Premium
Micro‐scale and rapid expression screening of highly expressed and/or stable membrane protein variants in Saccharomyces cerevisiae
Author(s) -
Shiroishi Mitsunori,
Moriya Mai,
Ueda Tadashi
Publication year - 2016
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2993
Subject(s) - saccharomyces cerevisiae , mutagenesis , tandem affinity purification , alanine scanning , mutant , membrane protein , biochemistry , protein engineering , biology , heterologous expression , high throughput screening , fusion protein , heterologous , protein purification , target protein , membrane , protein–protein interaction , chemistry , affinity chromatography , enzyme , gene , recombinant dna
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.