College of Health & Life Sciences, Aston University, Aston Triangle, Birmingham, UK.
Institute of Microbiology and Infection and School of Biosciences, University of Birmingham, Birmingham, UK.
Methods Mol Biol. 2022;2507:59-78. doi: 10.1007/978-1-0716-2368-8_4.
Over the decades, the bacterium Escherichia coli (E. coli) has become the cornerstone of recombinant protein production, used for heterologous synthesis of a variety of membrane proteins. Due to its rapid growth to high densities in cheap media, and its ease of manipulation and handling, E. coli is an excellent host cell for a range of membrane protein targets. Furthermore, its genetic tractability allows for a variety of gene constructs to be screened for optimal expression conditions, resulting in relatively high yields of membrane protein in a short amount of time. Here, we describe the general workflow for the production of membrane proteins in E. coli. The protocols we provide show how the gene of interest is modified, transferred to an expression vector and host, and how membrane protein yields can be optimized and analyzed. The examples we illustrate are well suited for scientists who are starting their journey into the world of membrane protein production.
几十年来，细菌大肠杆菌（E. coli）已成为重组蛋白生产的基石，用于各种膜蛋白的异源合成。由于其在廉价培养基中快速生长到高密度，以及易于操作和处理，大肠杆菌是一系列膜蛋白靶标的优秀宿主细胞。此外，其遗传可操作性允许筛选各种基因构建体以获得最佳表达条件，从而在短时间内产生相对较高产量的膜蛋白。在这里，我们描述了在大肠杆菌中生产膜蛋白的一般工作流程。我们提供的方案展示了如何修饰感兴趣的基因，将其转移到表达载体和宿主中，以及如何优化和分析膜蛋白的产量。我们举例说明的方法非常适合刚开始涉足膜蛋白生产领域的科学家。
