Molecular cloning of Reteplase and its expression in E. coli using tac promoter
نویسندگان
1 Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran
4 Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, Iran
doi
چکیده
Background and Aims: This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression.Materials and Methods: Reteplase cDNA was amplified by polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE.Results: Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG.Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli.
