Olfactory receptor expression libraries and methods of making and using them

• US 7,351,814 B2
• Filed: 10/25/2005
• Issued: 04/01/2008
• 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:
  US11/257,062
• Publication Number:
  US 20060094047A1
• Time in Patent Office: 889 Days
• Field of Search: None
• US Class Current: 536/23.5
• 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 nucleic acid construct that encodes a hybrid seven-transmembrane receptor which facilitates the translocation of a seven-transmembrane receptor across a cell membrane, said construct comprising:

(i) a first nucleic acid sequence that encodes a first seven-transmembrane receptor polypeptide that is the rhodopsin translocation peptide contained in SEQ ID NO;

3 or a nucleic acid sequence encoding a variant of said rhodopsin translocation peptide, wherein said variant is identical to said rhodopsin translocation peptide but for a single conservative amino acid substitution which substitutes a first amino residue in a first group for a second amino acid residue within the first group, wherein the first group is selected from the groups consisting of;

     1) Alanine (A), Serine (S), Threonine (T);

     2) Aspartic acid (D), Glutamic acid (E);

     3) Asparagine (N), Glutamine (Q);

     4) Arginine (R), Lysine (K);

     5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

     6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); and

(ii) a second nucleic acid sequence that encodes a second seven-transmembrane receptor polypeptide, wherein said nucleic acid construct when expressed in a cell results in a hybrid seven-transmembrane receptor polypeptide comprising the rhodopsin translocation peptide or said variant, and the second seven-transmembrane receptor polypeptide, wherein the rhodopsin translocation peptide or said variant directs the translocation of the second seven-transmembrane receptor polypeptide across the cell membrane, wherein said variant translocates the seven-transmembrane receptor polypeptide as efficiently as the rhodopsin translocation polypeptide.

2. The nucleic acid construct of claim 1 wherein said second seven-transmembrane receptor polypeptide is an olfactory receptor.

3. The nucleic acid construct of claim 2 wherein said first nucleic acid sequence encodes the rhodopsin translocation peptide contained in SEQ ID NO:

3.

4. The nucleic acid construct of claim 3 wherein said construct further comprises a transcriptional and/or a translational control element operably linked to said first nucleic acid sequence.

5. The nucleic acid construct of claim 1 which comprises a plasmid.

6. The nucleic acid construct of claim 1 wherein said first nucleic acid sequence encodes the rhodopsin translocation peptide contained in SEQ ID NO:

3.

7. The nucleic acid construct of claim 6 wherein said construct further comprises a transcriptional and/or a translational control element operably linked to said first nucleic acid sequence.

8. The nucleic acid construct of claim 1 wherein said construct further comprises a transcriptional and/or a translational control element operably linked to said first nucleic acid sequence.