Department of Chemistry and Biochemistry, UCLA-DOE Institute, Molecular Biology Institute, University of California, Los Angeles, CA, USA.
Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Methods Mol Biol. 2021;2178:439-467. doi: 10.1007/978-1-0716-0775-6_28.
G-protein-coupled receptors (GPCRs) are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of GPCRs' structure and function remains limited. The first bottleneck in GPCR studies is producing sufficient quantities of soluble, functional, and stable receptors. Currently, GPCR production largely depends on the choice of the host system and the type of detergent used to extract the GPCR from the cell membrane and stabilize the protein outside the membrane bilayer. Here, we present three protocols that we employ in our lab to produce and solubilize stable GPCRs: (1) cell-free in vitro translation, (2) HEK cells, and (3) Escherichia coli. Stable receptors can be purified using immunoaffinity chromatography and gel filtration, and can be analyzed with standard biophysical techniques and biochemical assays.
G 蛋白偶联受体（GPCRs）是细胞膜的整合蛋白，直接参与许多生物功能的调节和药物靶向。然而，我们对 GPCR 的结构和功能的了解仍然有限。GPCR 研究的第一个瓶颈是产生足够数量的可溶性、功能性和稳定的受体。目前，GPCR 的生产在很大程度上取决于宿主系统的选择和用于从细胞膜中提取 GPCR 并稳定跨膜双层外蛋白质的去污剂类型。在这里，我们介绍了我们在实验室中使用的三种生产和溶解稳定 GPCR 的方案：（1）无细胞体外翻译，（2）HEK 细胞，和（3）大肠杆菌。稳定的受体可以使用免疫亲和层析和凝胶过滤进行纯化，并可以使用标准的生物物理技术和生化测定进行分析。
