Direct Molecular Evolution of Detergent-Stable G Protein-Coupled Receptors Using Polymer Encapsulated Cells
Direct Molecular Evolution of Detergent-Stable G Protein-Coupled Receptors Using Polymer Encapsulated Cells
复制标题
DOI：
10.1016/j.jmb.2012.11.015
复制
发表时间：
2013-02-08
影响因子：
5.6
通讯作者：
Plueckthun, Andreas
中科院分区：
文献类型：
--
作者：
Scott, Daniel J.;Plueckthun, Andreas
G蛋白偶联受体（GPCR）是最大的一类药物蛋白质靶点，但药物开发受到缺乏有关其分子结构和构象动力学信息的阻碍。大多数机制和结构研究以及用纯化的受体进行的体外药物筛选需要GPCR的去污剂增溶，但通常，这些蛋白质在去污剂胶束中仅表现出低稳定性。我们已经开发出第一种定向进化方法，该方法允许从包含超过1亿个个体变体的文库中直接选择在所选洗涤剂中稳定的GPCR。关键的概念是封装库中的单个大肠杆菌细胞，每个细胞表达不同的GPCR变体，以形成耐洗涤剂的半渗透性纳米容器。与裸细胞不同，这些容器不会被洗涤剂溶解，使我们能够原位溶解GPCR蛋白，同时保持与蛋白质遗传信息的关联，这是定向进化的先决条件。控制孔径以允许GPCR配体渗透，但溶解的受体保留在纳米胶囊内。使用双标记的配体来结合纳米容器内的那些GPCR变体，所述变体在所测试的洗涤剂中保持活性。使用荧光激活细胞分选，可以鉴定来自两个不同家族A GPCR的洗涤剂稳定突变体，其中一些在短链洗涤剂中具有最高的稳定性。原则上，这种方法（称为细胞高通量封装、溶解和筛选）不限于工程化稳定的GPCR，而是可以用于稳定其他蛋白质以进行生物化学和结构研究。(C)2012爱思唯尔有限公司保留所有权利。
G protein-coupled receptors (GPCRs) are the largest class of pharmaceutical protein targets, yet drug development is encumbered by a lack of information about their molecular structure and conformational dynamics. Most mechanistic and structural studies as well as in vitro drug screening with purified receptors require detergent solubilization of the GPCR, but typically, these proteins exhibit only low stability in detergent micelles. We have developed the first directed evolution method that allows the direct selection of GPCRs stable in a chosen detergent from libraries containing over 100 million individual variants. The crucial concept was to encapsulate single Escherichia coli cells of a library, each expressing a different GPCR variant, to form detergent-resistant, semipermeable nano-containers. Unlike naked cells, these containers are not dissolved by detergents, allowing us to solubilize the GPCR proteins in situ while maintaining an association with the protein's genetic information, a prerequisite for directed evolution. The pore size was controlled to permit GPCR ligands to permeate but the solubilized receptor to remain within the nanocapsules. Fluorescently labeled ligands were used to bind to those GPCR variants inside the nano-containers that remained active in the detergent tested. With the use of fluorescence-activated cell sorting, detergent-stable mutants derived from two different family A GPCRs could be identified, some with the highest stability reported in short-chain detergents. In principle, this method (named cellular high-throughput encapsulation, solubilization and screening) is not limited to engineering stabilized GPCRs but could be used to stabilize other proteins for biochemical and structural studies. (C) 2012 Elsevier Ltd. All rights reserved.