978; Robbins, K. C. Summaria, L., Hsieh, B.; and Shah, R. S.: J. Biol. Chem. 242, p. 2333, 1967; Wiman, B.; Eur. J. Biochem, 76, p. 129, 1977).
However, plasmin can also exert its proteolytic effect on several plasma proteins among which the components of the coagulation pathway fibrinogen, factor V and VIII (Collen, D. and Lijnen, H. R., CRC Critical Reviews in oncology/hematology, 4, n. 3,p. 249, 1986; Verstraete, M. and Collen, D.: Blood, 67, n. 6,p. 1529, 1986; Wiman, B., Lijnen, H. R. and Collen, D.; Biochim, Biophys. Acta, 579, p. 142, 1979).
Activation of plasminogen may occur at the systemic level, leading to circulating plasmin that is rapidly neutralized by alfa2-antiplasmin and thus not available for fibrinolysis (Collen, D. and Lijnen, H. R., CRC Critical Reviews in oncology/hematology, 4, n. 3,p. 249, 1986; Verstraete, M. and Collen, D.: Blood, 67, n. 6,p. 1529, 1986).
When the alfa2-antiplasmin level is markedly reduced, plasmin is less rapidly neutralized and can exert its proteolytic effects not only on fibrin, but also on the blood coagulation proteins as described previously.
Excessive lowering in the plasma concentrations of fibrinogen, factor V and VIII, together with the inhibitory effects exerted by some of the fibrinogen degradation products on the hemostatic process, on platelet aggregation and on fibrin polymerisation lead to hemostatic deficiency and subsequently to high bleeding risk (Latallo, Z. S. and Lopaciuk, S.; Thrombos. Diath. Haemouh., 56, p. 253, 1973; Totty, W. G., Gilula, L. A., Mc. Clennman, M. Ahmed, P., and Sherman, L. Radiology, 143, p. 59, 1982). On the other hand, activation of plasminogen may occur at the fibrin level (fibrin-bound plasminogen activation) leading to fibrin-bound plasmin (Collen, D. and Lijnen, H. R., CRC Critical Reviews in oncology/hematology, 4, n. 3,p. 249, 1986; Verstraete, M. and Collen, D.: Blood, 67, n. 6,p. 1529, 1986) which is, instead, not affected by alfa2-antiplasmin and cannot induce systemic fibrinogenolysis.
Urokinase and streptokinase, the most commonly used plasminogen activators in conventional thrombolytic therapy in man, have no specific activity for fibrin. Both compounds activate relatively indiscriminately either circulating or fibrin-bound plasminogen (Zamarron, C., Lijnen, H. R., Van Hoef, B., and Collen, D., Thromb. Haemostas. 52, p. 19, 1984; Samama, M., and Kher, A. Sem. Hop. Paris. 61, n. 20. p. 1423, 1985). Therefore, the systemic haemostatic breakdown often encountered during treatment with streptokinase and urokinase and, consequently, the elevated bleeding risk have often hampered the widespread
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Brandazza Anna
Orsini Gaetano
Sarmientos Paolo
Railey II Johnny F.
Vascular Laboratory Inc.
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