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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 vomeronasalmore » receptors.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
913975
Report Number(s):
BNL-78543-2007-JA
TRN: US200804%%390
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Chem. Senses; Journal Volume: 31
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CHROMATOGRAPHY; DETECTION; ESCHERICHIA COLI; FUNCTIONALS; ODOR; POLYPEPTIDES; PROTEINS; PURIFICATION; LIGANDS; TASTE BUDS; national synchrotron light source

Citation Formats

Nie,Y., Hobbs, J., Vigues, S., Olson, W., Conn, G., and Munger, S. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors. United States: N. p., 2006. Web. doi:10.1093/chemse/bjj053.
Nie,Y., Hobbs, J., Vigues, S., Olson, W., Conn, G., & Munger, S. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors. United States. doi:10.1093/chemse/bjj053.
Nie,Y., Hobbs, J., Vigues, S., Olson, W., Conn, G., and Munger, S. Sun . "Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors". United States. doi:10.1093/chemse/bjj053.
@article{osti_913975,
title = {Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors},
author = {Nie,Y. and Hobbs, J. and Vigues, S. and Olson, W. and Conn, G. and Munger, S.},
abstractNote = {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.},
doi = {10.1093/chemse/bjj053},
journal = {Chem. Senses},
number = ,
volume = 31,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}