Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
Methods Mol Biol. 2022;2507:91-110. doi: 10.1007/978-1-0716-2368-8_6.
Functional and structural studies on membrane proteins are often hampered by insufficient yields, misfolding and aggregation during the production and purification process. Escherichia coli is the most commonly used expression host for the production of recombinant prokaryotic integral membrane proteins. However, in many cases expression hosts other than E. coli are more appropriate for certain target proteins. Here, we report a convenient, systematically developed expression system using the γ-proteobacterium Pseudomonas stutzeri as an alternative production host for over-expression of integral membrane proteins. P. stutzeri can be easily and inexpensively cultured in large quantities. The Pseudomonas expression vectors are designed for inducible expression of affinity-tagged fusion proteins controlled by the P promoter. This chapter provides detailed protocols of the different steps required to successfully produce and isolate recombinant membrane proteins with high yields in P. stutzeri.
功能和结构研究膜蛋白通常受到阻碍，产量不足，错误折叠和聚集在生产和纯化过程中。大肠杆菌是最常用的表达宿主生产重组原核整合膜蛋白。然而，在许多情况下，除了大肠杆菌以外的表达宿主对于某些目标蛋白更为合适。在这里，我们报告了一种方便的、系统开发的表达系统，使用γ-变形菌恶臭假单胞菌作为替代生产宿主，用于过表达整合膜蛋白。恶臭假单胞菌可以很容易且廉价地大量培养。假单胞菌表达载体设计用于诱导表达亲和标签融合蛋白，由 P 启动子控制。本章提供了详细的协议，需要成功生产和分离重组膜蛋白与高产量在恶臭假单胞菌。
