[Cloning and expression of the genes of glutathione synthetases].
The genes(gsh-I,gsh-II) for gamma-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 degrees C and pH 7.2 to OD550 = 0.5. The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/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.
Shen
L X
LX
State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry, East China University of Science & Technology, Shanghai 200237, China.
Wei
D Z
DZ
Zhao
Z F
ZF
Zhang
S L
SL
Wang
E L
EL
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005981:Glutathione Synthase / Q000096:biosynthesis / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D005981:Glutathione Synthase