Cloning, purification and biochemical characterization of two glutathione S-transferase isoforms from Rutilus frisii kutum

Glutathione S-transferases are an important multifunctional family of intracellular enzymes that their detoxification function has been reported in fishes since 1970, but no studies have been conducted on Rutilus frisii kutum GSTs yet. In the present study, RkGSTA and RkGSTM encoding genes were cloned and sequenced and their nucleotide sequences were submitted to NCBI GenBank. In order to reduce the expression challenges of recombinant proteins including low solubility, low yield and insufficient purity issues in E. coli, the pKJE7 chaperone plasmid was used to increase the recovery of expressed proteins in the soluble fractions. Best expression clone was selected for purification by Ni-NTA affinity chromatography. The three-dimensional structural models were constructed by I-TASSER. The optimum temperature of purified RkGSTA and RkGSTM was 35 and 30 degrees C, with optimum activity at pH 9.0 and 8.5, respectively. The thermostability and pH stability results indicated that RkGSTA is more heat-tolerant than RkGSTM though both of them retained more than 80% of their activities at pH 6.5 to 9.0. Overall, this study represents a comprehensive perspective on the structural and biochemical aspects of this enzyme that would be even used in further researches such as drug design studies in order to eliminate toxicant compounds from the body and environment of fishes to protect them against undesired harmful damages.