HETEROLOGOUS EXPRESSION OF RAT-LIVER MICROSOMAL GLUTATHIONE TRANSFERASE IN SIMIAN COS CELLS AND ESCHERICHIA-COLI

The cDNA coding for rat liver microsomal glutathione transferase was s ubcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). Th e activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 6 0-200 nmol/min per mg. Control and antisense-vector-treated cells disp layed very low activity (3-6 nmol/min per mg). A DNA fragment coding f or rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into E scherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL ). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the exp ression of significant amounts of enzymically active protein (4 mg/l o f culture as measured by Western blots). The recombinant protein was p urified and characterized and found to be indistinguishable from the r at liver enzyme with regard to enzymic activity, molecular mass and N- terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPT G, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to exp ress both human and rat microsomal glutathione transferase in an enzym ically active form in Escherichia coli.