Micro-scale and rapid expression screening of highly expressed and/or stable membrane protein variants in Saccharomyces cerevisiae

Mitsunori Shiroishi, Mai Moriya, Tadashi Ueda

研究成果: Contribution to journalArticle査読

2 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)1863-1872
ページ数10
ジャーナルProtein Science
DOI
出版ステータス出版済み - 10 1 2016

All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子生物学

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