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用于结构研究的酵母中重组膜蛋白的合成。

The synthesis of recombinant membrane proteins in yeast for structural studies.

作者信息

Routledge Sarah J, Mikaliunaite Lina, Patel Anjana, Clare Michelle, Cartwright Stephanie P, Bawa Zharain, Wilks Martin D B, Low Floren, Hardy David, Rothnie Alice J, Bill Roslyn M

机构信息

Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK; School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK.

School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK.

出版信息

Methods. 2016 Feb 15;95:26-37. doi: 10.1016/j.ymeth.2015.09.027. Epub 2015 Sep 30.

Abstract

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.

摘要

从历史上看,重组膜蛋白的生产一直是一项重大挑战,这意味着已发表的膜蛋白结构比可溶性蛋白的结构要少得多。然而,最近存入蛋白质数据库的膜蛋白结构数量几乎呈指数级增长。这表明现在已有经验方法可确保为这些难以攻克的目标提供所需的蛋白质供应。本综述聚焦于可用于在酵母中生产蛋白质的方法,酵母是重组真核膜蛋白的重要来源。我们概述了优化表达质粒、宿主细胞和培养条件的方法,以及为结构研究准备结晶试验而进行的功能性蛋白提取和纯化方法。

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