title: Expression and purification of functional ligand-binding domains of T1R3 taste receptors.
abstract: Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.
authors.affiliation: Department of Anatomy and Neurobiology, Graduate Programs in Life Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: Yiling
authors.initials: Y
authors.lastname: Nie
authors.forename: Jeanette R
authors.initials: JR
authors.lastname: Hobbs
authors.forename: Stephan
authors.initials: S
authors.lastname: Vigues
authors.forename: Wendy J
authors.initials: WJ
authors.lastname: Olson
authors.forename: Graeme L
authors.initials: GL
authors.lastname: Conn
authors.forename: Steven D
authors.initials: SD
authors.lastname: Munger
mesh_terms: D000818:Animals; D050260:Carbohydrate Metabolism; D002942:Circular Dichroism; D015870:Gene Expression / Q000235:genetics*; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008024:Ligands; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C431926:taste receptors, type 1