Production and characterization of glutathione-S-transferase fused with a poly-histidine tag

Schistosoma japonicum glutathione-S-transferase (SjGST) was genetically eng ineered with a poly-histidine tag at the C-terminus and highly expressed in Escherichia coli. Both SjGST and the tagged protein, SjCST/His, were purif ied with glutathione Sepharose 4B gels and subsequently studied for their a ctivities, antibody-binding abilities, and metal affinities. The production level of active SjGST/His was higher than that of SjGST. Both proteins had similar specific catalytic activities and binding abilities with anti-SjGS T antibody, while the antibody against poly-histidine recognized only SjGST /His. Proteolytic degradation was occasionally observed in aged dialyzed Sj CST/His preparation. Under a native condition, the Co2+-chelated TANOL gel (Co-TANOL) had a better binding specificity to the tagged protein than did the Ni2+-chelated nitriloacetic acid (Ni-NTA) agarose gel. However, the bin ding capacity of the Ni-NTA gel for SjGST/His was 2-fold higher than that o f the Co-TANOL one. To increase the native binding specificity of the Ni-NT A gel, 20 mM imidazole had to be added to the washing solution. In a denatu red state, both gels could only capture SjCST/His, and the binding capacity of the Ni-NTA gel was nearly 2-fold higher than that of the Co-TANOL gel. The binding association constants of both gels with SjGST/His did not diffe r greatly under either condition. The study demonstrated that the C-termina l addition of the poly-histidine tag to SjGST increased the metal affinity of the enzyme to the Co-TANOL gel under both native and denaturing conditio ns and to the Ni-NTA gel under denaturing conditions, whereas the enzymatic activity and antibody-binding ability were not affected. (C) 2000 Elsevier Science Inc. All rights reserved.