期刊
BIOSENSORS & BIOELECTRONICS
卷 29, 期 1, 页码 119-124出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2011.08.004
关键词
GPCR; Olfactory receptors; ODR-10; Diacetyl; Renilla luciferase; BRET
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. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.
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