Cloning, characterisation and bacterial expression of full length cDNA forthe mouse liver microsomal glutathione S-transferase

We have isolated a cDNA encoding full length microsomal glutathione S-trans ferase (MGST) from mouse liver. The cDNA was isolated by RT-PCR using prime rs designed from published cDNA sequence of rat MGST with the addition of 5 ' Nde-l and 3' HindIII sites, and cloned into bacterial expression vector p SP19T7LT. Deduced amino acid sequence (155 amino acids, calculated mol.mass 17512 Dalton) confirmed the identity of microsomal GST from mouse liver wh ich has sequence homology with that of rat and human liver MGST1. Recombina nt GST cDNA (GenBank accession # 159050) was expressed in BL21(DE3) in the presence of 1 mM IPTG at 30 degrees C. The expressed GST protein was found to be localised in the bacterial membrane as determined by measuring cataly tic activity using CDNB and cumene hydroperoxide substrates, SDS-PAGE and W estern blot analysis. We have demonstrated the cloning and expression of fu ll length cDNA for MGST from mouse liver and have characterised the functio nally active product as MGST protein. These results should facilitate studi es on the role of MGST in the regulation of chemical carcinogenesis and in the prevention of oxidative stress caused by endogenous and exogenous chemi cals.