NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
Methods Mol Biol. 2024;2797:253-260. doi: 10.1007/978-1-0716-3822-4_18.
Bioluminescence resonance energy transfer (BRET) is a valuable technique for studying protein-protein interactions (PPIs) within live cells (Pfleger and Eidne, Nat Methods 3:165-174, 2006). Among the various BRET methodologies, a recent addition called NanoBRET has emerged, leveraging advancements in donor and acceptor technologies (Machleidt and Woodroofe, ACS Chem Biol 10:1797-1804, 2015). In this study, we present a developed methodology designed to measure PPIs involving the RAS protein family and their effectors and interactors at the plasma membrane. By utilizing the NanoLuc and HaloTag BRET pair, we provide evidence of a saturable interaction between KRAS4b-G12D and full-length RAF1. Conversely, the RAF1 R89L mutant, known to impede RAF1 binding to active RAS, exhibits nonspecific interactions. The assay exhibits remarkable signal-to-background ratios and is highly suitable for investigating the interactions of RAS with effectors, as well as for high-throughput screening assays.
生物发光共振能量转移（BRET）是一种研究活细胞内蛋白质-蛋白质相互作用（PPIs）的有效技术（Pfleger 和 Eidne，Nat Methods 3:165-174, 2006）。在各种 BRET 方法中，最近出现了一种称为 NanoBRET 的新技术，它利用了供体和受体技术的进步（Machleidt 和 Woodroofe，ACS Chem Biol 10:1797-1804, 2015）。在这项研究中，我们提出了一种开发的方法，旨在测量涉及 RAS 蛋白家族及其在质膜上的效应物和相互作用物的 PPI。通过利用 NanoLuc 和 HaloTag BRET 对，我们提供了 KRAS4b-G12D 和全长 RAF1 之间存在可饱和相互作用的证据。相反，已知阻碍 RAF1 与活性 RAS 结合的 RAF1 R89L 突变体表现出非特异性相互作用。该测定法具有显著的信号与背景比，非常适合研究 RAS 与效应物的相互作用，也非常适合高通量筛选测定法。
