Molecular and Integrative Biosciences, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland.
Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, UK.
Methods Mol Biol. 2020;2168:3-49. doi: 10.1007/978-1-0716-0724-4_1.
A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.
生物物理技术研究膜蛋白的主要障碍是难以获得足够数量的用于功能和结构研究的材料。为了异源表达目标膜蛋白，特别是真核蛋白，关键步骤是找到最佳的宿主表达系统并进行后续的表达优化。在本章中，我们描述了使用细菌和昆虫细胞筛选膜蛋白生产、可溶性筛选、大规模生产以及常用亲和层析纯化方法的方案。我们讨论了一般的优化条件，如启动子和标签，并描述了当前可在任何实验室中使用的技术，而无需专门的昂贵设备。特别是对于昆虫细胞，GFP 融合特别有助于蛋白质在 SDS-PAGE 上的定位和胶内荧光检测。我们提供了详细的方案，可以用于筛选膜蛋白研究的最佳表达和纯化条件。
