Expression of recombinant flavin-containing monooxygenases in a baculovirus/insect cell system.
The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have used for the expression of human and mouse flavin-containing monooxygenases. Methods are described for the generation of recombinant baculoviral DNAs, via both site-specific transposition in Escherichia coli and site-specific recombination in vitro; adaptation of Spodopterafrugiperda (Sf) 9 cells to shaking culture and to serum-free medium; cryopreservation and transfection of Sf9 cells; amplification of baculovirus and determination of viral titer; analysis of baculoviral DNA; and expression and analysis of recombinant proteins.
Janmohamed
Azara
A
Department of Biochemistry and Molecular Biology, University College London, UK.
Thaunsukon
Prapimpuk
P
Shephard
Elizabeth A
EA
Phillips
lan R
lR
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D015774:Ganciclovir / Q000494:pharmacology; D010105:Oxygenases / Q000235:genetics*; D011995:Recombination, Genetic; D018411:Spodoptera; D010948:Viral Plaque Assay
D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D015774:Ganciclovir