Maryam Rafipour - Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran
Farzin Roohvand - Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran
Malihe Keramati - Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran
Arash Arashkia - Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran

Streptokinase (SK), a bacterial protein produced by streptococci, is one of the main treatments for thromboembolic disorders. However due to lack of fibrin specificity, its application is associated with the risk of hemorrhage. SKs from different strains exhibit different functional activities (plasminogen activation) and specificities which are attributed to the central domains (β-domain) of SK. In this study, chimeric SKs with predicted enhanced activity and improved specificitywere constructed by exchange of the central domains of SKs. Two SK molecules with significant different activities and specificities from two distinct streptococci strains were chosen for central domain exchange. Chimeric SKs were constructed by cross-ligation of BstEII/ BsiWI DNA segment encodingthe central fragment of sk genes. The assembled sk genes were cloned in pQE30 vector. The constructs were expressed inE.coli M15 host and induced by IPTG. The 6xHis-tagged recombinant SK proteins were purified by NI–NTAchromatography, analyzed by SDS-PAGE and Western blotting. For determination of specific activity, the functional activities were evaluated with chromogenic and clot lysis assay in presence and absence of fibrin. Gel electrophoresis confirmed digestion of the 320nt central domain. The constructs were confirmed by PCR, restrictionenzyme analysis and sequencing. The 47-kDa SKs molecules were expressed, purified and the biological activity of chimeric proteins were assayed. Application of SK is limited by shortcomings such as fibrin-non specificity. To address this concern, SK chimeras were constructed by exchange of central domains which seem to play a major role in SK activity and fibrin specificity

Streptokinase, thrombolytic agents, chimera