Lundstrom Kenneth
PanTherapeutics, Lutry, Switzerland.
Methods Mol Biol. 2010;601:149-63. doi: 10.1007/978-1-60761-344-2_10.
One of the major bottlenecks in drug screening and structural biology on membrane proteins has for a long time been the expression of recombinant protein in sufficient quality and quantity. The expression has been evaluated in all existing expression systems, from cell-free translation and bacterial systems to expression in animal cells. In contrast to soluble proteins, the expression levels have been relatively low due to the following reasons: The topology of membrane proteins requires special, posttranslational processing, folding, and insertion into membranes, which often are mammalian cell specific. Despite these strict demands, functional membrane proteins (G protein-coupled receptors, ion channels, and transporters) have been successfully expressed in bacterial, yeast, and insect cells. A general drawback observed in prokaryotic cells is that accumulation of foreign protein in membranes is toxic and results in growth arrest and therefore low yields of recombinant protein.In this chapter, the focus is on expression of recombinant mammalian membrane proteins in mammalian host cells, particularly applying Semliki Forest virus (SFV) vectors. Replication-deficient SFV vectors are rapidly generated at high titers in BHK-21 (Baby Hamster Kidney) cells, which then are applied for a broad range of mammalian and nonmammalian cells. The SFV system has provided high expression levels of topologically different proteins, especially for membrane proteins. Robust ligand-binding assays and functional coupling to G proteins and electrophysiological recordings have made the SFV system an attractive tool in drug discovery. Furthermore, the high susceptibility of SFV vectors to primary neurons has allowed various applications in neuroscience. Establishment of large-scale production in mammalian adherent and suspension cultures has allowed production of hundreds of milligrams of membrane proteins that has allowed their submission to serious structural biology approaches. In this context, a structural genomics program for SFV-based overexpression of 100 GPCRs was established.
长期以来，膜蛋白药物筛选和结构生物学的主要瓶颈之一是重组蛋白的高质量和高产量表达。人们已经在所有现有的表达系统中评估了这种表达，从无细胞翻译、细菌系统到动物细胞中的表达。与可溶性蛋白相比，由于以下原因，膜蛋白的表达水平相对较低：膜蛋白的拓扑结构需要特殊的翻译后加工、折叠以及插入膜中，而这些过程通常具有哺乳动物细胞特异性。尽管有这些严格要求，但功能性膜蛋白（G蛋白偶联受体、离子通道和转运蛋白）已成功在细菌、酵母和昆虫细胞中表达。原核细胞中普遍存在的一个缺点是，外源蛋白在膜中的积累具有毒性，会导致生长停滞，从而使重组蛋白产量较低。在本章中，重点是在哺乳动物宿主细胞中表达重组哺乳动物膜蛋白，特别是应用辛德毕斯病毒（SFV）载体。复制缺陷型SFV载体可在BHK-21（幼仓鼠肾）细胞中快速高滴度产生，然后应用于广泛的哺乳动物和非哺乳动物细胞。SFV系统已实现了拓扑结构不同的蛋白的高表达水平，尤其是膜蛋白。强大的配体结合测定以及与G蛋白的功能偶联和电生理记录使SFV系统成为药物发现中的一个有吸引力的工具。此外，SFV载体对原代神经元的高敏感性使其在神经科学中有多种应用。在哺乳动物贴壁和悬浮培养中建立大规模生产，使得能够生产数百毫克的膜蛋白，从而可以对其采用严格的结构生物学方法。在此背景下，建立了一个基于SFV过表达100种GPCR的结构基因组学计划。
