High-throughput analytical gel filtration screening of integral membrane proteins for structural studies
High-throughput analytical gel filtration screening of integral membrane proteins for structural studies
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DOI：
10.1016/j.bbagen.2013.02.001
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发表时间：
2013-06-01
影响因子：
3
通讯作者：
Nordlund, Par
中科院分区：
文献类型：
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作者：
Low, Christian;Moberg, Per;Nordlund, Par
工作背景：整合膜蛋白（IMP）的结构研究经常受到难以制备稳定的均质结晶样品的阻碍。为了克服这一障碍，筛选大量靶蛋白以找到合适的结晶候选物已成为常见的做法。为了使这种方法有效，一个有效的筛选策略是必不可少的。为此，已经开发了涉及使用绿色荧光蛋白（GFP）融合构建体的策略。然而，这些方法有两个缺点：不能测试具有易位C-末端的蛋白质，并且从分析纯化到制备纯化的规模扩大通常是不平凡的，并且可能需要重新cloning.Methods：在这里，我们提出了一种筛选方法，该方法基于三个标准优先考虑IMP靶点：表达水平，通过高通量小规模固定化金属亲和色谱法（IMAC）和自动化大小测定确定的去污剂溶解产率和均匀性。结果：在两种载体和两株大肠杆菌中筛选出48株原核IMP，验证了该策略的有效性。一组11种蛋白质通过了所有预设的质量控制检查点，并进行了结晶试验。其中四个结晶直接在初始稀疏矩阵screens，突出了鲁棒性的strategy.Conclusions：我们已经开发出一种快速和具有成本效益的筛选策略，可以用于所有IMP的拓扑结构无关。的分析步骤已被设计成是一个很好的模拟制备纯化，这大大有利于scale-up.General意义：这里提出的筛选方法的目的和预期有助于推动膜蛋白的结构生物学。(C)2013 Elsevier B. V.保留所有权利。
Background: Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks: proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning.Methods: Here we present a screening approach that prioritizes IMP targets based on three criteria: expression level, detergent solubilization yield and homogeneity as determined by high-throughput small-scale immobilized metal affinity chromatography (IMAC) and automated size-exclusion chromatography (SEC).Results: To validate the strategy, we screened 48 prokaryotic IMPs in two different vectors and two Escherichia coli strains. A set of 11 proteins passed all preset quality control checkpoints and was subjected to crystallization trials. Four of these crystallized directly in initial sparse matrix screens, highlighting the robustness of the strategy.Conclusions: We have developed a rapid and cost efficient screening strategy that can be used for all IMPs regardless of topology. The analytical steps have been designed to be a good mimic of preparative purification, which greatly facilitates scale-up.General significance: The screening approach presented here is intended and expected to help drive forward structural biology of membrane proteins. (C) 2013 Elsevier B.V. All rights reserved.