Department of Chemistry, University of Zurich, 8057 Zurich, Switzerland.
Institute of Medical Microbiology, University of Zurich, 8057 Zurich, Switzerland.
J Mol Biol. 2015 Feb 27;427(4):943-954. doi: 10.1016/j.jmb.2014.11.002. Epub 2014 Nov 9.
Functional overexpression of membrane proteins is essential for their structural and functional characterization. However, functional overexpression is often difficult to achieve, and frequently either no expression or expression as misfolded aggregates is observed. We present an approach for improving the functional overexpression of membrane proteins in Escherichia coli using transcriptional fusions. The method involves the use of a small additional RNA sequence upstream to the RNA sequence of the target membrane protein and results in the production of a bicistronic mRNA. In contrast to the common approach of translational fusions to enhance protein expression, transcriptional fusions do not require protease treatment and subsequent removal of the fusion protein. Using this strategy, we observed improvements in the quantity and/or the quality of the produced material for several membrane proteins to levels compatible with structural studies. Our analysis revealed that translation of the upstream RNA sequence was not essential for increased expression. Rather, the sequence itself had a large impact on protein yields, suggesting that alternative folding of the transcript was responsible for the observed effect.
功能过表达膜蛋白对于它们的结构和功能表征是必不可少的。然而，功能过表达通常很难实现，并且经常观察到要么没有表达，要么表达为错误折叠的聚集体。我们提出了一种使用转录融合来提高大肠杆菌中膜蛋白功能过表达的方法。该方法涉及在靶膜蛋白的 RNA 序列上游使用一小段额外的 RNA 序列，从而产生双顺反子 mRNA。与增强蛋白表达的常见翻译融合方法不同，转录融合不需要蛋白酶处理和随后去除融合蛋白。使用这种策略，我们观察到几种膜蛋白的产量和/或质量得到了改善，达到了与结构研究兼容的水平。我们的分析表明，上游 RNA 序列的翻译对于增加表达并不是必需的。相反，该序列本身对蛋白产量有很大的影响，这表明转录本的替代折叠是导致观察到的效果的原因。
