Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.
iScience
; 23(9): 101533, 2020 Sep 25.
Article
en En
| MEDLINE
| ID: mdl-33083762
ABSTRACT
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.
Texto completo:
1
Bases de datos:
MEDLINE
Tipo de estudio:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Revista:
IScience
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos