title: Expression of Recombinant Flavin-Containing Monooxygenases in a Baculovirus/Insect Cell System.
abstract: 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 Spodoptera frugiperda (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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University College London, London, UK.
authors.forename: Azara
authors.initials: A
authors.lastname: Janmohamed
authors.affiliation: School of Biological Sciences, Queen Mary, University of London, London, UK.
authors.forename: Prapimpuk
authors.initials: P
authors.lastname: Thaunsukon
authors.affiliation: Department of Biochemistry and Molecular Biology, University College London, London, UK.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Shephard
authors.affiliation: School of Biological Sciences, Queen Mary, University of London, London, UK.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Phillips
keywords: FMO4; Flavin-containing monooxygenase; baculovirus; cytochrome P450; heterologous expression; insect cells