A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems
A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems
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DOI：
10.1016/j.pep.2012.01.018
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发表时间：
2012-04-01
影响因子：
1.6
通讯作者：
Bernhard, Frank
中科院分区：
文献类型：
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作者：
Haberstock, Stefan;Roos, Christian;Bernhard, Frank
结构或功能分析需要大量的膜蛋白样品，并且第一个瓶颈通常是获得足够的生产效率。无细胞表达系统中蛋白质生产的复杂性降低导致效率问题与基本转录/翻译过程的频繁相关。我们提出了一种系统的标签变异策略，用于基于翻译起始的优化快速提高膜蛋白的无细胞表达效率。通过重叠PCR将少量合理设计的短表达标签连接到靶蛋白编码序列的5 ′端。在无细胞表达筛选中分析所产生的DNA模板库，并选择最有效的模板用于进一步制备规模的蛋白质生产。表达标签可以最小化到仅几个密码子，并且不需要对编码序列的进一步影响。整个过程仅需几天，合成的PCR片段可直接用作制备规模无细胞反应的模板。该策略的例子与生产的一组G-蛋白偶联受体和产量提高高达32倍，获得。所有蛋白质最终以足以用于进一步质量优化和初始结晶筛选的量合成。(C)2012 Elsevier Inc. All rights reserved.
High amounts of membrane protein samples are needed for structural or functional analysis and a first bottleneck is often to obtain sufficient production efficiencies. The reduced complexity of protein production in cell-free expression systems results in a frequent correlation of efficiency problems with the essential transcription/translation process. We present a systematic tag variation strategy for the rapid improvement of cell-free expression efficiencies of membrane proteins based on the optimization of translation initiation. A small number of rationally designed short expression tags is attached via overlap PCR to the 5-prime end of the target protein coding sequence. The generated pool of DNA templates is analyzed in a cell-free expression screen and the most efficient template is selected for further preparative scale protein production. The expression tags can be minimized to only a few codons and no further impact on the coding sequence is required. The complete process takes only few days and the synthesized PCR fragments can be used directly as templates for preparative scale cell-free reactions. The strategy is exemplified with the production of a set of G-protein coupled receptors and yield improvements of up to 32-fold were obtained. All proteins were finally synthesized in amounts sufficient for further quality optimization and initial crystallization screens. (C) 2012 Elsevier Inc. All rights reserved.