Mouillac Bernard, Banères Jean-Louis
Institut de Génomique Fonctionnelle, Montpellier, France.
Methods Mol Biol. 2010;601:39-48. doi: 10.1007/978-1-60761-344-2_3.
Integral membrane proteins, in particular receptors, regulate numerous physiological functions. The primary difficulty presented by their study in vitro is to obtain them in sufficient amounts in a functional state. Escherichia coli is a host of choice for producing recombinant proteins for structural studies. However, insertion of G-protein coupled receptors into its plasma membrane usually results in bacterial death. An alternative approach consists of targeting recombinant receptors to inclusion bodies, where they accumulate without affecting bacterial growth, and then fold them in vitro . We describe here a general approach that consists of accumulating the receptor in bacterial inclusion bodies, then purifying it under denaturing conditions. A simple assay is then described to screen for refolding conditions of the protein.
整合膜蛋白，尤其是受体，调节着众多生理功能。在体外研究它们时面临的主要困难是要获得足够数量的功能状态蛋白。大肠杆菌是用于生产重组蛋白以进行结构研究的首选宿主。然而，将G蛋白偶联受体插入其质膜通常会导致细菌死亡。另一种方法是将重组受体靶向至包涵体，在那里它们积累而不影响细菌生长，然后在体外进行折叠。我们在此描述一种通用方法，该方法包括在细菌包涵体中积累受体，然后在变性条件下对其进行纯化。随后描述了一种简单的检测方法来筛选该蛋白的复性条件。
