Molecular Cloning and Expression of Reteplase in Escherichia Coli Using tac Promoter
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...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | fas |
| Published: |
Isfahan University of Medical Sciences
2013-01-01
|
| Series: | مجله دانشکده پزشکی اصفهان |
| Online Access: | http://jims.mui.ac.ir/index.php/jims/article/view/2115 |
| Summary: | 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 |
|---|---|
| ISSN: | 1027-7595 1735-854X |