Olfactory receptor expression libraries and methods of making and using them
First Claim
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1. A method of determining whether a test compound binds to a mammalian olfactory receptor comprising the following steps:
- (i). expressing a nucleic acid under conditions permissive for translation of the nucleic acid to a receptor polypeptide, wherein the nucleic acid encodes a 7-transmembrane polypeptide that can transverse a plasma membrane seven times and wherein the nucleic acid comprises the following domains in 5′
to 3′
order;
a nucleic acid encoding an amino terminal plasma membrane translocation domain as shown in SEQ ID NO;
3;
a nucleic acid encoding a first transmembrane domain as shown in residues 2 1–
79 of SEQ ID NO;
55; and
a nucleic acid encoding an olfactory receptor ligand-binding region as shown in SEQ ID NO;
48;
(ii). contacting the translated polypeptide with the test compound; and
(iii). determining whether the test compound binds to the polypeptide.
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Abstract
This invention provides novel libraries of olfactory receptor odorant/ligand-binding domains and methods of making and using them. The invention also provides libraries of vectors and cells comprising these nucleic acid constructs. The compositions and methods of the invention are used to identify novel ligand-binding domains for olfactory neuron odorant receptors and their ligands. Thus, the compositions and methods of the invention can be used to generate novel odorants, to screen for toxic odorants, or to manipulate an animal'"'"'s olfactory response.
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Citations
6 Claims
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1. A method of determining whether a test compound binds to a mammalian olfactory receptor comprising the following steps:
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(i). expressing a nucleic acid under conditions permissive for translation of the nucleic acid to a receptor polypeptide, wherein the nucleic acid encodes a 7-transmembrane polypeptide that can transverse a plasma membrane seven times and wherein the nucleic acid comprises the following domains in 5′
to 3′
order;a nucleic acid encoding an amino terminal plasma membrane translocation domain as shown in SEQ ID NO;
3;a nucleic acid encoding a first transmembrane domain as shown in residues 2 1–
79 of SEQ ID NO;
55; anda nucleic acid encoding an olfactory receptor ligand-binding region as shown in SEQ ID NO;
48;(ii). contacting the translated polypeptide with the test compound; and (iii). determining whether the test compound binds to the polypeptide.
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2. A method of determining whether a test compound binds to a mammalian olfactory transmembrane receptor comprising the following steps:
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(i). culturing a cell stably or transiently transfected with a chimeric nucleic acid which encodes a 7-transmembrane polypeptide that can transverse a plasma membrane seven times, said chimeric nucleic acid comprising a first, second, and third nucleic acid sequence in 5′
to 3′
order;a first nucleic acid sequence encoding an amino terminal plasma membrane translocation domain as shown in SEQ ID NO;
3;a second nucleic acid sequence encoding a first transmembrane domain as shown in residues 21–
79 of SEQ ID NO;
55; anda third nucleic acid sequence encoding an olfactory receptor ligand-binding region as shown in SEQ ID NO;
48;under conditions permissive for translation of the nucleic acid to form a 7-transmembrane polypeptide on the cell'"'"'s plasma membrane outer surface; (ii). contacting the cell with the test compound; and (iii). determining whether the test compound binds to the 7-transmembrane polypeptide. - View Dependent Claims (3, 4, 5, 6)
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Specification