Identification, production and use of new staphylokinase derivat

Details Identification, production and use of new staphylokinase derivat Identification, production and use of new staphylokinase derivat

1997-01-16

1999-09-14

Wax, Robert A.

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

4241901, 4242001, 4351721, 4353201, 435220, A61K 3848, A61K 3902, C12N 1500, C12N 952

Patent

active

059519800

ABSTRACT:
Methods for the identification, production and use of derivatives of the invention obtained by preparing a DNA fragment comprising at least the part of the coding sequence of staphylokinase that provides for its biological activity; performing in vitro site-directed mutagenesis on the DNA fragment to replace one or more codons for wild-type amino acids by a codon for another amino acid; cloning the mutated DNA fragment in a suitable vector; transforming or transfecting a suitable host cell with the vector; and culturing the host cell under conditions suitable for expressing the DNA fragment. Preferably the DNA fragment is a 453 bp EcoRI-HindIII fragment of the plasmid pMEX602sakB, the in vitro site-directed mutagenesis is performed by spliced overlap extension polymerase chain reaction and the mutated DNA fragment is expressed in E. coli strain TG1 or WK6. The invention also relates to pharmaceutical compositions comprising at least one of the staphylokinase derivatives according to the invention together with a suitable excipient, for treatment of arterial thrombosis.

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Schlott et al. Functional properties of recombinant staphylokinase variants obtained by site-specific mutagenesis of methionine-26. Biochimica et Biophysica Acta. vol. 1204, pp. 235-242, 1994.
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Collen et al. "Primary structure and gene structure of staphylokinase." Fibrinolysis 6: 226-231 (1992).
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Collen et al. "Comparative immunogenicity and thrombolytic properties toward arterial and venous thrombi of streptokinase and recombinant staphylokinase in baboons." Circulation vol. 87: 996-1006 (1993).

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