P450 Fusion Proteins for Drug Metabolism Studies

With the increased understanding of the role of specific cytochrome P450 isoforms in drug metabolisgrowing need for improved in vitro methodology using purified P450s. The major drawbacks to currentinterference from contaminating enzymes present in heterogenous assay systemss and difficulties in rpurified P450s with there associated electron transfer proteins. We will extend technology developedEstabrook for the production of self-sufficient catalytic units composed of P450s linked to NADPH-P4incorporate the fusion proteins into a multi-well format. The goal of the research would be to develreductase fusion proteins, 2) assay methods for absorbance-based detection of substrate metabolism bTo demonstrate the feasibility of achieving these results, Phase I specific aims will be 1) producerecombinant fusion protein of human P450 3A4 linked to human NADPH-P450 reductase; 2) compare the meof the fusion protein with that of the native enzyme system; 3) examine the extent of coupling betweand substrate metabolism with the fusion protein.