Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence resonance energy transfer (BRET).
Biosens Bioelectron
; 29(1): 119-24, 2011 Nov 15.
Article
em En
| MEDLINE
| ID: mdl-21873043
ABSTRACT
Our goal is to develop a general transduction system for G-protein coupled receptors (GPCRs). GPCRs are present in most eukaryote cells and transduce diverse extracellular signals. GPCRs comprise not only the largest class of integral membrane receptors but also the largest class of targets for therapeutic drugs. In all cases studied, binding of ligand to a GPCR leads to a sub-nanometer intramolecular rearrangement. Here, we report the creation of a novel chimaeric BRET-based biosensor by insertion of sequences encoding a bioluminescent donor and a fluorescent acceptor protein into the primary sequence of a GPCR. The BRET(2)-ODR-10 biosensor was expressed in membranes of Saccharomyces cerevisiae. Assays conducted on isolated membranes indicated an EC(50) in the femtomolar range for diacetyl. The response was ligand-specific and was abolished by a single point mutation in the receptor sequence. Novel BRET-GPCR biosensors of this type have potential application in many fields including explosive detection, quality control of food and beverage production, clinical diagnosis and drug discovery.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
/
Receptores Acoplados a Proteínas G
/
Técnicas de Transferência de Energia por Ressonância de Bioluminescência
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Limite:
Animals
Idioma:
En
Revista:
Biosens Bioelectron
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Austrália