Chiu Mark L, Tsang Cindy, Grihalde Nelson, MacWilliams Maria P
Department of Structural Biology, Abbott Laboratories, R46Y AP10 LL8, 100 Abbott Park Road, Abbott Park, IL 60064-6098, USA.
Comb Chem High Throughput Screen. 2008 Jul;11(6):439-62. doi: 10.2174/138620708784911456.
With the advent of the recent determination of high-resolution crystal structures of bovine rhodopsin and human beta2 adrenergic receptor (beta2AR), there are still many structure-function relationships to be learned from other G protein-coupled receptors (GPCRs). Many of the pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergence from bovine rhodopsin and beta2AR. Structure determination of GPCRs can provide new avenues for engineering drugs with greater potency and higher specificity. Several obstacles need to be overcome before membrane protein structural biology becomes routine: over-expression, solubilization, and purification of milligram quantities of active and stable GPCRs. Coordinated iterative efforts are required to generate any significant GPCR over-expression. To formulate guidelines for GPCR purification efforts, we review published conditions for solubilization and purification using detergents and additives. A discussion of sample preparation of GPCRs in detergent phase, bicelles, nanodiscs, or low-density lipoproteins is presented in the context of potential structural biology applications. In addition, a review of the solubilization and purification of successfully crystallized bovine rhodopsin and beta2AR highlights tools that can be used for other GPCRs.
随着牛视紫红质和人β2肾上腺素能受体（β2AR）高分辨率晶体结构的最新测定结果的出现，仍有许多结构-功能关系有待从其他G蛋白偶联受体（GPCR）中了解。许多具有药学意义的GPCR由于其氨基酸序列与牛视紫红质和β2AR存在差异而无法建模。GPCR的结构测定可为设计更高效力和更高特异性的药物提供新途径。在膜蛋白结构生物学成为常规方法之前，需要克服几个障碍：毫克量活性和稳定的GPCR的过量表达、溶解和纯化。需要协同的迭代努力来实现任何显著的GPCR过量表达。为了制定GPCR纯化工作的指导方针，我们回顾了已发表的使用去污剂和添加剂进行溶解和纯化的条件。在潜在的结构生物学应用背景下，讨论了在去污剂相、双分子层、纳米圆盘或低密度脂蛋白中制备GPCR样品的方法。此外，对成功结晶的牛视紫红质和β2AR的溶解和纯化的回顾突出了可用于其他GPCR的工具。
