Molecular Microbiology and Structural Biochemistry, UMR 5086 CNRS/University of Lyon, Lyon, France.
Methods Mol Biol. 2022;2507:41-58. doi: 10.1007/978-1-0716-2368-8_3.
Overexpression of properly folded membrane proteins is a mandatory step for their functional and structural characterization. One of the most used expression systems for the production of proteins is Escherichia coli. Many advantageous strains combined with T7 expression systems have been developed over the years. Recently, we showed that the choice of the strain is critical for the functionality of membrane proteins, even when the proteins are successfully incorporated in the membrane (Mathieu et al. Sci Rep. 2019; 9(1):2654). Notably, the amount and/or activity of the T7-RNA polymerase, which drives the transcription of the genes of interest, may indirectly affect the folding and functionality of overexpressed membrane proteins. Moreover, we reported a general trend in which mild detergents mainly extract the population of active membrane proteins, whereas a harsher detergent like Fos-choline 12 could solubilize them irrespectively of their functionality. Based on these observations, we provide some guidelines to optimize the quality of membrane proteins overexpressed in E. coli.
正确折叠的膜蛋白的过度表达是其功能和结构表征的必要步骤。用于生产蛋白质的最常用的表达系统之一是大肠杆菌。多年来，已经开发出许多具有优势的菌株，并结合 T7 表达系统。最近，我们表明，即使蛋白质成功地整合到膜中，菌株的选择对于膜蛋白的功能也是至关重要的（Mathieu 等人，《科学报告》2019 年；9（1）：2654）。值得注意的是，T7-RNA 聚合酶的数量和/或活性，其驱动目的基因的转录，可能会间接影响过度表达的膜蛋白的折叠和功能。此外，我们报告了一个普遍的趋势，即温和的去污剂主要提取活性膜蛋白的群体，而像 Fos-choline 12 这样更苛刻的去污剂可以将它们溶解，而不管它们的功能如何。基于这些观察结果，我们提供了一些指南，以优化在大肠杆菌中过度表达的膜蛋白的质量。
