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.
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Acknowledgments
We thank R. M. Philpot for membranes from bacteria expressing human FMO3 and antisera to rabbit FMO3, and R. W. Pickersgill for His-tagged internalin C. P. T. is a recipient of a studentship from the Thai Government Pharmaceutical Organization. This work was supported by a grant from the Wellcome Trust (no. 053590).
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Janmohamed, A., Thaunsukon, P., Shephard, E.A., Phillips, I.R. (2006). Expression of Recombinant Flavin-Containing Monooxygenases in a Baculovirus/Insect Cell System. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-998-2:39
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DOI: https://doi.org/10.1385/1-59259-998-2:39
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