Mhurchú Niamh Ní, Zoubak Lioudmila, McGauran Gavin, Linse Sara, Yeliseev Alexei, O'Connell David J
School of Biomolecular and Biomedical Science, Conway Institute , University College Dublin , Dublin D04 V1W8 , Ireland.
National Institute on Alcohol Abuse and Alcoholism , National Institutes of Health , 5625 Fishers Lane , Bethesda , Maryland 20892 , United States.
Biochemistry. 2018 Jul 31;57(30):4383-4390. doi: 10.1021/acs.biochem.8b00469. Epub 2018 Jul 17.
The process of isolating recombinant G protein-coupled receptors from membrane preparations is challenging because the process requires solubilization in detergent micelles and multistep affinity chromatography protocols. Solubilization buffers contain high concentrations of salts, detergents, and glycerol that create stringent conditions necessary to stabilize the receptor but in which affinity chromatography resins perform poorly, and these resins also require the addition of eluting agents that complicate downstream assays. To simplify this process we have developed a high affinity fragment complementation molecular switch as a highly specific system for receptor capture in solubilization buffer with a calcium chelation-based elution step releasing functional protein in a simple buffer. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G protein-coupled receptor CB, in comparison with commercially available systems. This powerful tool may be applied to any recombinant membrane bound protein and can be further optimized to enhance the yield and purity of the most challenging protein targets for study.
从膜制剂中分离重组G蛋白偶联受体的过程具有挑战性，因为该过程需要在去污剂胶束中溶解，并采用多步亲和色谱方案。溶解缓冲液含有高浓度的盐、去污剂和甘油，这些成分创造了稳定受体所需的严格条件，但在这种条件下亲和色谱树脂的性能较差，而且这些树脂还需要添加洗脱剂，这会使下游分析变得复杂。为了简化这一过程，我们开发了一种高亲和力片段互补分子开关，作为一种高度特异性的系统，用于在溶解缓冲液中捕获受体，并通过基于钙螯合的洗脱步骤在简单缓冲液中释放功能蛋白。在此，我们详细描述了这种新型亲和色谱系统在大麻素G蛋白偶联受体CB1的分离和纯化中的设计、方法、解释及局限性，并与市售系统进行了比较。这个强大的工具可应用于任何重组膜结合蛋白，并且可以进一步优化，以提高最具挑战性的研究蛋白靶点的产量和纯度。
