Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli

The bacterial expression and purification of human pi class glutathione S-t ransferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cD NA which codes for the protein into the expression vector pET-15b. The expr essed protein was purified by either glutathione or metal (Co2+) affinity c olumn chromatography, which produced the pure and fully active enzyme in on e step with a yield of more than 30 mg/liter culture. The activity of the p urified protein was 130 units mg(-1) from the GSH affinity column and 112 u nits mg(-1) from the Co2+ affinity column chromatography. The purity of the protein was assessed by electrospray ionization mass spectrometry and size -exclusion chromatography. It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column. Our expression system allows the expression and pur ification of active hexahistidine-tagged hGST P1-1 in high yield with no ne ed of removal of the hexahistidine tag and gives pure protein in one purifi cation step allowing further study of this enzyme. (C) 1999 Academic Press.