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 beta-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and used to express CYP2D6 in Spodoptera 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. The K(m) and K(cat) values for bufuralol metabolism were estimated as 4.7 microM and 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.
Paine
M J
MJ
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Scotland.
Gilham
D
D
Roberts
G C
GC
Wolf
C R
CR
D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004983:Ethanolamines / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011802:Quinidine / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013053:Spectrophotometry; D018411:Spodoptera / Q000166:cytology / Q000821:virology
D000319:Adrenergic beta-Antagonists; D004983:Ethanolamines; D011994:Recombinant Proteins; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6; D009251:NADPH-Ferrihemoprotein Reductase; D011802:Quinidine