ISOLATION AND CONDITIONING OF RECOMBINANT STAPHYLOKINASE FOR USE IN MAN

Staphylokinase(STA), a M(r) 18000 protein produced by Staphylococcus a ureus is known to have profibrinolytic properties for more than 40 yea rs (Lack CH, Nature 1948; 161: 559-560) but its potential for thrombol ytic therapy has not been adequately investigated. Therefore we have e laborated procedures for the large scale production of recombinant STA (STAR) from the culture broth of E. coli cells transformed with the r ecombinant plasmid pUC19 which contains a 2.9 kb insert obtained by Hi n dIII restriction enzyme digestion of genomic DNA obtained from a sel ected Staphylococcus aureus strain. STAR, purified from 12 litre batch es by chromatography on SP-Sephadex with pH gradient elution, SP-Sepha dex with NaCl gradient elution and Sephacryl S-300 superfine gel filtr ation, with a recovery of 19 +/- 4 mg and a yield of 35 +/- 15 percent , contained a single band on SDS-polyacrylamide gel electrophoresis wi th NH2-terminal sequence -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 filte rs, the preparations were sterile under aerobic and anaerobic bacteria l culture conditions and virus free by routine screening for human pat hogenic viruses. The material remained active after incubation at 37-d egrees-C for several days. Bolus injection of STAR at a dose of 3 mg/k g in mice did not produce weight loss within 8 days. Thus, these mater ials appear to be suitable for the investigation, on a pilot scale, of the pharmacokinetic and thrombolytic properties of STAR in patients w ith thromboembolic disease.