Expression and Activity Evaluation of Reteplase in Escherichia coli TOP10

ABSTRACT

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