Evaluation of the α۲-Antipalsmin-Resistance of a Domain Exchanged-Chimeric Streptokinase from Two Streptococci Groups

Aims: The chimeric domain-exchanged streptokinase (SKch) between two sk genes from groups G and A streptococci (SK۲aG۸۸ and SK۲bALAB۴۹, respectively) was constructed to evaluate the role of SK-domains (α, β, γ) in α۲-antipalsmin-resistance variations of SK. Materials and Methods: In this experimental study, PCR-amplified genes of streptococci (skg, ska) were cloned into pET۲۶b vector to produce pET۲۶-SKG۸۸ and pET۲۶-SKALAB۴۹. For domain exchange, the amplicon containing β and g domains of SK۲bALAB۴۹ was replaced for that of the SK۲aG۸۸ within pET-SK۲aG۸۸ (pET۲۶-SKch; α۲aG۸۸β۲bALABγ۲bALAB). All constructs were confirmed by restriction analyses/agarose-gel electrophoresis and DNA sequencing, transformed into E.coli Rosetta, and induced by IPTG for protein expression. Proteins were purified by Ni-NTA chromatography, quantified by Bradford method, and analyzed by SDS-PAGE/Western blotting assays. The α۲-antipalsmin-resistance was measured by S۲۲۵۱ colorimetric assay for plasminogen activation. Findings: SDS-PAGE and western blotting results indicated the expression of proteins with the size of ۴۷kD. At the highest concentration of α۲-antiplasmin, SK۲aG۸۸ remained ۸۰% active, whereas the SK۲bALAB۴۹ and SKch retained ۵۵% of their activity. Conclusion: SKch shows similar activity reduction, indicating the minor role of the α domain compared to β and g domains for α۲-antipalsmin-resistance.

کلیدواژه ها:

Streptokinase ، Domain exchange ، Chimera ، α۲-Antiplasmin ، استرپتوکیناز ، تبادل دومین ، کایمر ، آلفا دو آنتی پلاسمین