High Yield Production and Purification of Recombinant Staphylokinase for Thrombolytic Therapy

Abstract

Recombinant plasmids were constructed in which the signal sequence of the sak42D and the sakSTAR staphylokinase genes were replaced by an ATG 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 fermentor produced intracellular staphylokinase representing 10 to 15% of total cell protein. Gram quantities of highly purified recombinant staphylokinase were obtained 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 reactions or affect body weight in mice. Intravenous administration of 10 mg SakSTAR over 30 minutes in five patients with acute myocardial infarction induced complete coronary artery recanalization, without associated fibrinogen degradation. However, neutralizing antibodies appeared in the plasma of all patients within 12 to 20 days. Thus, the present expression and purification method for recombinant staphylokinase yields large amounts of highly purified mature protein (approximately 200 mg per liter fermentation broth) suitable for a more detailed clinical investigation of its potential as a thrombolytic agent.

A fully functional deletion staphylokinase derivative crystallizes in two non-isomorphous monoclinic modifications

“…All other methods used for the construction of the expression vector pMEXÁ10Sak have been described previously (Sambrook et al, 1989). A Á10Sak gene was constructed by the polymerase chain reaction (PCR), using Vent polymerase (New England Biolabs, Leusden, The Netherlands) and the expression vector pMEX.SakSTAR (Schlott et al, 1994) as template. After a denaturation step of 3 min at 367 K, the fragment was ampli®ed by cycling 30 times (1 s at 367 K, 1 s at 323 K, 10 s at 345 K) followed by a ®nal elongation step of 2 min at 345 K, using the 5 H -primer LY272 and the 3 H -primer 818D.…”

The thermostability of natural variants of bacterial plasminogen‐activator staphylokinase

“…Wild-type staphylokinase has sequence G W E L L S P R ; [R36G, R43Hlstaphylokinase has sequence GKGNELLSPH; [G34S, R36G, R34Hlstaphyloki-nase has sequence SKGNELLSPH (positions 34, 36 and 43 are underlined). These staphylokinase variants are stable at ambient temperature [lo, 131 but have different sensitivities to thermal inactivation [13].In the present study, we have measured the effects of temperature and concentration on the structural and functional properties of these variants and the recovery of activity after renaturation. …”

“…Wild-type staphylokinase has sequence G W E L L S P R ; [R36G, R43Hlstaphylokinase has sequence GKGNELLSPH; [G34S, R36G, R34Hlstaphyloki-nase has sequence SKGNELLSPH (positions 34, 36 and 43 are underlined). These staphylokinase variants are stable at ambient temperature [lo, 131 but have different sensitivities to thermal inactivation [13].…”

“…The Escherichia coli strain TG1, used as the host for production and purification of the staphylokinase proteins, has been described elsewhere [15]. The procedures for expression and purification of the wild-type staphylokinase, [G34S, R36G, R43Hlstaphylokinase and [R36G, R43Hlstaphylokinase proteins have been described elsewhere [13].…”

“…The parameters determining thermostability are not well defined and approaches to increase the stability of proteins are lacking [l]. The structural basis of the thermostability of proteins with known three-dimensional structure may be investigated either by specific amino acid replacements via site-directed mutagenesis [2-61, [13].…”