Olfactory receptor expression libraries and methods of making and using them

• US 20030175744A1
• Filed: 09/30/2002
• Published: 09/18/2003
• Est. Priority Date: 12/17/1998
• Status: Active Grant

• Current Assignee: Dietmar Krautwurst, King-Wai Yau, Randall R. Reed
• Original Assignee: Dietmar Krautwurst, King-Wai Yau, Randall R. Reed
• Inventors: Krautwurst, Dietmar, Yau, King-Wai, Reed, Randall R.
• Granted Patent: US 7,138,242 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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. An amplification primer sequence pair for amplifying a nucleic acid encoding an olfactory receptor ligand-binding region comprising a first primer comprising a sequence 5′

-GGGGTCCGGAG(A/G)(C/G) (A/G)TA(A/G/T)AT(A/G/P)A(A/G/P)(A/G/P)GG-3′

(SEQ ID NO;

1) and a second primer comprising a sequence 5′

-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T) (C/T)T(A/C/G/T)TT(C/T)(C/T)T-3′

(SEQ ID NO;

2).

2. The amplification primer sequence pair of claim 1, wherein the receptor ligand-binding region comprises olfactory receptor transmembrane domains II through VII.

3. A method for generating nucleic acid sequence that encodes a ligand-binding region of an olfactory receptor, the method comprising amplification of a nucleic acid using a primer pair as set forth in claim 1.

4. The method of claim 3, wherein the amplified nucleic acid is genomic DNA, mRNA or cDNA derived from olfactory neurons or olfactory epithelium.

5. The method of claim 3, wherein the amplification comprises the following conditions and steps in the following order:

about one cycle at about 94°

C. for about 2 min; and

about 30 cycles of about 45°

C. to about 65°

C. for about 1 min, followed by about 72°

C. for about one min. followed by about 94°

C. for about 1 min.

6. The method of claim 5, wherein the PCR amplification further comprises the following conditions and steps in the following order:

about one cycle of about 45°

C. to about 65°

C. for about 10 min; and

about one cycle of about 72°

C. for about 10 min.

7. A kit for amplification of olfactory receptor sequences comprising the primer pair of claim 1.

8. A library of olfactory receptor ligand-binding regions consisting essentially of olfactory receptor transmembrane domain regions II through VII, II through VI, III through VII, or III through VI.

9. The library of claim 8, wherein the olfactory receptor ligand-binding regions are generated by polymerase chain reaction using degenerate primer pairs.

10. A library of chimeric nucleic acid sequences comprising the following domains in 5′

to 3′

order;

a nucleic acid encoding an amino terminal plasma membrane translocation domain;

a nucleic acid encoding a first transmembrane domain; and

a nucleic acid encoding an olfactory receptor ligand-binding region,

wherein the chimeric nucleic acid sequence encodes a 7-transmembrane polypeptide that can transverse a plasma membrane seven times.

11. The library of claim 10, wherein the amino terminal plasma membrane translocation domain comprises a sequence as set forth in SEQ ID NO:

3.

12. The library of claim 10, wherein the first transmembrane receptor is a 7-transmembrane receptor region I domain.

13. The library of claim 12, wherein the 7-transmembrane receptor transmembrane region I domain comprises a sequence as set forth in SEQ ID NO:

4.

14. The library of claim 10, wherein the olfactory receptor ligand-binding region comprises olfactory receptor transmembrane domain regions II through VII, II through VI, III through VII, or III through VI.

15. The library of claim 10, wherein the olfactory receptor ligand-binding regions are generated by polymerase chain reaction using degenerate primer pairs.

16. The library of claim 14, wherein the nucleic acid sequence encoding the transmembrane domain regions II through VII is generated by polymerase chain reaction (PCR) amplification of nucleic acid using a first primer comprising a sequence 5′

-GGGGTCCGGAG(A/G)(C/G)(A/G)TA(A/G/T)AT(A/G/P)A(A/G/P)(A/G/P)GG-3′

(SEQ ID NO;

1) and a second primer comprising a sequence 5′

-GGGGCTGCA GACACC(A/C/G/T)ATGTA(C/T)(C/T)T(A/C/G/T)TT(C/T)(C/T)T-3′

(SEQ ID NO;

2).

17. The library of claim 15, wherein the PCR-amplified nucleic acid is genomic DNA, mRNA or cDNA derived from olfactory neurons or olfactory epithelium.

18. The library of claim 10, wherein the ligand-binding region comprising transmembrane domains II through VII is an amino acid sequence encoded by a nucleic acid selected from the group consisting of SEQ ID NO:

11, SEQ ID NO;

13, SEQ ID NO;

15, SEQ ID NO;

17, SEQ ID NO;

19, SEQ ID NO;

21, SEQ ID NO;

23, SEQ ID NO;

25, SEQ ID NO;

27, SEQ ID NO;

29, SEQ ID NO;

31, SEQ ID NO;

33, SEQ ID NO;

35, SEQ ID NO;

37, SEQ ID NO;

39, SEQ ID NO;

41, SEQ ID NO;

43, SEQ ID NO;

45 and SEQ ID NO;

47, or an amino acid sequence selected from the group consisting of SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

20, SEQ ID NO;

22, SEQ ID NO;

24, SEQ ID NO;

26, SEQ ID NO;

28, SEQ ID NO;

30, SEQ ID NO;

32, SEQ ID NO;

34, SEQ ID NO;

36, SEQ ID NO;

38, SEQ ID NO;

40, SEQ ID NO;

42, SEQ ID NO;

44, SEQ ID NO;

46, SEQ ID NO;

48.

19. The library of claim 15, wherein the PCR amplification comprises the following conditions and steps in the following order:

about one cycle at about 94°

C. for about 2 min;

about 30 cycles of about 55°

C. for about 1 min, followed by about 72°

C. for about one min. followed by about 94°

C. for about 1 min;

about one cycle of about 55°

C. for about 10 min; and

about one cycle of about 72°

C. for about 10 min.

20. The library of claim 10, further comprising a carboxy terminal 7-transmembrane receptor transmembrane region VII domain.

21. The library of claim 20, wherein the 7-transmembrane receptor transmembrane region VII domain comprises a sequence as set forth in SEQ ID NO:

5.

23. An expression vector comprising a nucleic acid sequence derived from a library of nucleic acid sequences as set forth in claim 8 or claim 10.

24. A transformed or isolated infected cell comprising a nucleic acid sequence derived from a library of nucleic acid sequences as set forth in claim 8 or claim 10 or an expression vector as set forth in claim 23.

25. A transgenic animal comprising a nucleic acid sequence derived from a library of nucleic acid sequences as set forth in claim 8 or claim 10 or an expression vector as set forth in claim 23.

27. A library of recombinant polypeptides translated or derived from the library of nucleic acids as set forth in claim 8 or claim 10.

28. An isolated polypeptide isolated or derived from the library of polypeptides as set forth in claim 27.

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

(i) expressing a nucleic acid derived from a nucleic acid library as set forth in claim 8 or claim 10 under conditions permissive for translation of the nucleic acid to a receptor polypeptide;

(ii) contacting the translated polypeptide with the test compound; and

(iii) determining whether the test compound specifically binds to the polypeptide.

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

(i) contacting a cell stably or transiently transfected with a nucleic acid derived from a nucleic acid library as set forth in claim 8 or claim 10;

(ii) culturing the cell under conditions permissive for translation of the nucleic acid to a receptor polypeptide with the test compound; and

(iii) determining whether the test compound specifically binds to the receptor polypeptide.

31. The method of claim 30, wherein the receptor polypeptide is expressed as a transmembrane receptor with a ligand binding site on the cell'"'"'s plasma membrane outer surface.

32. The method of claim 30, wherein the specific binding of the test compound to the 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 specifically binds to the polypeptide.

33. The method of claim 32, wherein the measured cell activity is a change in the calcium ion (Ca²⁺) or cAMP concentration in the cell.

34. The method of claim 33, 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.

35. The method of claim 30, wherein the cell is a human cell or a Xenopus oocyte.

36. A method of determining whether a test compound specifically binds to a mammalian olfactory transmembrane receptor polypeptide in vivo comprising the following steps:

(i) contacting a non-human animal stably or transiently infected with a nucleic acid derived from the library as set forth in claim 8 or claim 10 or an expression vector as set forth in claim 23 with the test compound; and

(ii) determining whether the animal reacts to the test compound by specifically binding to the receptor polypeptide, wherein the specific binding of the test compound to the polypeptide is determined by measuring a change in a physiologic activity of the animal, wherein a change in a receptor-encoding vector-infected animal'"'"'s activity measured in the presence of the test compound compared to a bare vector-infected animal'"'"'s activity in the presence of the test compound provides a determination that the test compound specifically binds to the mammalian olfactory transmembrane receptor polypeptide.

37. The method of claim 36, wherein the measured physiologic activity is measured by an electroolfactogram.

38. The method of claim 36, wherein the vector is an adenovirus expression vector.

39. A method of determining whether a test compound is neurotoxic to an olfactory neuron expressing an olfactory transmembrane receptor polypeptide comprising the following steps:

(i) contacting an olfactory neuron cell stably or transiently infected with a nucleic acid derived from a library as set forth in claim 8 or claim 10 or an expression vector as set forth in claim 23 with the test compound; and

(ii) measuring 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 is toxic.

40. The method of claim 39, wherein toxicity is indicated by abnormal calcium ion, cAMP or plasma membrane homeostasis.

22. A library of nucleic acid sequences comprising the following domains in 5′

to 3′

order

a nucleic acid encoding an amino terminal plasma membrane translocation domain comprising a sequence as set forth in SEQ ID NO;

3,

a nucleic acid encoding a transmembrane region I domain comprising a sequence as set forth in SEQ ID NO;

4,

a nucleic acid sequence generated by polymerase chain reaction (PCR) amplification of mRNA or cDNA derived from olfactory epithelium using a first primer comprising a sequence 5′

-GGGGTCCGGAG(A/G)(C/G)T(A/G)A(A/G/T)AT (A/G/P)A(A/G/P)(A/G/P)GG-3′

(SEQ ID NO;

1) and a second primer comprising a sequence 5′

-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)C/T)T(A/C/G/T) TT(C/T)C/T)T-3′

(SEQ ID NO;

2), and

a nucleic acid encoding a 7-transmembrane receptor transmembrane region VII domain comprising a sequence as set forth in SEQ ID NO;

5.

26. The transgenic animal of claim 22, wherein the expression vector is a mammalian expression vector that can be expressed in olfactory epithelium or olfactory neurons.

41. A peptide domain for the efficient translocation of a newly translated protein to a plasma membrane comprising an amino acid sequence as set forth in SEQ ID NO:

3 or an amino acid sequence having conservative amino acid residue substitutions based on SEQ ID NO;

3.

42. The peptide translocation domain of claim 41, wherein the translocation domain is about 20 amino acids in length.

43. The peptide translocation domain of claim 41, wherein the polypeptide translocation domain is SEQ ID NO:

3.

44. The peptide translocation domain of claim 41, wherein the newly translated protein is a transmembrane protein.

45. The peptide translocation domain of claim 41, wherein the transmembrane protein is a 7-transmembrane protein receptor.

46. The peptide translocation domain of claim 45, wherein the 7-transmembrane protein receptor is an olfactory receptor.

47. A hybrid polypeptide comprising an amino terminal amino acid sequence comprising a peptide translocation domain as set forth in claim 41 and a second polypeptide sequence, wherein the second polypeptide sequence is not a rhodopsin polypeptide sequence.

48. The hybrid polypeptide of claim 47, wherein the second polypeptide sequence is a transmembrane protein.

49. The hybrid polypeptide of claim 48, wherein the transmembrane protein is a 7-transmembrane protein receptor.

50. The hybrid polypeptide of claim 49, wherein the 7-transmembrane protein receptor is an olfactory receptor.

51. An isolated or recombinant nucleic acid sequence encoding the hybrid polypeptide of claim 47.