title: Screening of stable G-protein-coupled receptor variants in Saccharomyces cerevisiae.
abstract: 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 yeast Saccharomyces cerevisiae enables 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 and S. cerevisiae.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan, shiroish@phar.kyushu-u.ac.jp.
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Shiroishi
authors.forename: Takuya
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D002850:Chromatography, Gel; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D059765:Homologous Recombination; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins