Aloia Amanda L, Glatz Richard V, McMurchie Edward J, Leifert Wayne R
School of Biology, Flinders University, Bedford Park, SA, Australia.
Methods Mol Biol. 2009;552:115-29. doi: 10.1007/978-1-60327-317-6_8.
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 6 x histidine-tagged mammalian M(2) 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.
利用重组杆状病毒在昆虫细胞中表达蛋白质已在G蛋白偶联受体（GPCR）领域得到广泛应用。该表达系统能高产功能性受体，能够进行翻译后修饰，并且易于适应大规模培养。在此，我们描述了使用杆状病毒感染的昆虫细胞表达GPCR的通用方法，包括昆虫细胞培养的维持。文中给出了多角体蛋白启动子驱动的C端带有6个组氨酸标签的哺乳动物M(2)毒蕈碱受体在Sf9细胞中表达的数据。结果表明，使用放射性标记配体结合可检测和定量表达的受体，感染后约72小时表达量最高，并且通过向感染昆虫细胞的培养物中添加各种配体可改变表达水平。
