EVALUATION OF SOLUBLE RECOMBINANT RETEPLASE PRODUCTION IN Escherichia coli USING T7 AND gadA PROMOTER

• Main Author: Imelda, Widya
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/40485
• Institution: Institut Teknologi Bandung

Coronary heart disease and ischemic stroke are the leading causes of death in the world including Indonesia. These diseases can be treated by thrombolytic agents such as reteplase. Recombinant reteplase is a therapeutic protein with plasminogen activator activity that approved by FDA and EMA. However, reteplase has never been produced and marketed in Indonesia. The problem of reteplase production is 9 disulfide bonds which make reteplase difficult to produce in soluble form using E. coli system. The aim of this study was to evaluate the strategy of increasing the solubility of recombinant reteplase through production by using weak promoter (gadA) and strong promoter (T7). The production with gadA promoter was done using pMCD_ret plasmid which was constructed in this study. Overproduction performed using ¼LB-Succinate medium for 11.5 hours. Evaluation of recombinant reteplase production was done using SDS-PAGE method. Production of reteplase with T7 promoter was performed using plasmid pET24b_ret on LB media at 37°C and 0.1 mM IPTG as inducer. Reteplase produced by T7 promoter was in inclusion body, which then solubilized with 8 M urea and the refolding was done by reducing the concentration of urea gradually. The solubilized reteplase was analyzed by SDS-PAGE and Western Blot. Purification of recombinant reteplase was done using ion exchange and gel filtration chromatography. The activity assay was performed using casein zymography and caseinolysis-radial. The pMCD_ret plasmid was successfully constructed, but has not been able to produce recombinant reteplase with the conditions that used in this study. Production, solubilization, and refolding of reteplase from the inclusion body were successfully done, but the purification has not been succeeded yet. The renaturated reteplase has not shown activity as plasminogen activator. The conclusion of this study is construction of pMCD_ret for reteplase production with weak promoter has been done successfully. Optimal condition for produce reteplase in soluble and active form using gadA dan T7 promoter have not been found yet.