Xu Hui, Clairfeuille Thomas, Jao Christine C, Ho Hoangdung, Sweeney Zachary, Payandeh Jian, Koth Christopher M
Department of Structural Biology, Genentech Inc., South San Francisco, CA, USA.
Department of Discovery Chemistry, Genentech Inc., South San Francisco, CA, USA.
Methods Mol Biol. 2019;2025:389-402. doi: 10.1007/978-1-4939-9624-7_18.
Integral membrane proteins (MP) are implicated in many disease processes and are the primary targets of numerous marketed drugs. Despite recent advances in the areas of MP solubilization, stabilization, and reconstitution, it remains a time-consuming task to identify the combination of constructs and purification conditions that will enable MP structure-function studies outside of the lipid bilayer. In this chapter, we describe a strategy for rapidly identifying and optimizing the solubilization and purification conditions for nearly any recombinant MP, based on the use of a noninvasive fluorescent probe (His-Glow) that specifically binds to the common hexahistidine affinity tag of expressed targets. This His-Glow approach permits fluorescent size-exclusion chromatography (FSEC) without the need for green fluorescent protein (GFP) fusion. A two-stage detergent screening strategy is employed at the solubilization stage, whereby appropriate detergent families are identified first, followed by optimization within these families. Screening up to 96 unique combinations of solubilization conditions and constructs can be achieved in less than 24 h. At the outset of each new project, we screen six different detergents for each construct and the subsequent implementation of a simple thermostability challenge further aids in the identification of constructs and conditions suitable for large-scale production. Our strategy streamlines the parallel optimization of appropriate production conditions for multiple MP targets to rapidly enable downstream biochemical, immunization, or structural studies.
整合膜蛋白（MP）与许多疾病过程相关，并且是众多已上市药物的主要靶点。尽管在MP的溶解、稳定和重组方面取得了最新进展，但要确定能够在脂质双层之外进行MP结构功能研究的构建体和纯化条件的组合，仍然是一项耗时的任务。在本章中，我们描述了一种基于使用非侵入性荧光探针（His-Glow）的策略，该探针特异性结合表达靶点的常见六聚组氨酸亲和标签，用于快速鉴定和优化几乎任何重组MP的溶解和纯化条件。这种His-Glow方法允许进行荧光尺寸排阻色谱（FSEC），而无需绿色荧光蛋白（GFP）融合。在溶解阶段采用两阶段去污剂筛选策略，首先确定合适的去污剂家族，然后在这些家族内进行优化。在不到24小时内可以筛选多达96种独特的溶解条件和构建体组合。在每个新项目开始时，我们为每个构建体筛选六种不同的去污剂，随后进行简单的热稳定性挑战，这进一步有助于鉴定适合大规模生产的构建体和条件。我们的策略简化了对多个MP靶点合适生产条件的并行优化，以便快速开展下游生化、免疫或结构研究。
