ISOLATION AND CONDITIONING OF RECOMBINANT STAPHYLOKINASE FOR USE IN MAN

Staphylokinase(STA), a M(r) 18000 protein produced by Staphylococcus aureus is known to have profibrinolytic properties for more than 40 years (Lack CH, Nature 1948; 161: 559-560) but its potential for thrombolytic therapy has not been adequately investigated. Therefore we have elaborated procedures for the large scale production of recombinant STA (STAR) from the culture broth of E. coli cells transformed with the recombinant plasmid pUC19 which contains a 2.9 kb insert obtained by Hin dIII restriction enzyme digestion of genomic DNA obtained from a selected Staphylococcus aureus strain. STAR, purified from 12 litre batches by chromatography on SP-Sephadex with pH gradient elution, SP-Sephadex with NaCl gradient elution and Sephacryl S-300 superfine gel filtration, with a recovery of 19 +/- 4 mg and a yield of 35 +/- 15 percent, contained a single band on SDS-polyacrylamide gel electrophoresis with NH2-terminal sequence Ser-Ser-Ser-Phe-Asp-Lys-Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala-. It was obtained at a concentration of approximately 1 mg/ml with a specific activity of 185 000 +/- 35 000 units/mg with an endotoxin content of 10 +/- 7 U/mg. After filtration on 0.22 mum Millipore filters, the preparations were sterile under aerobic and anaerobic bacterial culture conditions and virus free by routine screening for human pathogenic viruses. The material remained active after incubation at 37-degrees-C for several days. Bolus injection of STAR at a dose of 3 mg/kg in mice did not produce weight loss within 8 days. Thus, these materials appear to be suitable for the investigation, on a pilot scale, of the pharmacokinetic and thrombolytic properties of STAR in patients with thromboembolic disease.