Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.
A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.
Chen
H M
HM
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei.
Luo
S L
SL
Chen
K T
KT
Lii
C K
CK
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D008670:Metals; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D008670:Metals; D011994:Recombinant Proteins; D005982:Glutathione Transferase