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-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b. The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture. The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) 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 purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme.
Chang
M
M
Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois 60612-7231, USA.
Bolton
J L
JL
Blond
S Y
SY
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013058:Mass Spectrometry; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D007527:Isoenzymes; D011994:Recombinant Proteins; D006639:Histidine; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase