Isfahan University of Medical Sciences Journal of Isfahan Medical School 1027-7595 30 213 2012 12 21 Molecular Cloning and Expression of Reteplase in Escherichia Coli Using tac Promoter Molecular Cloning and Expression of Reteplase in Escherichia Coli Using tac Promoter 1911 1918 13938 FA Safieh Aghabdollahian Pharm D Student, Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran Mohammad Rabbani Professor, Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran Kamran Ghaedi Assistant Professor, Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran Hamid Mir Mohammad Sadeghi Professor, Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran Journal Article 2012 08 18 Background: This study was aimed to clone and express the reteplase complementary deoxyribonucleic acid (cDNA), a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in escherichia coli using tac promoter. Methods: Reteplase cDNA was amplified using polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57 plasmid. The cloned DNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion and determination of the nucleotide sequence. By using 0.2, 0.5, and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), reteplase was induced in escherichia coli TOP10 cells and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Findings: Electrophoresis of PCR product as well as double digested recombinant pTZ57 plasmid showed a 1068 base pair band of reteplase. SDS-PAGE analysis showed a 60 kilodalton band of protein product induced by IPTG. Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector was used for the optimization of the expression of reteplase in escherichia coli. Keywords: Reteplase, Tissue plasminogen activator, Genetic promoter regions, Molecular cloning, Escherichia coli Background: This study was aimed to clone and express the reteplase complementary deoxyribonucleic acid (cDNA), a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in escherichia coli using tac promoter. Methods: Reteplase cDNA was amplified using polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57 plasmid. The cloned DNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion and determination of the nucleotide sequence. By using 0.2, 0.5, and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), reteplase was induced in escherichia coli TOP10 cells and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Findings: Electrophoresis of PCR product as well as double digested recombinant pTZ57 plasmid showed a 1068 base pair band of reteplase. SDS-PAGE analysis showed a 60 kilodalton band of protein product induced by IPTG. Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector was used for the optimization of the expression of reteplase in escherichia coli. Keywords: Reteplase, Tissue plasminogen activator, Genetic promoter regions, Molecular cloning, Escherichia coli https://jims.mui.ac.ir/article_13938_cf3b784c8593890043b17e24088125d4.pdf