Cloning, identification and functional characterization of a pi-class glutathione-S-transferase from the freshwater mussel Cristaria plicata

Glutathione-S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione and play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). The piGST cDNA was cloned and sequenced after rapid amplification of cDNA ends (RACE) from the freshwater mussel Cristaria plicata. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzymes belonged to the pi-class and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. The Cp-piGST cDNA is 816 nucleotides (nt) in length and contained a 615 nt open reading frame (ORF) encoding 205 amino acid residues, and has 19 nt of 5' untranslated region (UTR) and a 3' UTR of 182 nt including a tailing signal (AATAAA) and a poly (A) tail. The molecular weight of the predicted piGST is 23.4 kDa, with the calculated PI being 5.2. The mRNA transcript of Cp-piGST could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of Cp-piGST in hepatopancreas and gill showed similar trend that were significantly increased after bacterial challenge compared to the control group at 12 h. Furthermore, the recombinant Cp-piGST with high enzyme activity was induced to be expressed as a soluble form by IPTG at 20 degrees C for 8 h, and then was purified by using the native Ni2+ affinity chromatography. The specific activity of the purified soluble Cp-piGST enzyme into pET30 was 2.396 mu mol/min/mg, and which into pET32 was 1.706 mu mol/min/mg. The recombinant Cp-piGST had a maximum activity at approximately pH 8.0, and its optimum temperature was 37 degrees C. The recombinant Cp-piGST enzyme activity became lower gradually with the denaturant concentration increasing. (C) 2011 Elsevier Ltd. All rights reserved.