Materials and methods for detecting interaction of CFTR polypeptides

• US 7,238,474 B1
• Filed: 10/06/2000
• Issued: 07/03/2007
• Est. Priority Date: 10/06/1999
• Status: Expired due to Fees

• Current Assignee: Florida State University (State University System of Florida)
• Original Assignee: Florida State University (State University System of Florida)
• Inventors: Teem, John L.
• Primary Examiner(s): Guzo; David
• Assistant Examiner(s): Joike; Michele K.
• Application Number:
  US10/089,875
• Time in Patent Office: 2,461 Days
• Field of Search: None
• US Class Current: 435/6.16
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/4712 Cystic fibrosis
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2565/201 Two hybrid system
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Abstract

The subject invention concerns materials and methods for detecting the interaction of CFTR proteins. In one embodiment, the method can be used to determine whether one CFTR polypeptide interacts with a second CFTR polypeptide. The subject invention also concerns materials and methods for screening for drugs or compositions that can restore or enhance interaction of CFTR proteins containing mutation(s) that reduce or prevent dimerization of the proteins. The assay of the present invention can be used to screen a large number of compounds in a high throughput format. The subject invention also pertains to host cells useful in the methods of the invention. The subject invention also concerns compositions and methods for treating patients afflicted with cystic fibrosis.

Claims

1. A method for detecting or determining the interaction of a first CFTR polypeptide with a second CFTR polypeptide, said method comprising:

(a) providing a first polynucleotide encoding a fusion protein comprising all or a portion of a first CFTR polypeptide and a DNA binding domain of a transcriptional activator that can bind to a site on a detectable gene, wherein said first CFTR polypeptide comprises a first nucleotide binding domain (NBD

     1) of a CFTR polypeptide, or a functional fragment of said NBD 1;

(b) providing a second polynucleotide encoding a fusion protein comprising all or a portion of a second CFTR polypeptide and a transcriptional activation domain of a transcriptional activator that can activate transcription of said detectable gene, wherein said second CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD 1;

(c) incorporating said first and second polynucleotide into a host cell comprising said detectable gene wherein transcription of said detectable gene is under control of said transcriptional activator;

(d) expressing said polynucleotide encoding said first CFTR polypeptide and expressing said polynucleotide encoding said second CFTR polypeptide under conditions in which said detectable gene is expressed when said NBD 1 of said first CFTR polypeptide and said NBD 1 of said second CFTR polypeptide interact; and

(e) detecting transcription of said detectable gene or expression of the gene product of said detectable gene.

2. The method according to claim 1, wherein said host cell is a yeast cell.

3. The method according to claim 2, wherein said yeast cell is Saccharomyces.

4. The method according to claim 1, wherein the host cell is a mammalian cell.

5. The method according to claim 1, wherein said CFTR polypeptide is a mammalian CFTR polypeptide.

6. The method according to claim 1, wherein one or both of said CFTR polypeptides comprises amino acid residues 351 through 650 of the human CFTR protein sequence.

7. The method according to claim 1, wherein said detectable gene is selected from the group consisting of lacZ, LEU2 and HIS3.

8. The method according to claim 1, wherein said DNA binding domain comprises the DNA binding domain of GAL4 protein.

9. The method according to claim 1, wherein said transcriptional activation domain comprises the transcriptional activation domain of GAL4 protein.

10. The method according to claim 1, wherein one or both of said CFTR polypeptides are mutant CFTR polypeptides.

11. The method according to claim 1, wherein one or both of said CFTR polypeptides comprises a mutation in said first nucleotide binding domain (NBD1).

12. The method according to claim 10, wherein one or both of said mutant CFTR polypeptides contains a Δ

F508 mutation.

13. The method according to claim 1, wherein one or both of said CFTR polypeptides has a wild type CFTR amino acid sequence.

14. The method according to claim 1, wherein said DNA binding domain and said transcriptional activation domain are from an organism other than yeast.

15. The method according to claim 12, wherein said host cell is incubated at a non-permissive temperature.

16. A method for detecting or determining the interaction of a first CFTR polypeptide with a second CFTR polypeptide, said method comprising (a) providing a fusion protein comprising all or a portion of a first CFTR protein and a DNA binding domain of a transcriptional activator that can bind to a site on a detectable marker gene, wherein said first CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1;

(b) providing a second fusion protein comprising all or a portion of a second CFTR polypeptide and a transcriptional activation domain of a transcriptional activator that can activate transcription of the detectable marker gene, wherein said second CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1;

(c) contacting said first fusion protein and said second fusion protein under conditions where if said NBD1 of said first fusion protein and said NBD1 of said second fusion protein interact then said interaction causes said transcriptional activation domain to activate transcription of said detectable marker gene; and

(d) detecting transcription of said detectable marker gene or expression of the gene product of said detectable marker gene.

17. The method according to claim 16, wherein said CFTR polypeptide is a mammalian CFTR polypeptide.

18. The method according to claim 16, wherein one or both of said CFTR polypeptides comprises amino acid residues 351 through 650 of the human CFTR protein sequence.

19. The method according to claim 16, wherein said detectable gene is selected from the group consisting of lacZ, LEU2 and HIS3.

20. The method according to claim 16, wherein said DNA binding domain comprises the DNA binding domain of GAL4 protein.

21. The method according to claim 16, wherein said transcriptional activation domain comprises the transcriptional activation domain of GAL4 protein.

22. The method according to claim 16, wherein one or both of said CFTR polypeptides are mutant CFTR polypeptides.

23. The method according to claim 16, wherein one or both of said CFTR polypeptides comprises a mutation in the first nucleotide binding domain (NBD1).

24. The method according to claim 22, wherein one or both of said mutant CFTR polypeptides contains a Δ

F508 mutation.

25. The method according to claim 16, wherein one or both of said CFTR polypeptides has a wild type CFTR amino acid sequence.

26. The method according to claim 16, wherein said DNA binding domain and said transcriptional activation domain are from an organism other than yeast.

27. The method according to claim 24, wherein said host cell is incubated at a non-permissive temperature.

28. A host cell comprising a polynucleotide encoding a fusion protein comprising all or a portion of a first CFTR protein and a DNA binding domain of a transcriptional activator that can bind to a site on a detectable gene, wherein said first CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1;

and a polynucleotide encoding a fusion protein comprising all or a portion of a second CFTR protein and a transcriptional activation domain of a transcriptional activator that can activate transcription of said detectable gene, wherein said second CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1.

29. The host cell according to claim 28, wherein said host cell is a yeast cell.

30. The host cell according to claim 29, wherein said yeast cell is Saccharomyces.

31. The host cell according to claim 28, wherein the host cell is a mammalian cell.

32. The host cell according to claim 28, wherein said CFTR polypeptide is a mammalian CFTR polypeptide.

33. The host cell according to claim 28, wherein one or both of said CFTR polypeptides comprises amino acid residues 351 through 650 of the human CFTR protein sequence.

34. The host cell according to claim 28, wherein said detectable gene is selected from the group consisting of lacZ, LEU2 and HIS3.

35. The host cell according to claim 28, wherein said DNA binding domain comprises the DNA binding domain of GAL4 protein.

36. The host cell according to claim 28, wherein said transcriptional activation domain comprises the transcriptional activation domain of GAL4 protein.

37. The host cell according to claim 28, wherein one or both of said CFTR polypeptides are mutant CFTR polypeptides.

38. The host cell according to claim 28, wherein one or both of said CFTR polypeptides comprises a mutation in the first nucleotide binding domain (NBD1).

39. The host cell according to claim 37, wherein one or both of said mutant CFTR polypeptides contains a Δ

F508 mutation.

40. The host cell according to claim 28, wherein one or both of said CFTR polypeptides has a wild type CFTR amino acid sequence.

41. The host cell according to claim 28, wherein said DNA binding domain and said transcriptional activation domain are from an organism other than yeast.