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T1r hetero-oligomeric taste receptors, cell lines that express said receptors, and taste compounds

a taste receptor and heterooligomeric technology, applied in the field of t1r heterooligomeric taste receptors, cell lines that express said receptors, and taste compounds, can solve the problems of difficult and expensive to isolate and purify from natural sources, little progress has been made in identifying artificial substitutes for msg, and adverse reactions of msg in some people, etc., to achieve high throughput cell-based screening and high throughput

Inactive Publication Date: 2007-05-10
SENOMYX INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MSG is known to produce adverse reactions in some people, but very little progress has been made in identifying artificial substitutes for MSG.
For example the naturally occurring nucleotide compounds inosine monophosphate (IMP) or guanosine monophosphate (GMP) are known to have a multiplier effect on the savory taste of MSG, but IMP and GMP are very difficult and expensive to isolate and purify from natural sources, or synthesize, and hence have only limited practical application to most commercial needs in food or medicinal compositions.

Method used

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  • T1r hetero-oligomeric taste receptors, cell lines that express said receptors, and taste compounds
  • T1r hetero-oligomeric taste receptors, cell lines that express said receptors, and taste compounds
  • T1r hetero-oligomeric taste receptors, cell lines that express said receptors, and taste compounds

Examples

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example 1

Production of Intronless HT1R Expression Constructs

[0347] Intronless hT1R expression constructs were cloned by a combination of cDNA-based and genomic DNA-based methods. To generate the fall-length hT1R1 expression construct, two 5′ coding exons identified in a cloned hT1R1 interval (accession # AL159177) were combined by PCR-overlap, and then joined to a 5′-truncated testis cDNA clone. The hT1R2 expression construct was generated from a partially sequenced hT1R2 genomic interval. Two missing hT1R2 5′ exons were identified by screening shotgun libraries of the cloned genomic interval using probes derived from the corresponding rat coding sequence. Coding exons were then combined by PCR-overlap to produce the full-length expression construct. The hT1R3 expression construct was generated by PCR-overlap from a sequenced hT1R3 genomic interval (accession # AL139287). Rat T1R3 was isolated from a rat taste tissue-derived cDNA library using an rT1R3 exon fragment generated by hT1R3-base...

example 2

Sequence Alignment of Human and Rat T1Rs

[0357] Cloned T1R sequences selected from those identified above were aligned against the corresponding rat T1Rs. As shown in FIG. 1, human T1R1, human T1R2 and human T1R3 and rat T1R3 were aligned with previously described T1Rs (rT1R1 having Accession # AAD 18069 and rT1R2 having Accession # AAD 18070), the rat mGluR1 metabotropic, glutamate receptor (Accession # P23385); and the human calcium-sensing receptor (Accession #P41180). For clarity of the comparison, the mGluR1 and calcium-sensing receptor C-termini are truncated. The seven potential transmembrane segments are boxed in blue. Residues that contact the glutamate side-chain carbutylate in the mGluRl crystal structure are boxed in red, and residues that contact the glutamate α-amino acid moiety are boxed in green. The mGluR1 and calcium-sensing receptor cysteine residues implicated in intersubunit disulfide-based formation are circled in purple. These cysteines are not conserved in T...

example 3

Demonstration by RT-PCR that hT1R2 and hT1R3 are Expressed in Taste Tissue

[0358] As shown in FIG. 2, hT1R2 and hT1R3 are expressed in taste tissue: expression of both genes can be detected by RT-PCR from resected human circumvallate papillae.

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Abstract

The invention relates to compounds that specifically bind a T1R1 / T1R3 or T1R2 / T1R3 receptor or fragments or sub-units thereof. The present invention also relates to the use of hetero-oligomeric and chimeric taste receptors comprising T1R1 / T1R3 and T1R2 / T1R3 in assays to identify compounds that respectively respond to umami taste stimuli and sweet taste stimuli. Further, the invention relates to the constitutive of cell lines that stably or transiently co-express a combination of T1R1 and T1R3; or T1R2 and T1R3; under constitutive or inducible conditions. The use of these cells lines in cell-based assays to identify umami and sweet taste modulatory compounds is also provided, particularly high throughput screening assays that detect receptor activity by use of fluorometric imaging.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application Ser. No. 60 / 494,071 filed on Aug. 6, 2003, and U.S. Provisional Application Ser. No. 60 / 552,064 filed Mar. 9, 2004, both of which are incorporated by reference in their entirety.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention; in part relates to the discovery that the T1R receptors assemble to form functional taste receptors. Particularly, it has been discovered that co-expression of T1R1 and T1R3 results in a taste receptor that responds to umami taste stimuli, including monosodium glutamate. Also, it has been discovered that co-expression of the T1R2 and T1R3 receptors results in a taste receptor that responds to sweet taste stimuli including naturally occurring and artificial sweeteners. [0004] Also, the present invention relates to the use of hetero-oligomeric taste receptors comprising T1R1 / T1R3 and T1R2 / T1R3 in assays to iden...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K16/28A23L23/00A23L27/00A23L27/20A23L27/30A23L33/20A61K9/00A61K47/00C07C233/65C07C235/54C07C275/30C07C275/34C07D307/84C07D317/68G01N33/566G01N33/74
CPCA23L1/22614A23L1/2265A23L1/22678C07C233/65C07C235/54C07C275/30G01N33/5041C07D307/84C07D317/68G01N33/566G01N33/74G01N2333/726G01N33/543C07C275/34A23L27/202A23L27/203A23L27/2054C07D263/56C07D213/81C07D213/85C07D215/48C07D217/06C07D231/14C07D333/24C07D333/38C07D401/04C07D409/12C07D417/12C07D261/18C07D271/12C07D275/03C07D209/08C07D277/64C07D209/42C07D285/135C07D213/40C07D307/52C07D307/68C07D307/79C07D307/82C07D405/12A23L2/56A23V2002/00A61K8/4973A61K8/55A61K47/22A61K47/24C07F9/65517A23L27/88
Inventor LI, XIAODONGSTASZEWSKI, LENAXU, HONG
Owner SENOMYX INC
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