Method for assaying protein-protein interaction

• US 20060147975A1
• Filed: 02/02/2006
• Published: 07/06/2006
• 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: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Kevin J., Barnea, Gilad, Axel, Richard, Strapps, Walter
• Granted Patent: US 8,017,398 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 nucleic acid molecule which comprises:

(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, and

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

(b) a nucleic acid molecule which comprises;

(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.

2-27. -27. (canceled)

28. Recombinant cell, transformed or transfected with:

(a) a nucleic acid molecule which comprises;

(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, and

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

(b) a nucleic acid molecule which comprises;

(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.

29. The recombinant cell of claim 28, wherein one or both of said nucleic acid molecules are stably incorporated into the genome of said cell.

30. The recombinant cell of claim 28, wherein said cell has been transformed or transfected with said reporter gene.

31. The recombinant cell of claim 28, wherein said first test protein is a membrane bound protein.

32. The recombinant cell of claim 31, wherein said membrane bound protein is a transmembrane receptor.

33. The recombinant cell of claim 32, wherein said transmembrane receptor is a GPCR.

34. The recombinant cell of claim 28, wherein said protease or portion of a protease is tobacco etch virus nuclear inclusion A protease.

35. The recombinant cell of claim 28, wherein said protein which activates said reporter gene is a transcription factor.

36. The recombinant cell of claim 31, wherein said membrane bound protein is ADBR2, AVPR2, HTR1A, CHRM2, CCR5, DRD2, or OPRK.

37. The recombinant cell of claim 28, wherein said transcription factor is tTA or GAL4.

38. The recombinant cell of claim 28, wherein said second protein is an inhibitory protein.

39. The recombinant cell of claim 38, wherein said inhibitory protein is an arrestin, and said first protein is a transmembrane receptor.

40. The recombinant cell of claim 28, wherein said cell is a eukaryote.

41. The recombinant cell of claim 28, wherein said cell is a prokaryote.

42. The recombinant cell of claim 28, wherein said reporter gene is an exogenous gene.

43. The recombinant cell of claim 42, wherein said exogenous gene encodes ÿ

-galactosidase or luciferase.

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

45. The recombinant cell of claim 44, 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.

46. The recombinant cell of claim 44, wherein the 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.

47. An isolated nucleic acid molecule which comprises, in 5′

to 3′

order,

(i) a nucleotide sequence which encodes a 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.

48-55. -55. (canceled)

56. An isolated nucleic acid molecule which comprises:

(i) nucleotide sequence which encodes a test protein whose interaction with another test protein in the presence of a 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.

56-76. -76. (canceled)