Optimizing of Nutrients for High Level Expression of Recombinant Streptokinase Using pET32a Expression System

摘要

ABSTRACTStreptokinase (SK) is being increasingly used in the treatment of acute myocardial infarction and ischemic stroke. The feeding control method using substrate balance equations is vital to maintain the preferred specific growth rate for the high-level expression of recombinant proteins. In this study, initial experiments on chemical and temperature inducible expression systems were carried out to identify appropriate expression conditions to improve production of recombinant Streptokinase. Streptokinase gene of group C Streptococci was cloned into prokaryotic expression vector pET32a. Gene expression was optimized by changing levels of glucose, tryptone, and MgSO4 in the media and temperature-inducible expression system and recombinant protein was confirmed by western blot analysis with anti streptokinase sera of immunized rats. Among the various expression systems used, the quantity of recombinant streptokinase produced in the medium containing 2.4% glucose was more than two-fold compared to the medium containing 0.2% glucose. In addition, temperature induction system (37°C) was found to result in higher productivities compared to room temperature. In conclusion we have demonstrated that significant improvement in the streptokinase yield can be obtained by modifying the media and feeding of substrate. These results indicate that efficient process control strategy is important for the mass production of streptokinase.