Expression of calf prochymosin gene in Escherichia coli

The expression plasmid pTaAC containing Tac promoter and calf prochymosin B gene was constructed and transformed into E.coli JM105. Addition of 0.1 mM IPTG into the culture at logarithmic phase induced the production of prochymosin markedly. The expression of prochymosin gene was regulated by temperature in addition to the inducer IPTG. At 30 degrees C in the presence of IPTG prochymosin was barely detected, whereas at 42 degrees C in the absence of IPTG a relatively high level of prochymosin was found. The expressed protein was estimated to be 12-19% of total cell proteins by electrophoresis analysis or 80-100 mg/l by ELISA. The yield of active chymosin was 14-20 mg/l after denaturation, renaturation and activation.