EVALUATION OF RECOMBINANT RETEPLASE PROTEIN PRODUCTION FUSED WITH PELB AND TORA SIGNAL PEPTIDES

• Main Author: Fitriyani, Alfi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/85336
• Institution: Institut Teknologi Bandung

Reteplase, a recombinant protein derivative of tissue plasminogen activator (tPA), is one of the key fibrinolytic enzymes used in thrombolytic therapy for the treatment of acute myocardial infarction (AMI). However, the production of reteplase in Escherichia coli often results in the formation of insoluble protein aggregates known as inclusion bodies. Additionally, reteplase contains several disulfide bonds that are critical for its stability and biological activity, which often pose challenges in achieving correct disulfide bond formation when produced in the cytoplasm of E. coli. This study aims to investigate the effects of the PelB and TorA signal peptides on the solubility and activity of reteplase, and to determine the optimal production conditions for reteplase fused with these signal peptides. In this research, genes encoding the pelB and torA signal peptides were cloned into the expression vector pET24b_ret to construct pET24b_ret_pelB and pET24b_ret_torA, and transformed into E. coli BL21(DE3). Protein expression was induced by IPTG at concentrations of 0.5 and 1.0 mM at 20°C and 37°C to determine the optimal conditions for producing soluble reteplase. The activity of the soluble reteplase was tested using a radial caseinolysis method in the presence of blood plasma as a source of plasminogen. The results demonstrated that optimal reteplase expression was achieved at 37°C with 0.5 mM IPTG. The activity assays indicated that reteplase fused with the PelB signal peptide resulted in soluble reteplase with activity as evidenced by the radial caseinolysis test, whereas reteplase fused with the TorA signal peptide did not exhibit activity.