Functional High Level Expression of Cytochrome P450 CYP2D6 Using Baculoviral Expression Systems

Cytochrome P-450 CYP2D6 plays a central role in the metabolism of many widely used therapeutic drugs including β-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and used to express CYP2D6 inSpodoptera frugiperda(Sf9) cells. High levels of recombinant protein have been produced using either polyhedrin or basic protein promoters (0.05–0.20 nmol/mg cell protein; 0.05–0.15 nmol/liter). The enzyme is catalytically active toward CYP2D6 substrates such as bufuralol and metoprolol. In order to optimize catalytic activity human reductase was coexpressed with CYP2D6 in Sf9 cells; reductase activity was in the region of 1000–1500 units per mg cell protein, while spectrally active CY2D6 was in the range 10–20 pmol/mg cell protein. TheKmandKcatvalues for bufuralol metabolism were estimated as 4.7 μMand 12.23 min−1, respectively. The use of the conventional very late promoters such as the polyhedrin promoter generate a large proportion of inactive CYP2D6. The problem was to a degree circumvented using the “late” basic promoter which is active earlier in the baculovirus infection cycle. The yield of functional CYP2D6 was at least as high as with very late promoters, but the proportion of inactive protein was reduced. Bufuralol hydroxylase activity could be measured directly by HPLC analysis of cell culture media supplemented with bufuralol, and we have developed a plate assay system which provides a simple method for the analysis of drug metabolism reactions using Sf9 cells. Expression using baculovirus provides a valuable source of functional CYP2D6 for work aimed at elucidating the structure and function of the enzyme.