Expression and Activity Evaluation of Reteplase in Escherichia coli TOP10

Reteplase is a part of tissue plasminogen activator [t-PA] used for the removal of thrombi in blood vessels. In the present study we express the Reteplase gene in Escherichia coli TOP10 and then its thrombolytic activity was measured. The recombinant plasmid pBADgIIIA was transformed into the competent Escherichia coli TOP10 and then transformed bacteria was seeded into bioreactor containing 1.5 L LB medium and induced by 0.02% L-Arabinoseat 37 degreeC, pH 7, and 180 rpm until OD 600 of 0.6 was reached. Samples were analyzed by SDS-PAGE and western blotting and the expression of Reteplase was examined. Finally the activity of this recombinant protein was evaluated using Chromogenic Activity Assay Kit. The presence of Reteplase in transformed Escherichia coli TOP10 was examined by western blotting which revealed that the target protein in form inclusion body was expressed as a unique band at 39 and the refolded Reteplase was 66 KDa. The amount of protein produced was 90.5 microg/mL and its activity was determined as 0.8 units. In this study, the expression of Reteplase in Escherichia coli TOP10 was scaled up under optimum condition. Furthermore we earned Reteplase with partially suitable thrombolytic activity

Molecular cloning and expression of rat micro -opioid receptor in Escherichia coli [BL21]

The micro [mu] opioid receptors, which mediate the effects of morphine, are widely distributed in brain. The purpose of this study was to design a simple expression system for rat micro -receptor in Escherichia coli [BL21]. In this laboratory study, rat micro -receptor cDNA was isolated from pcDNA3 vector using Xba1 and Hind3 restriction enzymes. pET-15b was digested by Nco1 restriction enzyme. micro -receptor cDNA and pET-15b formed a recombinant DNA that was transformed to Escherichia coli [BL21]. The insert presence was proved by Rsa1 restriction enzyme and the induction of its expression was performed using IPTG. Finally, the presence of desired insert was confirmed using RSA1, and the colonies that had correct orientation in gene containing plasmid were used for further studies. On the SDS-page gel electrophoresis, a 33 kDa band was observed when IPTG was used at 0.5 and 1 mM concentrations, that is equal to calculated molecular weight of rat micro -receptor. At the end of this project, the expression of rat micro -receptor by IPTG induction was successfully performed