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Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study
Maryam Rafipour , Malihe Keramati , Mohammad Mehdi Aslani , Arash Arashkia , Farzin Roohvand *
Abstract:  
Background: Streptokinase (SK), a heterogeneous plasminogen (PG) activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SKg). Based on the variable region of SKβ, GAS-SKs (ska) are clustered as SK1 and SK2 (including SK2a/SK2b), which show low and high fibrinogen (FG)-dependent PG activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SKs (skcg) variants display properties similar to SK1. Herein, by SKβ exchange between GGS (G88) and GAS-SK2a (STAB902) variants, the potential roles of SK domains in amidolytic/proteolytic activity and FG-bound-PG activation are represented. Methods: Two parental SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKβ-exchanged constructs (SKC1: αG88STABG88 and SKC2; αSTABG88STAB) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SKs were expressed in E. coli and purified by nickel-nitriloacetic acid chromatography. PA potencies of SKs were measured by colorimetric assay. Results: SDS-PAGE and Western-blot analyses confirmed the proper expression of 47-kDa SKs. Analyses indicated that the catalytic efficiency (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SKβ, respectively. The increase of FG-bound-PG activation for SKSTAB902/SKC1 containing SK2aβ was around six times, whereas for SKG88/SKC2 containing skcgβ, it was four times. Conclusion: Although SKβ has noticeable contribution in FG-bound-PG activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK.
Keywords: Plasminogen, Streptokinase, Thrombolytic therapy
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Type of Study: Full Length | Subject: Related Fields


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