Evaluation of the α2-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 (SK2aG88 and SK2bALAB49, respectively) was constructed to evaluate the role of SK-domains (α, β, γ) in α2-antipalsmin-resistance variations of SK. Materials and Methods: In this experimental study, PCR-amplified genes of streptococci (skg, ska) were cloned into pET26b vector to produce pET26-SKG88 and pET26-SKALAB49. For domain exchange, the amplicon containing β and g domains of SK2bALAB49 was replaced for that of the SK2aG88 within pET-SK2aG88 (pET26-SKch; α2aG88β2bALABγ2bALAB). 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 α2-antipalsmin-resistance was measured by S2251 colorimetric assay for plasminogen activation. Findings: SDS-PAGE and western blotting results indicated the expression of proteins with the size of 47kD. At the highest concentration of α2-antiplasmin, SK2aG88 remained 80% active, whereas the SK2bALAB49 and SKch retained 55% of their activity. Conclusion: SKch shows similar activity reduction, indicating the minor role of the α domain compared to β and g domains for α2-antipalsmin-resistance.

Evaluation of the α2-Antipalsmin-Resistance of a Domain Exchanged-Chimeric Streptokinase from Two Streptococci Groups Keywords:

Streptokinase , Domain exchange , Chimera , α2-Antiplasmin , استرپتوکیناز , تبادل دومین , کایمر , آلفا دو آنتی پلاسمین