期刊
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
卷 1830, 期 6, 页码 3497-3508出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbagen.2013.02.001
关键词
Structural genomics; Membrane protein structure; Membrane protein over-expression; Analytical gel filtration; Detergent screen
资金
- Danish Council for Independent Research (Medical Sciences) [271-09-0187]
- European Molecular Biology Organization (EMBO) postdoctoral fellowship
- Swedish Research council
- Swedish Cancer Society
- integrated EU project EDICT (European drug initiative on channels and transporters)
- Singapore NRF-CRP (National Research Foundation - Competitive Research Programme) grant
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据