HIGH-YIELD PRODUCTION AND PURIFICATION OF RECOMBINANT STAPHYLOKINASE FOR THROMBOLYTIC THERAPY

Recombinant plasmids were constructed in which the signal sequence of the sak42D and the sakSTAR staphylokinase genes were replaced by an AT G start codon and which express staphylokinase under the control of a tac promoter and two Shine-Dalgarno sequences in tandem. Induction of transfected E. coli TGl cells in a bacterial fermenter produced intrac ellular staphylokinase representing 10 to 15% of total cell protein. G ram quantities of highly purified recombinant staphylokinase were obta ined from cytosol fractions by chromatography, at room temperature, on SP-Sepharose and on phenyl-Sepharose columns, with yields of 50 to 70 percent. The material, at a dose of 4 mg/kg, did not produce acute re actions or affect body weight in mice. Intravenous administration of 1 0 mg SakSTAR over 30 minutes in five patients with acute myocardial in farction induced complete coronary artery recanalization, without asso ciated fibrinogen degradation. However, neutralizing antibodies appear ed in the plasma of all patients within 12 to 20 days. Thus, the prese nt expression and purification method for recombinant staphylokinase y ields large amounts of highly purified mature protein (approximately 2 00 mg per liter fermentation broth) suitable for a more detailed clini cal investigation of its potential as a thrombolytic agent.