Drug Resistance and Membrane Proteins Team, CNRS - Lyon 1 University UMR 5086, Institut de Biologie et Chimie des Protéines, Lyon, France.
Calixar, Lyon, France.
Methods Mol Biol. 2022;2507:175-185. doi: 10.1007/978-1-0716-2368-8_9.
The production and purification are the first steps required in any functional or structural study of a protein of interest. In the case of membrane proteins, these tasks can be difficult due to low expression levels and the necessity to extract them from their membrane environment. This chapter describes a convenient method based on GFP tagged to the membrane protein to facilitates these steps. Production is carried out in the yeast S. cerevisiae and purification steps are carried out and monitored taking advantage of an anti-GFP nanobody. We show how GFP can be a very helpful tool for controlling the correct addressing of the protein and for probing and optimizing purification. These methods are described here for producing and purifying CaCdr1p, an ABC exporter conferring multiantifungal resistance to C. albicans. This purification method can be amenable to any other GFP-tagged protein.
生产和纯化是任何对感兴趣的蛋白质进行功能或结构研究的第一步。在膜蛋白的情况下，由于表达水平低，以及必须从膜环境中提取它们，这些任务可能很困难。本章描述了一种基于 GFP 标记膜蛋白的方便方法，可简化这些步骤。生产在酵母 S. cerevisiae 中进行，纯化步骤利用抗 GFP 纳米抗体进行，并进行监测。我们展示了 GFP 如何成为控制蛋白质正确定位以及探测和优化纯化的非常有用的工具。这些方法是为生产和纯化 CaCdr1p 而描述的，CaCdr1p 是一种 ABC 外排泵，使 C. albicans 具有多种抗真菌抗性。这种纯化方法适用于任何其他 GFP 标记的蛋白质。
