Isfahan University of Medical Sciences

Science Communicator Platform

Stay connected! Follow us on X network (Twitter):
Share this content! On (X network) By
Molecular Cloning and Expression of Reteplase in Escherichia Coli Using Tac Promoter



Sadeghi HMM1 ; Aghabdollahian S2 ; Rabbani M1 ; Ghaedi K3
Authors
Show Affiliations
Authors Affiliations
  1. 1. Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
  2. 2. Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran
  3. 3. Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran

Source: Journal of Isfahan Medical School Published:2013

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.
Related Docs
View other Related Docs
1. Reteplase: Structure, Function, and Production, Advanced Biomedical Research (2019)
2. Expression of the Recombinant Plasminogen Activator (Reteplase) by a Non-Lytic Insect Cell Expression System, Research in Pharmaceutical Sciences (2013)
3. Cloning and Expression of Functional Reteplase in Escherichia Coli Top10, Avicenna Journal of Medical Biotechnology (2013)
4. Optimization of the Expression of Reteplase in Escherichia Coli, Research in Pharmaceutical Sciences (2011)
5. Bacterial Staphylokinase As a Promising Third-Generation Drug in the Treatment for Vascular Occlusion, Molecular Biology Reports (2020)
6. Comparison of the Cytoplasmic and Periplasmic Production of Reteplase in Escherichia Coli, Preparative Biochemistry and Biotechnology (2013)
7. Improvement of Soluble Production of Reteplase in Escherichia Coli by Optimization of Chemical Chaperones in Lysis Buffer, Advanced Biomedical Research (2019)
8. Optimization of the Expression of Reteplase in Escherichia Coli Top10 Using Arabinose Promoter, Jundishapur Journal of Natural Pharmaceutical Products (2015)
Experts (# of related papers)
View all Related Experts
Hamid Mirmohammad Sadeghi (10)
Mohammad Rabbani (7)
Other Related Docs
9. Optimization of the Production and Purification of Taq Polymerase Enzyme Containing N666e Mutation in Its O-Helix Region, Journal of Isfahan Medical School (2012)
10. Construction of Tenecteplase Coding Sequence by a Rapid Megaprimer Pcr Method and Its Cloning in a Mammary Gland Tissue Specific Expression Vector, Research in Pharmaceutical Sciences (2009)
11. Cloning and Expression of a Human Tissue Plasminogen Activator Variant: K2s in Escherichia Coli, Pakistan Journal of Biological Sciences (2007)
12. Investigation of Supercharging As a Strategy to Enhance the Solubility and Plasminogen Cleavage Activity of Reteplase, Iranian Journal of Biotechnology (2020)