High-Throughput Nano-Scale Characterization of Membrane Proteins Using Fluorescence-Detection Size-Exclusion Chromatography.
High-Throughput Nano-Scale Characterization of Membrane Proteins Using Fluorescence-Detection Size-Exclusion Chromatography.
复制标题
使用荧光检测尺寸排阻色谱法对膜蛋白进行高通量纳米级表征。
DOI：
10.1007/978-1-4939-9624-7_17
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Stroud,RobertM
中科院分区：
文献类型：
--
作者：
Vecchio,AlexJ;Stroud,RobertM
结构生物学已经揭示，从蛋白质的一级结构预测其异源表达水平、同质性和稳定性是极其困难的。特别是膜蛋白，提出了许多挑战，使获得毫克量的质量样品的问题。然而，对于这些分子的结构和功能研究，这是所需要的。荧光尺寸排阻色谱法（F-SEC）是一种将生物学目的蛋白质与绿色荧光蛋白（GFP）融合并进行监测的技术，它规避了膜蛋白结构生物学固有的许多瓶颈。体内表达产率以及体外均一性和稳定性可以利用纳克量的未纯化蛋白质快速评估。在这一章中，我们描述了我们最新的协议，表达筛选和膜蛋白的生化特性，使用F-SEC，因为它涉及到一个高通量（HTP）的结晶管道。其中，所述方法和工作流程被设计和优化用于真核整合膜蛋白的结构-功能阐明，但是可以应用于具有微小修饰的原核或水溶性蛋白，从而使其成为有用的通用方法。
Structural biology has revealed predicting heterologous expression levels, homogeneity, and stability of a protein from its primary structure are exceedingly difficult. Membrane proteins, in particular, present numerous challenges that make obtaining milligram quantities of quality samples problematic. For structural and functional investigation of these molecules, however, this is what is required. Fluorescence size-exclusion chromatography (F-SEC), a technique where a protein of biological interest is fused to green fluorescent protein (GFP) and monitored, circumvents many bottlenecks inherent to membrane protein structural biology. In vivo expression yields, as well as in vitro homogeneity and stability, can be rapidly evaluated utilizing nanogram quantities of unpurified protein. In this chapter we describe our most current protocols for expression screening and biochemical characterization of membrane proteins using F-SEC, as it pertains to a high-throughput (HTP) crystallographic pipeline. Therein, the methods and workflow were designed and optimized for structure–function elucidation of eukaryotic integral membrane proteins, but may be applied to prokaryotic or water-soluble proteins with minor modifications, thus making it a useful general approach.
使用毕赤酵母中绿色荧光蛋白融合伴侣体内监测人白细胞介素 2 的细胞内表达
影响因子：
2.7
作者：
H. Cha;N. G. Dalal;W. Bentley
通讯作者：
W. Bentley