Thermal precipitation fluorescence assay for protein stability screening
Thermal precipitation fluorescence assay for protein stability screening
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DOI：
10.1016/j.jsb.2011.05.003
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发表时间：
2011-09-01
影响因子：
3
通讯作者：
Zhang, Xuejun C.
中科院分区：
文献类型：
--
作者：
Fan, Junping;Huang, Bo;Zhang, Xuejun C.
一种简单可靠的蛋白质稳定性评估方法对于重组蛋白的高通量表达筛选来说是理想的。在这里，我们描述了一种称为热沉淀荧光（TPF）的测定法，可用于比较直接来自细胞裂解物上清液的重组蛋白样品的热稳定性。在此测定中，靶膜蛋白表达为带有绿色荧光蛋白标签的重组融合物，并用去污剂溶解，荧光信号用于报告细胞裂解物可溶部分中融合蛋白的数量。热激后，通过离心除去不溶性蛋白质聚集体。随后，通过凝胶内荧光分析对上清液中剩余蛋白质的量进行定量，并与未经热激处理的样品进行比较。在存在和不存在几种常用去垢剂的情况下，对来自大肠杆菌的 60 多种重组膜蛋白进行了筛选，并对结果进行了分析。由于不需要复杂的蛋白质纯化，因此该 TPF 技术适用于重组膜蛋白以及可溶性膜蛋白的高通量表达筛选，并且可用于根据目标蛋白的热稳定性对目标蛋白进行优先级排序，以进行后续的大规模表达和结构研究。 (C) 2011 Elsevier Inc. 保留所有权利。
A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. (C) 2011 Elsevier Inc. All rights reserved.