Method for assaying protein—protein interaction

• US 7,049,076 B2
• Filed: 07/09/2004
• Issued: 05/23/2006
• Est. Priority Date: 07/09/2003
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University), Sentigen Biosciences, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Kevin J., Axel, Richard, Barnea, Gilad, Strapps, Walter
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): Joike, Michele K.
• Application Number:
  US10/888,313
• Publication Number:
  US 20050100934A1
• Time in Patent Office: 683 Days
• Field of Search: None
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/9406 Neurotransmitters

Abstract

The invention relates to a method for determining if a test compound, or a mix of compounds, modulates the interaction between two proteins of interest. The determination is made possible via the use of two recombinant molecules, one of which contains the first protein a cleavage site for a proteolytic molecules, and an activator of a gene. The second recombinant molecule includes the second protein and the proteolytic molecule. If the test compound binds to the first protein, a reaction is initiated whereby the activator is cleaved, and activates a reporter gene.

Claims

1. A method for determining if a test compound modulates a specific protein/protein interaction of interest, comprising contacting said compound to a cell which has been transformed or transfected with:

(a) a first nucleic acid molecule which encodes a first, fusion protein, said first nucleic acid molecule comprising;

(i) a nucleotide sequence which encodes a first test protein,

(ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and

(iii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell, and

(b) a second nucleic acid molecule which encodes a second, fusion protein, said second nucleic acid molecule comprising;

(i) a nucleotide sequence which encodes a second test protein whose interaction with said first test protein in the presence of said test compound is to be measured, and

(ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for said cleavage site,

and determining activity of said reporter gene as a determination of whether said compound modulates said protein/protein interaction.

2. The method of claim 1, wherein said first test protein is a membrane bound protein.

3. The method of claim 1, wherein said protease or portion of a protease is tobacco etch virus nuclear inclusion A protease.

4. The method of claim 1, wherein said protein which activates said reporter gene is a transcription factor.

5. The method of claim 1, wherein said second protein is an inhibitory protein.

6. The method of claim 1, wherein said cell is a eukaryote.

7. The method of claim 1, wherein said reporter gene is an exogenous gene.

8. The method of claim 1, wherein the nucleotide sequence encoding said first test protein is modified to increase interaction with said second test protein.

9. The method of claim 1, comprising contacting more than one compound to a plurality of samples of cells, each of said samples being contacted by one or more of said compounds, wherein each of said cell samples have been transformed or transfected with (a) and (b), and determining activity of reporter genes in said plurality of said samples to determine if any of said compounds modulates said specific, protein/protein interaction.

10. The method of claim 2, wherein said membrane bound protein is a transmembrane receptor.

11. The method of claim 2, wherein said membrane bound protein is ÿ

2-adrenergic receptor (ADRB2), arginine vasopressin receptor 2 (AVPR2), serotonin receptor 1a (HTR1A), m2 muscarinic acetylcholine receptor ( CHRM2), chemokine (C-C motif) receptor 5 (CCR5), dopamine D2 receptor (DRD2), kappa opioid receptor (OPRK), or ADRA1A.

12. The method of claim 10, wherein said transmembrane receptor is a GPCR.

13. The method of claim 4, wherein said transcription factor is tTA or GAL4.

14. The method of claim 5, wherein said inhibitory protein is an arrestin, and said first protein is a transmembrane receptor.

15. The method of claim 7, wherein said exogenous gene encodes ÿ

-galactosidase or luciferase.

16. The method of claim 8, wherein said modification comprises replacing all or part of the nucleotide sequence of the C-terminal region of said first test protein with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for said second test protein than the original sequence.

17. The method of claim 16, wherein the nucleotide sequence of said C-terminal region is replaced by a nucleotide sequence encoding all or a part of the C-terminal region of AVPR2, AGTRLI, GRPR, F2PL1, CXCR2/IL-8B, CCR4, or GRPR.

18. The method of claim 9, comprising contacting each of said samples with one compound, each of which differs from all others.

19. The method of claim 9, comprising contacting each of said samples with a mixture of said compounds.

20. The method of claim 19, wherein said mixture of compounds comprises a biological sample.

27. The method of claim 20, wherein said biological sample is cerebrospinal fluid, urine, blood, serum, pus, ascites, synovial fluid, a tissue extract, or an exudate.

21. A method for determining if a test compound modulates one or more of a plurality of protein interactions of interest, comprising contacting said test compound to a plurality of samples of cells, each of which has been transformed or transfected with

(a) a first nucleic acid molecule which encodes a first, fusion proteins, said first nucleic acid molecule comprising:

(i) a nucleotide sequence which encodes a first test protein,

(ii) a nucleotide sequence encoding a cleavage site for a protease, and

(iii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell,

(b) a second nucleic acid molecule which encodes a second, fusion protein said second nucleic acid molecule comprising;

(i) a nucleotide sequence which encodes a second test protein whose interaction with said first test protein in the presence of said test compound of interest is to be measured,

(ii) a nucleotide sequence which encodes a protease or a protease which is specific for said cleavage site, wherein said first test protein differs from other first test proteins in each of said plurality of samples,

and determining activity of said reporter gene in at one or more of said plurality of samples as a determination of modulation of one or more protein interactions of interest.

22. The method of claim 21, wherein said second test protein is different in each sample.

23. The method of claim 21, wherein said second test protein is the same in each sample.

24. The method of claim 21, wherein all of said samples are combined in a common receptacle, and each samples comprises a different pair of first and second test proteins.

25. The method of claim 21, wherein each sample is tested in a different receptacle.

26. The method of claim 21, wherein the reporter gene in a given sample differs from the reporter gene in other samples.

28. A test kit useful for determining if a test compound modulates a specific protein/protein interaction of interest comprising a separate portion of each of:

(a) a first nucleic acid molecule which encodes a first, fusion protein, said first nucleic acid molecule comprising;

(i) a nucleotide sequence which encodes said first test protein,

(ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease,

(iii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell, and

(b) a second nucleic acid molecule which encodes a second, fusion protein, said second nucleic acid molecule comprising;

(i) a nucleotide sequence which encodes a second test protein whose interaction with said first test protein in the presence of said test compound is to be measured,

(ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for said cleavage site, and

(c) container means for holding each of (a) and (b) separately from each other.

29. The test kit of claim 28, wherein said first test protein is a membrane bound protein.

30. The test kit of claim 28, wherein said protease or portion of a protease is tobacco etch virus nuclear inclusion A protease.

31. The test kit of claim 28, wherein said protein which activates said reporter gene is a transcription factor.

32. The test kit of claim 28, wherein said second protein is an inhibitory protein.

33. The test kit of claim 28, further comprising a separate portion of an isolated nucleic acid molecule which encodes a reporter gene.

34. The test kit of claim 28, wherein the nucleotide sequence encoding said first test protein is modified to increase interaction with said second test protein.

35. The test kit of claim 29, wherein said membrane bound protein is a transmembrane receptor.

36. The test kit of claim 29, wherein said membrane bound protein is ADRB2, AVPR2, HTR1A, CHRM2, CCR5, DRD2, or OPRK.

37. The test kit of claim 35, wherein said transmembrane receptor is a GPCR.

38. The test kit of claim 31, wherein said transcription factor is tTA or GAL4.

39. The test kit of claim 32, wherein said inhibitory protein is an arrestin, and said first protein is a transmembrane receptor.

40. The test kit of claim 33, wherein said reporter gene encodes ÿ

-galactosidase or luciferase.

41. The test kit of claim 34, wherein said modification comprises replacing all or part of the nucleotide sequence of the C-terminal region of said first test protein with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for said second test protein than the original sequence.

42. The test kit of claim 41, wherein said nucleotide sequence of said C-terminal region is replaced by a nucleotide sequence encoding the C-terminal region of AVPR2, AGTRLI, GRPR, F2PL1, CXCR2/IL-8B or CCR4.