Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

基于镧系元素的 Förster 共振能量转移 (LRET) 生物传感器能够对活细胞中的蛋白质-蛋白质相互作用 (PPI) 进行灵敏的时间选通发光 (TGL) 成像或多孔板分析。我们制备了表达多肽的稳定细胞系，该多肽由侧翼为 Tb(III) 复合物结合结构域、GFP 和每个末端两个相互作用结构域的 α 螺旋接头组成。检查的 PPI 包括 FKBP12 和 m-Tor 雷帕霉素结合域 (FRB) 之间以及 p53 (1-92) 和 HDM2 (1-128) 之间的 PPI。 TGL 显微镜显示生物传感器的开放（未结合）状态和闭合（结合）状态之间以供体或受体命名的 Tb(III) 与 GFP LRET 比率存在显着差异 (>500%)。当我们在 96 或 384 孔板中培养细胞并使用 TGL 读板器分析 PPI 变化时，我们观察到更大的信号变化 (>2,500%) 和 0.5 或更高的 Z' 因子。基于镧系元素的 LRET 生物传感器的模块化设计和卓越的动态范围将促进 PPI 的多功能成像和基于细胞的筛选。