Volume 22, Issue 2 (2019)
mjms 2019, 22(2): 77-84 |
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Rafipour M, Keramati M, Aslani M, Arashkia A, Roohvand F. Evaluation of the α2-Antipalsmin-Resistance of a Domain Exchanged-Chimeric Streptokinase from Two Streptococci Groups. mjms 2019; 22 (2) :77-84
URL: http://mjms.modares.ac.ir/article-30-27264-en.html
URL: http://mjms.modares.ac.ir/article-30-27264-en.html
1- Virology & Microbiology Department, Pasteur Institute of Iran, Tehran, Iran
2- Nano Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran
3- Microbiology Department, Pasteur Institute of Iran, Tehran, Iran
4- Virology Department, Pasteur Institute of Iran, Tehran, Iran
5- Virology Department, Pasteur Institute of Iran, Tehran, Iran , rfarzin@pasteur.ac.ir
2- Nano Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran
3- Microbiology Department, Pasteur Institute of Iran, Tehran, Iran
4- Virology Department, Pasteur Institute of Iran, Tehran, Iran
5- Virology Department, Pasteur Institute of Iran, Tehran, Iran , rfarzin@pasteur.ac.ir
Abstract: (6816 Views)
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.
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.
Article Type: Original Research |
Subject:
Biologic Products
Received: 2018/11/18 | Accepted: 2019/02/12
Received: 2018/11/18 | Accepted: 2019/02/12
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