METHOD FOR ASSAYING PROTEIN-PROTEIN INTERACTION

• US 20120077706A1
• Filed: 08/18/2011
• Published: 03/29/2012
• Est. Priority Date: 07/09/2003
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Axel, Richard, LEE, Kevin J., Strapps, Walter, Barnea, Gilad
• Application Number:
  US13/212,965
• Publication Number:
  US 20120077706A1
• Time in Patent Office: Days
• US Class Current: 506/11
• 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.

62. A test kit useful for determining if a test compound modulates a specific protein/protein interaction of interest comprising the recombinant cell according to claim 28.

29-46. -46. (canceled)

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. The isolated nucleic acid molecule of claim 47, wherein said test protein is a membrane bound protein.

49. The isolated nucleic acid molecule of claim 48, wherein said membrane bound protein is a transmembrane receptor.

50. The isolated nucleic acid molecule of claim 49, wherein said transmembrane receptor is a GPCR.

51. The isolated nucleic acid molecule of claim 47, wherein said protease or portion of a protease is tobacco etch virus nuclear inclusion A protease.

52. The isolated nucleic acid molecule of claim 47, wherein said protein which activates said reporter gene is a transcription factor.

53. The isolated nucleic acid molecule of claim 52, wherein said transcription factor is tTA or GAL4.

54. The isolated nucleic acid molecule of claim 48, wherein said membrane bound protein is ADBR2, AVPR2, HTR1A, CHRM2, CCR5, DRD2, or OPRK.

55. Expression vector comprising the isolated nucleic acid molecule of claim 47, operably linked to a promoter.

60. A fusion protein produced by expression of the isolated nucleic acid molecule of claim 47.

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.

57. The isolated nucleic acid molecule of claim 56, wherein said test protein is an inhibitory protein.

58. The isolated nucleic acid molecule of claim 57, wherein said inhibitory protein is an arrestin.

59. Expression vector comprising the isolated nucleic acid molecule of claim 56, operably linked to a promoter.

61. A fusion protein produced by expression of the isolated nucleic acid molecule of claim 56.

63-76. -76. (canceled)