Expression, purification and functional analysis of hexahistidine-tagged human glutathione S-transferase P1-1 and its cysteinyl mutants.
The bacterial expression and purification of human glutathione S-transferase P1-1(hGST P1-1), as a hexahistidine-tagged polypeptide was performed. Site-directed mutagenesis was used to construct mutants in which alanine replaced two (C47A/C101A), three (C14A/C47A/C101A) or all four (C14A/C47A/ C101A/C169A) cysteine residues using the plasmid for the wild type enzyme. Analysis of their catalytic activities and kinetic parameters suggested that cysteins are not essential for the catalytic activity but may contribute to some extent to the catalytic efficiency. Moreover, on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, hexahistidine-tagged hGST P1-1 (His(6)-hGST P1-1) treated with 1 mM H(2)O(2) showed at least three extra bands, in addition to the native His(6)-hGST P1-1 subunit band. These extra bands were not detected in the cysteinyl mutants. Thus, it indicated that disulfide bonds were formed mainly within subunits between cysteine residues, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed.
Wu
Yifan
Y
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, PR China.
Shen
Jiayin
J
Yin
Zhimin
Z
D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D005982:Glutathione Transferase; D003545:Cysteine