Chen Ting, Pham Ha, Mohamadi Ali, Miller Lawrence W
Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA.
iScience. 2020 Sep 6;23(9):101533. doi: 10.1016/j.isci.2020.101533. eCollection 2020 Sep 25.
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.
基于镧系元素的荧光共振能量转移（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）进行灵敏的时间分辨发光（TGL）成像或多孔板分析。我们制备了稳定的细胞系，这些细胞系表达的多肽由一个α螺旋连接子组成，该连接子两侧分别是一个Tb(III)复合物结合结构域、绿色荧光蛋白（GFP），以及在每个末端的两个相互作用结构域。所检测的PPI包括FKBP12与m-Tor的雷帕霉素结合结构域（FRB）之间的相互作用，以及p53（1-92）与HDM2（1-128）之间的相互作用。TGL显微镜显示，在生物传感器的开放（未结合）和封闭（结合）状态之间，以供体或受体为单位的Tb(III)到GFP的LRET比率存在显著差异（>500%）。当我们在96孔或384孔板中培养细胞，并使用TGL酶标仪分析PPI变化时，我们观察到更大的信号变化（>2500%）和Z'因子为0.5或更高。基于镧系元素的LRET生物传感器的模块化设计和出色的动态范围将有助于对PPI进行多功能成像和基于细胞的筛选。
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