title: Micro-scale and rapid expression screening of highly expressed and/or stable membrane protein variants in Saccharomyces cerevisiae.
abstract: Purification of milligram quantities of target proteins is required for structural and biophysical studies. However, mammalian membrane proteins, many of which are important therapeutic targets, are too unstable to be expressed in heterologous hosts and to be solubilized by detergents. One of the most promising ways to overcome these limitations is to stabilize the membrane proteins by generating variants via introduction of truncated flexible regions, fusion partners, and site-directed mutagenesis. Therefore, an effective screening strategy is a key to obtaining successful protein stabilization. Herein, we report the micro-scale and high-throughput screening of stabilized membrane protein variants using Saccharomyces cerevisiae as a host. All steps of the screening, including cultivation and disruption of cells, solubilization of the target protein, and the pretreatment for fluorescence-detected size exclusion chromatography (FSEC), could be performed in a 96-well microplate format. We demonstrated that the dispersion among wells was small, enabling detection of a small but important improvement in the protein stability. We also demonstrated that the thermally stable mutants of a human G protein-coupled receptor could be distinguished based on an increase of the peak height in the FSEC profile, which was well correlated with increased ligand binding activity of the protein. This strategy represents a significant platform for handling numerous mutants, similar to alanine scanning.
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.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Moriya
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ueda
mesh_terms: D015870:Gene Expression; D006801:Humans; D055550:Protein Stability; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins
keywords: 96-well microplate; Saccharomyces cerevisiae; expression; fluorescence-detection size-exclusion chromatography (FSEC); screening; stabilization; unstable membrane protein