SBIR-STTR Award

The plan is to develop and optimize a method for high level production of functional, catalytically active human cytochromes P450 using baculovirus expression in insect cells. Cytochromes P450 are the principal enzymes for the metabolism of many drugs, protoxins, and environmental pollutants. This proposed method includes enhancement of cytochrome P450 catalytic activity through the co-expression of cytochrome P450:NADPH oxidoreductase and/or cytochrome b5. Human cytochromes P4502A6, P4502E1, and P4503A4 will be used as prototype enzymes for method development. Catalytic constants, apparent Km, and turnover number will be measured for the optimized baculovirus-expressed enzymes and compared to the same enzyme isolated from human liver or produced using other heterologous expression systems. The eventual goal is to develop a complete panel of baculovirus expressed human hepatic cytochrome P450 isoforms. The commercial applications of this system would be for the study of human xenobiotic metabolism.National Institute of Environmental Health Sciences (NIEHS)

Cytochromes P450 are the principal enzymes for the metabolism of many drugs, protoxins and environmental pollutants. We propose to further develop and optimize a method for high level production of functional, catalytically active human cytochromes P450 using baculovirus-expression in insect cells. This proposed method includes enhancement of cytochrome P450 catalytic activity through the co-expression of cytochrome P450:NADPH oxidoreductase and cytochrome b5. Human cytochromes CYP2C9, CYP3A4, CYP2E1 and CYP2D6 will be used as prototype enzymes for method development. Catalytic constants, apparent Km and turnover number, will be measured for the optimized baculovirus-expressed enzymes and compared to the same enzyme isolated from human liver or produced using other heterologous expression systems. Our eventual goal is to develop a complete panel of baculovirus-expressed human cytochrome P450 isoforms. The commercial applications of this system would be for the study of human xenobiotic metabolism.Proposed commercial application:Production of large quantities of catalytically active enzyme to allow structural determination of metabolites, study metabolism of substrates with low turnover, determine P450 form specific metabolism and physical studies of ligand/substrate interaction with specific cytochrome P450 enzymes.Thesaurus termsNAD(P)H oxidoreductase, chimeric protein, cytochrome P450, cytochrome b5 reductase, enzyme activity, gene expression, tissue /cell culture drug design /synthesis /production, isozyme, metabolism, method development, microsome, protein engineering, recombinant protein Baculoviridae, Insecta, cell line, molecular cloning, transfectionNational Institute of Environmental Health Sciences (NIEHS)