A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells.
PLoS One
; 5(2): e9344, 2010 Feb 22.
Article
em En
| MEDLINE
| ID: mdl-20179761
ABSTRACT
BACKGROUND:
Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.RESULTS:
Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.CONCLUSION:
The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Virais
/
Mapeamento de Interação de Proteínas
/
Transferência Ressonante de Energia de Fluorescência
/
Citometria de Fluxo
Tipo de estudo:
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article