Expression and characterization of ligand-binding domain of T1R1 taste receptor

Umami is the typical taste induced by monosodium glutamate,which is thought to be detected by a heterodimeric G-proteincoupled receptors, T1R1 and T1R3. The most unique feature ofumami taste is its potentiation by purine nucleotidemonophosphates (IMP, GMP), which also elicit umami taste bytheir own. Zhang et al. (Proc. Natl Acad. Sci. USA, 2008) haverecently proposed a cooperative ligand-binding model involvingT1R1 N-terminal domain (NTD), where L-glutamate (L-glu)binds close to the hinge region, and purine nucleotides bind to anadjacent site close to the opening of the Venus flytrap domain. Tofurther understand the structural basis of umami stimulirecognition by T1R1, a large amount of purified T1R1 NTD issuitable for biochemical and structural studies. Here, we reportthe successful expression and purification of the soluble NTD ofhuman T1R1. The protein was expressed as insoluble aggregatedprotein (inclusion bodies) expressed in high level in Escherichiacoli. The protein was solubilized and in vitro refolded usingsuitable buffer and additives. We then measured the binding ofumami stimuli to T1R1 NTD using fluorescence spectroscopy.Fluorescent assay demonstrated that T1R1 NTD is properlyrefolded and able to bind L-glu with physiological relevantaffinity. The mode of interaction was specific since T1R1 NTDdid not bind sweet stimuli like fructose, sucralose or glycine. Inaddition, we observed that L-glu binding affinity is enhanced inpresence of IMP. To further validate our results, we havegenerated single amino-acid changes in L-Glu and IMP bindingsites. In summary, our expression system will allow large scaleproduction of active protein suitable for structural and functionalstudies.