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8. GPCR Expression Using Baculovirus-Infected Sf9 Cells
Abstract
Expression of proteins in insect cells using recombinant baculoviruses has gained wide use in the G protein-coupled receptor (GPCR) community. This expression system produces high yields of functional receptor, is able to perform post-translational modifications, and is readily adaptable to large-scale culture. Here, we describe the generic methods for expressing a GPCR using baculovirus-infected insect cells, including the maintenance of insect cell culture. Data are presented for polyhedrin promoter-driven expression of a C-terminal 6Ă—histidine-tagged mammalian M2 muscarinic receptor in Sf9 cells. Results demonstrate that expressed receptor could be detected and quantified using radiolabeled ligand binding, that expression was maximal at approximately 72 h post-infection, and that expression levels could be altered by addition of various ligands to cultures of infected insect cells.
Affiliation(s): (2) School of Biology, Flinders University, Bedford Park, SA, Australia
(3) SARDI Entomology, Adelaide, SA, Australia
(4) CSIRO Molecular and Health Technologies, Adelaide, SA, Australia
(5) CSIRO Human Nutrition, Adelaide, SA, Australia
Series: Methods in Molecular Biology  |  Volume: 552  |  Pub. Date: Aug-01-2009  |  Page Range: 115-129  |  DOI: 10.1007/978-1-60327-317-6_8
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