Olfactory receptor expression libraries and methods of making and using them

• US 7,138,242 B2
• Filed: 09/30/2002
• Issued: 11/21/2006
• Est. Priority Date: 12/17/1998
• Status: Expired due to Term

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Krautwurst, Dietmar, Yau, King-Wai, Reed, Randall R.
• Primary Examiner(s): Martinell, James
• Application Number:
  US10/259,423
• Publication Number:
  US 20030175744A1
• Time in Patent Office: 1,513 Days
• Field of Search: 435/7.1
• US Class Current: 435/7.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/10 Mammal
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/32 fusions with soluble part of a cell surface receptor, "decoy receptors"
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: G01N 2500/20 cell-free systems
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

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.

Claims

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.

2. A method of determining whether a test compound binds to a mammalian olfactory transmembrane receptor comprising the following steps:

(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; and

a 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.

3. The method of claim 2, wherein the binding of the test compound to the 7-transmembrane polypeptide is determined by measuring a change in the physiologic activity of the cell, wherein a change in the cell'"'"'s activity measured in the presence of the test compound compared to the cell'"'"'s activity in the absence of the test compound provides a determination that the test compound binds to the 7-transmembrane polypeptide.

4. The method of claim 3, wherein the measured cell activity is a change in the calcium ion (Ca²⁺) or cAMP concentration in the cell.

5. The method of claim 4, wherein the calcium ion concentration is measured by loading the cell with a calcium ion-sensitive fluorescent dye before contacting the cell with the test compound.

6. The method of claim 2, wherein the cell is a human cell or a Xenopus oocyte.