Screening of stable G-protein-coupled receptor variants in Saccharomyces cerevisiae.
Screening of stable G-protein-coupled receptor variants in Saccharomyces cerevisiae.
复制标题
酿酒酵母中稳定的 G 蛋白偶联受体变体的筛选。
DOI：
10.1007/978-1-4939-2230-7_9
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Kobayashi T.
中科院分区：
文献类型：
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作者：
Shiroishi M;Kobayashi T.
G蛋白偶联受体（GPCR）不仅是最大的蛋白质家族，而且作为一个整体，它们代表了最大的治疗药物靶点组。最近在GPCR结构测定中的成功依赖于受体的稳定化以克服表达和纯化中的困难。虽然需要大量纯化的蛋白质用于结构测定，但大多数野生型GPCR太不稳定而不能大规模表达和纯化。因此，快速筛选高表达的稳定受体“变体”至关重要。已经证明，将绿色荧光蛋白（GFP）融合到靶膜蛋白上有助于评价洗涤剂中膜蛋白的物理性质。此外，芽殖酵母Saccharomyces aeParty能够通过其自身高效的同源重组系统快速构建表达载体。在此，我们描述了使用GFP和S快速构建和筛选稳定的GPCR变体的方案。啤酒。
G-protein-coupled receptors (GPCRs) are not only the largest protein family, but as a whole, they represent the largest group of therapeutic drug targets. Recent successes in the determination of GPCR structures have relied on the stabilization of receptors to overcome the difficulties in expression and purification. Although a large quantity of purified protein is needed for structural determination, the majority of wild-type GPCRs are too unstable to express and purify on a large scale. Therefore, rapid screening of highly expressed stable receptor “variants” is crucial. It has been demonstrated that fusing green fluorescent protein (GFP) to a target membrane protein facilitates the evaluation of the physical properties of the membrane protein in detergent. Furthermore, the budding yeastSaccharomyces cerevisiaeenables rapid construction of an expression vector via its own efficient homologous recombination system. Herein, we describe the protocols for rapid construction and screening of stable GPCR variants using GFP andS. cerevisiae.