Method for assaying protein-protein interaction

• US 8,017,398 B2
• Filed: 02/02/2006
• Issued: 09/13/2011
• 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), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Kevin J., Barnea, Gilad, Axel, Richard, Strapps, Walter
• Primary Examiner(s): JOIKE, MICHELE K
• Application Number:
  US11/346,759
• Publication Number:
  US 20060147975A1
• Time in Patent Office: 2,049 Days
• Field of Search: None
• US Class Current: 435/455
• 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 recombinant cell, said recombinant cell comprising a reporter gene and:

(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, wherein said test protein is a transmembrane receptor,

(ii) a nucleotide sequence which encodes a proteolytic cleavage site for a protease or a portion of a protease not expressed endogenously in said cell, and

(iii) a nucleotide sequence which encodes a protein capable of activating said 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 a test compound is to be measured, and

(ii) a nucleotide sequence which encodes said protease or said portion of a protease capable of specific proteolytic cleavage of said cleavage site.

2. The recombinant cell of claim 1, wherein one or both of said nucleic acid molecules are stably incorporated into the genome of said cell.

3. The recombinant cell of claim 1, wherein said cell has been transformed or transfected with said reporter gene.

4. The recombinant cell of claim 1, wherein said transmembrane receptor is a GPCR.

5. The recombinant cell of claim 1, wherein said protease or portion of a protease is tobacco etch virus nuclear inclusion A protease.

6. The recombinant cell of claim 1, wherein said protein capable of activating said reporter gene is a transcription factor.

7. The recombinant cell of claim 1, wherein said first test protein is ADBR2, AVPR2, HTRIA, CHRM2, CCR5, DRD2, or OPRK.

8. The recombinant cell of claim 6, wherein said transcription factor is tTA or GAL4.

9. The recombinant cell of claim 1, wherein said second test protein is an inhibitory protein.

10. The recombinant cell of claim 9, wherein said inhibitory protein is an arrestin.

11. The recombinant cell of claim 1, wherein said cell is a eukaryote.

12. The recombinant cell of claim 1, wherein said cell is a prokaryote.

13. The recombinant cell of claim 1, wherein said reporter gene is an exogenous gene.

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

15. The recombinant cell of claim 14, 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.

16. The recombinant cell of claim 15, 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.

17. The recombinant cell of claim 13, wherein said exogenous gene encodes an enzyme.

18. The recombinant cell of claim 17, wherein said enzyme is β

-galactosidase, luciferase, chloramphenicol acetyl transferase, beta-glucouronidase, or alkaline phosphatase.

19. The recombinant cell of claim 13, wherein said exogenous gene encodes a protein which provides a detectable signal when expressed and is a fluorescent or chemiluminescent protein.

20. The recombinant cell of claim 19, wherein said fluorescent protein is green fluorescent protein, red fluorescent protein, or cyan fluorescent protein.

21. The recombinant cell of claim 1, wherein the presence of said test compound enhances association of said first and second test proteins.

22. The recombinant cell of claim 1, wherein said test compound is a ligand of the first test protein.

23. The recombinant cell of claim 1, wherein said test compound is an agonist of said first test protein.