National Institute on Alcoholism and Alcohol Abuse, NIH, Bethesda, MD, 20892, USA.
ThermoFisher Scientific, 7335 Executive Way, Frederick, MD, 21704, USA.
Sci Rep. 2020 Oct 8;10(1):16805. doi: 10.1038/s41598-020-73813-7.
Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E. coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB. Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.
针对整联膜 G 蛋白偶联受体 (GPCR) 的药物的合理设计需要通过对纯化蛋白的高分辨率结构研究，彻底了解配体结合和激活机制。由于 GPCR 固有的构象灵活性，将这些从细胞膜中溶解到去污剂中的蛋白质稳定下来是一项具有挑战性的任务。在这里，我们利用天然存在的翻译后修饰来稳定去污剂胶束中的纯化 GPCR。重组大麻素 CB 受体在 Expi293F 哺乳动物细胞培养物中以高产率表达，在 Façade 去污剂中溶解和纯化。我们报告了与大肠杆菌表达的受体相比，哺乳动物细胞表达的受体具有更高的稳定性，这是由于 N 端糖基化和 C 端棕榈酰化的贡献。最后，我们证明了哺乳动物 Exp293F 氨基酸标记试剂盒适用于制备多毫克量的高质量、选择性稳定同位素标记的 GPCR，用于核磁共振研究。
