BACULOVIRUS-MEDIATED HIGH-LEVEL EXPRESSION OF A HUMAN THIOPURINE METHYL TRANSFERASE

We have expressed the human thiopurine methyltransferase cDNA in a bac ulovirus vector in Sf21 (Spodoptera frugiperda) cells. This system exp resses the enzyme at levels such that the thiopurine methyltransferase enzyme may be readily visualised by Coomassie blue stained sodium dod ecyl sulphate-polyacrylamide gel electrophoresis. The expressed enzyme catalysed the methylation of 6-mercaptopurine with an apparent Km of 892 mu M, similar to that observed in human liver cytosol ie. 657 mu M however, the Vmax was 13,500 pmole/mg/min, which is approximately 400 times higher than the Vmax observed in human liver cytosol ie. 33 pmo le/mg/min. The thiopurine methyltransferase inhibitors 6-thioxanthine, p-methoxybenzoic acid and 3,5-dimethoxy benzoic acid were found to be potent inhibitors of the expressed enzyme.