Journal of Chromatography A, 852 (1999) 151–159 Affinity purification of Schistosoma japonicum glutathione-S- transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography a, a a b * Hsiu-Mei Chen , San-Lin Luo , Kai-Ti Chen , Chong-Kuei Lii a Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan b Department of Nutrition, Chung Shan Medical College, Taichung 402, Taiwan Abstract 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 21 were used to purify these four enzymes. All of them were purified with equal efficiency by Ni -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.  1999 Elsevier Science B.V. All rights reserved. Keywords: Affinity chromatography; Schistosoma japonicum; Immobilized metal affinity chromatography; Gluatathione-S- transferase; Enzymes 1. Introduction peutic targets in disease [2]. For example, Schis- tosoma japonicum GSTs, the major detoxification Glutathione-S-transferases (GSTs) are a family of enzymes in S. japonicum, have promising vaccine multifunctional proteins that can catalyze the nu- potential against Schistosomiasis [3,4], and their cleophilic addition of the thiol group of glutathione inhibitors are novel antischistosomal drugs [5]. The (GSH) to a variety of electrophiles or bind a range of gene of M 26 000 GST from S. japonicum (SjGST) r hydrophobic ligands [1]. They are important thera- has been cloned [3,6], highly expressed in various host cells, and successfully used as a gene fusion *Corresponding author. system for high expression and affinity purification 0021-9673 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0021-9673(99)00490-2