[Prokaryotic expression, purification and identification of recombinant staphylokinase-HC fusion protein].

Aim: To construct the prokaryotic expression plasmid pEGX-6P-1-SAK-HC, express it in E.coli, and identify its biological activity.

Methods: The fusion gene (SAK-HC) was obtained by overlap-extension PCR and then inserted into prokaryotic soluble pEGX-6P-1 vector with GST tag to construct expression plasmid (pEGX-6P-1-SAK-HC). GST-SAK-HC was expressed by E.coli B834 (DE3) under the induction of IPTG and purified by Glutathion-Sepharose 4B (GST) affinity chromatography and negative-ion exchange column (DEAE) chromatography. PreScission protease was used to remove the GST tag. The purity of the fusion protein was analyzed by SDS-PAGE and the fibrinolytic activity of SAK-HC in vitro was characterized by soluble fibrin plate method.

Results: PCR, sequencing and restriction enzyme digestion analysis demonstrated that the recombinant plasmid was constructed successfully. The fusion protein was expressed in E.coli B834 (DE3), M(r); being 36 000 as shown by SDS-PAGE. After purified by GST affinity and DEAE chromatography, SAK-HC fusion protein of high purity was obtained from the cell supernantants. In vitro experiments showed that the fibrinolytic activity of the recombinant SAK-HC was about 9.4×10();4 IU/mg.

Conclusion: The SAK-HC fusion protein we obtained was successfully expressed in E.coli and exhibited a fibrinolytic activity as high as the urokinase standard, which offers a base for the identification of immunogenicity of the fusion protein.