MOLECULAR-CLONING, EXPRESSION AND CHARACTERIZATION OF A RECOMBINANT GLUTATHIONE-S-TRANSFERASE FROM ECHINOCOCCUS-MULTILOCULARIS

We report the identification and characterization of the first cestode glutathione S-transferase (GST) cDNA sequence. A fragment of an Echin ococcus multilocularis glutathione S-transferase cDNA was isolated by the polymerase chain reaction. Subsequently, a Lambda zap cDNA library prepared from mRNA from protoscolices of E. multilocularis was screen ed with this PCR fragment. A complete cDNA clone was isolated and the nucleotide sequence determined. Analysis of the E. multilocularis GST- deduced amino acid sequence indicates that it is clearly related to th e mammalian mu-class GSTs. The E. multilocularis GST cDNA was expresse d in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. The 25.5-kDa enzyme subunit was purified to homogeneity using glutathione-sepharose chromatography. Ge l filtration demonstrated that this GST is enzymatically active as a h omodimer. The recombinant enzyme had conjugating activity with organic hydroperoxides and with members of the trans,trans-2,4 alkadienal and trans-2-alkenal series, which are secondary products of lipid peroxid ation.