Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study
| Autor: | Mohammad Mehdi Aslani, Arash Arashkia, Malihe Keramati, Maryam Rafipour, Farzin Roohvand |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
XhoI
Streptokinase Clinical Biochemistry Full Length lcsh:Medicine Fibrinogen General Biochemistry Genetics and Molecular Biology 03 medical and health sciences 0302 clinical medicine Plasmid Protein Domains medicine Enzyme kinetics 0303 health sciences Fibrin Expression vector biology 030306 microbiology Chemistry lcsh:R Biochemistry (medical) Streptococcus Plasminogen Molecular biology Amides Thrombolytic therapy Kinetics 030220 oncology & carcinogenesis NdeI Proteolysis biology.protein Plasminogen activator medicine.drug |
| Zdroj: | Iranian Biomedical Journal Iranian Biomedical Journal, Vol 24, Iss 1, Pp 15-23 (2020) |
| ISSN: | 2008-823X 1028-852X |
| Popis: | Background Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SK). Based on the variable region of SKβ, GAS-SK (ska) are clustered as SK1 and SK2 (including cluster2-streptokinase (SK2a)/SK2b), which show low and high fibrinogen (FG)-dependent plasminogen (Plg) activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (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-Plg 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: αG88-βSTAB-γG88 and SKC2; αSTAB-βG88-γSTAB) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SK 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 SK. 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-Plg 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-Plg activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK. |
| Databáze: | OpenAIRE |
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