Site-Directed Mutation, Cloning and Expression of Streptokinase for Producing a New Suitable Molecule for PEGylation

Abstract

Abstract Background: Streptokinase is one of the most common and cost effective fibrinolytic drugs for treatment of heart attacks and vein thrombosis. Unlike many advantages over other thrombolytic drugs, administration of streptokinase can produce some complications such as immunologic reactions, hemorrhage and incomplete treatment due to relative short half life. Pegylation is one of the most common methods for improving of these shortcomings. Materials and Methods: In this study, designing a proper candidate for specific pegylation with cysteine was done by means of SPDBviewer software. After a meaning ful mutation by SOEing PCR method, mutated (sk45cys) and intact SK (ski) genes were cloned in pET26-b vector and the structures were transformed in E.coli. Clones, Afrer growing, were expressed by IpTG and exptression of proteins was confirmed by SDS-PAGE and western blotting. The proteins were purified by affinity chromatography with NiNTA columns and amidolytic activity of purified proteins was assayed using chromogenic method and different concentrations of S2251 substrate. Results: Results of activity assays showed that amidolytic activity of SK45cys had about 10% increase in comparison to Ski, after 30 minutes of complex formation with plasminogen. Conclusion: Generally, it was concluded that, considering cys45 as a superficial aminoacid and also relative increase of activity, SK45cys can be considered a suitable protein for specific pegylation.

Keywords

• Activity assay
• Mutation
• Pegylation
• Streptokinase