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The influence of the enzyme source such as phospholipid and expression ratio of NADPHcytochrome P450 reductase on midazolam 1'-hydroxylation activity of cytochrome P450 3A4 was investigated in reconstituted systems and in membranes using bicistronic expressions. Preferable ratio of P450 reductase over human P450 3A4 would be a determinant factor for the drug metabolism studies.