NanoBRET--A Novel BRET Platform for the Analysis of Protein-Protein Interactions.
ACS Chem Biol
; 10(8): 1797-804, 2015 Aug 21.
Article
en En
| MEDLINE
| ID: mdl-26006698
ABSTRACT
Dynamic interactions between proteins comprise a key mechanism for temporal control of cellular function and thus hold promise for development of novel drug therapies. It remains technically challenging, however, to quantitatively characterize these interactions within the biologically relevant context of living cells. Although, bioluminescence resonance energy transfer (BRET) has often been used for this purpose, its general applicability has been hindered by limited sensitivity and dynamic range. We have addressed this by combining an extremely bright luciferase (Nanoluc) with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The small size (19 kDa), high emission intensity, and relatively narrow spectrum (460 nm peak intensity) make Nanoluc luciferase well suited as an energy donor. By selecting an efficient red-emitting fluorophore (635 nm peak intensity) for attachment onto the HaloTag, an overall spectral separation exceeding 175 nm was achieved. This combination of greater light intensity with improved spectral resolution results in substantially increased detection sensitivity and dynamic range over current BRET technologies. Enhanced performance is demonstrated using several established model systems, as well as the ability to image BRET in individual cells. The capabilities are further exhibited in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Mapeo de Interacción de Proteínas
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Luciferasas de Renilla
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Mapas de Interacción de Proteínas
/
Colorantes Fluorescentes
Límite:
Humans
Idioma:
En
Revista:
ACS Chem Biol
Año:
2015
Tipo del documento:
Article
País de afiliación:
Estados Unidos