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.