Marino Jacopo, Holzhüter Katharina, Kuhn Benedikt, Geertsma Eric R
University of Zurich, Zurich, Switzerland.
Goethe University Frankfurt, Frankfurt/Main, Germany.
Methods Enzymol. 2017;594:139-164. doi: 10.1016/bs.mie.2017.05.011. Epub 2017 Jul 25.
Escherichia coli is one of the most widely used expression hosts for membrane proteins. However, establishing conditions for its recombinant production of membrane proteins remains difficult. Attempts to produce membrane proteins frequently result in either no expression or expression as misfolded aggregates. We developed an efficient pipeline for improving membrane protein overexpression in E. coli that is based on two approaches. The first involves transcriptional fusions, small additional RNA sequences upstream of the target open reading frame, to overcome no or poor overall expression levels. The other is based on a tunable promoter in combination with a fusion to green fluorescent protein serving as a reporter for the folding state of the target membrane protein. The latter combination allows adjusting the membrane protein expression rate to the downstream folding capacity, in order to decrease the formation of protein aggregates. This pipeline has proven successful for the efficient and parallel optimization of a diverse set of membrane proteins.
大肠杆菌是用于膜蛋白表达的最广泛使用的宿主之一。然而，为其重组生产膜蛋白建立条件仍然很困难。生产膜蛋白的尝试常常导致无表达或表达为错误折叠的聚集体。我们基于两种方法开发了一种用于提高大肠杆菌中膜蛋白过表达的高效流程。第一种方法涉及转录融合，即在目标开放阅读框上游添加小的额外RNA序列，以克服整体表达水平低或无表达的问题。另一种方法基于可调节启动子，并与绿色荧光蛋白融合，用作目标膜蛋白折叠状态的报告基因。后一种组合允许将膜蛋白表达速率调整到下游折叠能力，以减少蛋白质聚集体的形成。该流程已被证明可成功地对多种膜蛋白进行高效且并行的优化。
