Tarbiat Modares University Pathobiology Research 2538-3000 22 2 2019 03 01 Evaluation of the α2-Antipalsmin-Resistance of a Domain Exchanged-Chimeric Streptokinase from Two Streptococci Groups بررسی مقاومت استرپتوکیناز کایمری حاصل از جابه‌جایی دومین‌ها میان دو گروه استرپتوکوکی نسبت به آلفا دو آنتی‌پلاسمین 77 84 19345 EN M. Rafipour Virology & Microbiology Department, Pasteur Institute of Iran, Tehran, Iran M. Keramati Nano Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran M.M. Aslani Microbiology Department, Pasteur Institute of Iran, Tehran, Iran A. Arashkia Virology Department, Pasteur Institute of Iran, Tehran, Iran F. Roohvand Virology Department, Pasteur Institute of Iran, Tehran, Iran Journal Article <strong>Aims:</strong> The chimeric domain-exchanged streptokinase (SKch) between two <em>sk</em> genes from groups G and A streptococci (SK2aG88 and SK2bALAB49, respectively) was constructed to evaluate the role of SK-domains (α<sub>, </sub>β<sub>, </sub>γ) in α<sub>2</sub>-antipalsmin-resistance variations of SK.<br> <strong>Materials and Methods:</strong> In this experimental study, PCR-amplified genes of streptococci (<em>skg, ska</em>) 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; α<sub>2aG88</sub>β<sub>2bALAB</sub>γ<sub>2bALAB</sub>). All constructs were confirmed by restriction analyses/agarose-gel electrophoresis and DNA sequencing, transformed into <em>E.coli</em> 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 α<sub>2</sub>-antipalsmin-resistance was measured by S2251 colorimetric assay for plasminogen activation.<br> <strong>Findings:</strong> SDS-PAGE and western blotting results indicated the expression of proteins with the size of 47kD. At the highest concentration of α<sub>2</sub>-antiplasmin, SK2aG88 remained 80% active, whereas the SK2bALAB49 and SKch retained 55% of their activity.<br> <strong>Conclusion: </strong>SKch shows similar activity reduction, indicating the minor role of the α domain compared to β and g domains for α<sub>2</sub>-antipalsmin-resistance. Streptokinase Domain exchange Chimera α2-Antiplasmin https://mjms.modares.ac.ir/article_19345_437fcf36c72ca2d5d6179539a1eee748.pdf