High-level expression and secretion of streptokinase in escherichia coli

Abstract

The high-level expression and secretion vector for streptokinase has been constructed. The streptokinase structural gene(skc) was amplified with polymerase chain reaction(PCR) and subcloned into the secretion vector for E. coli, pSTO-E, which contains the strong $T_7$ promotor, ompA signal sequence and $T_7$ terminator. The resulting streptokinase secretion vector pTOS306 was not stably maintained in E. coli BL21(DE3) strain which contains the $T_7$ RNA polymerase gene. Therefore, the ompA signal sequence and streptokinase structural gene was cleaved and subcloned into the another expression vector pKK223-3 which contains the strong tac promoter and rrnB transcription terminator. This newly constructed vector was named as pKOS 307. To test for streptokinase secretion on agar plate. E. coli JM109 carrying pKOS307 was grown on LB-ampicillin plate and the skim milk/human plasminogen overlay test was carried out after IPTG induction. About 1hr after overlay, the clear zone was shown around the colony. When the streptokinase activities were compared in LB-ampicillin media between E. coli JM109 carrying pKOS307 and E. coli JM109 carrying pKS601 without ompA signal sequence, E. coli JM109 carrying pKS601 without ompA signal sequence, E. coli JM109 carrying pKOS307 produced about four times more streptokinases that E. coli JM109 carrying pKS601. After induction of streptokinase expression in E. cole JM109 carrying pKOS307, about 47 kd and 44kd proteins were found to be newly accumulated in cytoplasmic, periplasmic and extracellular fractions and they were identified as streptokinases by western blotting. The growth of E. coli JM109 carrying pKOS307 was not interfered by the expression of streptokinase. When the streptokinase prooduction with growth phase was observed, periplasmic fraction showed the peak production 1hr after induction and about 80\% of total streptokinases were secreted into periplasmic and extracellular fractions. In this case, the streptokinase activ...