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生物发光共振能量转移的应用,以量化膜蛋白的细胞表面表达

Jordan M Mattheisen¹, Victoria A Rasmussen¹, Emilie Ceraudo²

¹Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, NY, 10065, USA; Tri-Institutional PhD Program in Chemical Biology, New York, NY, 10065, USA.

Analytical biochemistry

|October 21, 2023

概括

我们开发了一种新的生物发光共振能量转移 (BRET) 试验,以准确测量细胞表面的工程膜蛋白. 这种方法为量化蛋白质局部化提供了一个强大的,高通量替代方案.

科学领域:

生物化学生物化学
细胞生物学细胞生物学
生物物理学的生物物理.

背景情况:

量化膜蛋白定位对于理解细胞过程至关重要.
现有的方法,如光共振能量转移 (FRET) 有局限性,包括光漂白和自光.
准确评估细胞表面蛋白质表达对于药物发现和生物研究至关重要.

研究的目的:

开发和验证一种新的生物发光共振能量转移 (BRET) 试验,用于量化细胞表面与细胞内膜蛋白.
为了克服传统的细胞内光共振能量转移 (FRET) 试验的局限性.
为测量膜蛋白表面表达提供一个强大的,定量和高通量方法.

主要方法:

通过NanoLuc luciferase (NLuc) 和HaloTag的融合,设计了G蛋白合受体 (GPCR).
采用脉冲追踪标签策略,使用光谱明显的,细胞不透的光染料.
测量BRET从NLuc化学发光到HaloTag结合的光体,以区分表面和细胞内受体.

主要成果:

在BRET测试准确量化工程膜蛋白质在细胞表面与细胞内部的部分.
证明了细胞表面和细胞内局部受体之间的定量差异化.
与酶相关免疫吸收试验 (ELISA) 和免疫涂抹试验相比,该试验显示出强度和定量准确性.

结论:

开发的BRET试验提供了一种敏感和可靠的方法来测量膜蛋白表面表达.
这种测试尽量减少了与细胞内FRET相关的局限性,例如光漂白和自光.
微型板格式显著增加了吞吐量,使其适合大规模选和研究.

关键词:

生物发光共振能量转移 (BRET) 是指生物发光共振能量转移.G蛋白结合受体 (GPCR) 是一种这是HaloTag的标签.表面表达的表达方式纳米BRETET是一个纳米BRETET.

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