Molecular Cloning and Expression of Reteplase in Escherichia Coli Using tac Promoter

Document Type : Original Article (s)

Authors

1 Pharm D Student, Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran

2 Professor, Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

3 Assistant Professor, Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran

Abstract

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
Journal of Isfahan Medical School
Volume 30, Issue 213 - Serial Number 213
September and October 2013
Pages 1911-1918

Files

History

  • Receive Date: 18 August 2012
  • Revise Date:
  • Accept Date: 06 April 2022

Share

How to cite

Statistics