Rafipour, M., Keramati, M., Aslani, M.M., Arashkia, A., Roohvand, F. (2019). Evaluation of the α2-Antipalsmin-Resistance of a Domain Exchanged-Chimeric Streptokinase from Two Streptococci Groups. , 22(2), 77-84.
M. Rafipour; M. Keramati; M.M. Aslani; A. Arashkia; F. Roohvand. "Evaluation of the α2-Antipalsmin-Resistance of a Domain Exchanged-Chimeric Streptokinase from Two Streptococci Groups". , 22, 2, 2019, 77-84.
Rafipour, M., Keramati, M., Aslani, M.M., Arashkia, A., Roohvand, F. (2019). 'Evaluation of the α2-Antipalsmin-Resistance of a Domain Exchanged-Chimeric Streptokinase from Two Streptococci Groups', , 22(2), pp. 77-84.
Rafipour, M., Keramati, M., Aslani, M.M., Arashkia, A., Roohvand, F. Evaluation of the α2-Antipalsmin-Resistance of a Domain Exchanged-Chimeric Streptokinase from Two Streptococci Groups. , 2019; 22(2): 77-84.

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

Pathobiology Reserach
Article 3, Volume 22, Issue 2, 2019, Pages 77-84    PDF (847.2 K)
Document Type: Original Research
Authors
M. Rafipour1; M. Keramati2; M.M. Aslani3; A. Arashkia4; F. Roohvand* 4
1Virology & Microbiology Department, Pasteur Institute of Iran, Tehran, Iran
2Nano Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran
3Microbiology Department, Pasteur Institute of Iran, Tehran, Iran
4Virology Department, Pasteur Institute of Iran, Tehran, Iran
Abstract
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.
Keywords
Streptokinase; Domain exchange; Chimera; α2-Antiplasmin
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