The genes(gsh\|I,gsh\|II)for γ\|glutamyl\|cysteine synthetase(GSH\|I) and glutathione synthetase(GSH\|II)from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively.The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc\|gsh. E.coli BL21 was transformed by pTrc\|gsh for expression of the related enzymes.Analysis of SDS\|PAGE showed that the expected products were expressed. E.coli BL21(pTrc\|gsh) were incubated at 37℃ and pH 7.2 to OD 550 =0 5.The conditions were then switched to 34℃ and pH6.7 after the addition of 0.1mmol/L IPTG.The expressed products were up to 25% of the total protein of the bacteria.Acetone\|treated cells of the engineered strain could synthesize GSH efficiently.
