Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions

iScience

Ha Pham¹  Ali Mohamadi¹  Lawrence W. Miller¹  Ting Chen¹ 

[1] Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA;

关键词: Sensor;    Molecular Spectroscopy Techniques;    Molecular Interaction;    Biomolecular Engineering;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: 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.

【 授权许可】

Unknown