An adaptable luminescence resonance energy transfer assay for measuring and screening protein-protein interactions and their inhibition.
Chembiochem
; 13(4): 553-8, 489, 2012 Mar 05.
Article
en En
| MEDLINE
| ID: mdl-22271654
ABSTRACT
Protein-protein interactions (PPIs) are central to biological processes and represent an important class of therapeutic targets. Here we show that the interaction between FK506-binding protein 12 fused to green fluorescent protein (GFP-FKBP) and the rapamycin-binding domain of mTor fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) can be sensitively detected (signal-to-background ratio (S/B)>100) and accurately quantified within an impure cell lysate matrix using a luminescence resonance energy transfer (LRET) assay. Ascomycin-mediated inhibition of GFP-FKBP-rapamycin-FRB-eDHFR complex formation was also detected at high S/B ratio (>80) and Z'-factor (0.89). The method leverages the selective, stable binding of trimethoprim (TMP)-terbium complex conjugates to eDHFR, and time-resolved, background-free detection of the long-lifetime (â¼ms) terbium-to-GFP LRET signal that indicates target binding. TMP-eDHFR labeling can be adapted to develop high-throughput screening assays and complementary, quantitative counter-screens for a wide variety of PPI targets with a broad range of affinities that may not be amenable to purification.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Tetrahidrofolato Deshidrogenasa
/
Proteínas de Unión a Tacrolimus
/
Transferencia Resonante de Energía de Fluorescencia
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Proteínas Fluorescentes Verdes
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Serina-Treonina Quinasas TOR
Tipo de estudio:
Diagnostic_studies
/
Screening_studies
Límite:
Humans
Idioma:
En
Revista:
Chembiochem
Asunto de la revista:
BIOQUIMICA
Año:
2012
Tipo del documento:
Article
País de afiliación:
Estados Unidos