Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2023;2706:137-148. doi: 10.1007/978-1-0716-3397-7_10.
Protein-protein interactions (PPIs) are increasingly recognized for their roles in functional cellular networks and their importance in disease-targeting contexts. Assessing PPI in the native cellular environment is challenging and requires specific and quantitative methods. Bioluminescence resonance energy transfer (BRET) is a biophysical process that can be used to quantify PPI. With Nanoluciferase bioluminescent protein as a donor and a fluorescent chloroalkane ligand covalently bound to HaloTag protein as an acceptor, NanoBRET provides a versatile and robust system to quantitatively measure PPI in living cells. BRET efficiency is proportional to the distance between the donor and acceptor, allowing for the measurement of PPI in real time. In this paper, we describe the use of NanoBRET to study specific interactions between proteins of interest in living cells that can be perturbed by using small-molecule antagonists and genetic mutations. Here, we provide a detailed protocol for expressing NanoLuc and HaloTag fusion proteins in cell culture and the necessary optimization of NanoBRET assay conditions. Our example results demonstrate the reliability and sensitivity of NanoBRET for measuring interactions between proteins, protein domains, and short peptides and quantitating the PPI antagonist compound activity in living cells.
蛋白质-蛋白质相互作用（PPIs）在功能细胞网络中的作用及其在疾病靶点中的重要性越来越受到重视。在天然细胞环境中评估 PPI 具有挑战性，需要特定和定量的方法。生物发光共振能量转移（BRET）是一种可以用来定量 PPI 的生物物理过程。使用 Nanoluciferase 生物发光蛋白作为供体，将荧光氯烷烃配体共价结合到 HaloTag 蛋白上作为受体，NanoBRET 提供了一种通用且强大的系统，可以在活细胞中定量测量 PPI。BRET 效率与供体和受体之间的距离成正比，允许实时测量 PPI。在本文中，我们描述了使用 NanoBRET 来研究活细胞中感兴趣的蛋白质之间的特定相互作用，这些相互作用可以通过使用小分子拮抗剂和基因突变来干扰。在这里，我们提供了在细胞培养中表达 NanoLuc 和 HaloTag 融合蛋白的详细方案，并对 NanoBRET 测定条件进行了必要的优化。我们的示例结果证明了 NanoBRET 用于测量蛋白质、蛋白质结构域和短肽之间相互作用以及定量活细胞中 PPI 拮抗剂化合物活性的可靠性和敏感性。
