Method for assaying protein-protein interaction

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.

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 . (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 - 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. 
     
     
         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. 
     
     
         60 . A fusion protein produced by expression of the isolated nucleic acid molecule of  claim 47 . 
     
     
         61 . A fusion protein produced by expression of the isolated nucleic acid molecule of  claim 56 . 
     
     
         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 . 
     
     
         63 - 76 . (canceled)