Optimizing synthesis and expression of transmembrane peptides and proteins
Optimizing synthesis and expression of transmembrane peptides and proteins
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DOI：
10.1016/j.ymeth.2006.07.003
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发表时间：
2007-04-01
期刊：
影响因子：
4.8
通讯作者：
Deber, Charles M.
中科院分区：
文献类型：
--
作者：
Cunningham, Fiona;Deber, Charles M.
全长膜蛋白的疏水性和在各种体系中的低表达阻碍了对其结构的研究。然而，膜蛋白折叠的一个简化方面是，已经观察到单个跨膜片段或膜蛋白片段代表独立的折叠结构域，因此，有助于研究TM螺旋之间的堆积相互作用，并收集有关膜蛋白的结构信息。本文综述了两类可用于制备跨膜蛋白片段的技术--总肽合成技术和细菌表达技术。首先，合成带有赖氨酸等增溶残基的N-和/或C-标记的疏水性跨膜多肽可以改善在各种生物物理实验中对跨膜核心的操纵。在此背景下，我们描述了在这些多肽的合成、裂解和纯化阶段的一般方案考虑，以确定适当的参数，结合以提高疏水性多肽的产量。第二，细菌表达膜蛋白片段是产生大量疏水蛋白片段的有用工具。靶向蛋白在大肠杆菌中的表达可以促进纯化，而将疏水构建物连接到亲水性融合蛋白上可以放大表达。我们表明，调整蛋白质结构以符合表达宿主规范，并结合对表达条件的彻底探索，如用于表达的介质类型、温度和细胞菌株，可以显著提高蛋白质产量。(C)2006 Elsevier Inc.保留所有权利。
Structural studies of full-length membrane proteins have been hindered by their hydrophobicity and low expression in a variety of systems. However, a simplifying aspect of membrane protein folding is that individual transmembrane segments or membrane protein fragments have been observed to represent independent folding domains, and as such, can facilitate the study of packing interactions between TM helices, and the collection of structural information regarding membrane proteins. This review focuses on two categories of techniques-total peptide synthesis and bacterial expression-that can each be optimized for preparation of transmembrane protein segments. First, synthesis of hydrophobic transmembrane peptides that are N- and/or C-tagged with solubilizing residues such as lysine can improve manipulation of the transmembrane core in a variety of biophysical experiments. In this context, we describe general protocol considerations during the synthesis, cleavage, and purification stages of these peptides to identify appropriate parameters that combine to improve yields of hydrophobic peptides. Second, bacterial expression of membrane protein fragments is a useful tool for producing large quantities of hydrophobic protein segments. Targeting protein expression within Escherichia coli can facilitate purification, while attaching the hydrophobic construct to a hydrophilic fusion protein can amplify expression. We show that adapting protein constructs to comply with expression host specifications, in concert with thorough exploration of expression conditions such as the type of media used for expression, temperature, and cell strain, can significantly improve protein yields. (c) 2006 Elsevier Inc. All rights reserved.