Method for assaying protein-protein interaction
View Patent ↗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.
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.