Drug discovery and protein-protein interaction assay using fluorescent protein exchange

Abstract

A novel assay for determining a molecular process using a fluorescent protein exchange assay, and a composition for use thereof, are provided. The assay provides first and second signalling proteins and an exchange protein, wherein the exchange protein interacts with the first signalling protein to form a complex, then introducing the second signalling protein, wherein in response to the molecular process, the exchange protein dissociates from the first protein and associates with the second protein. The change in signal in response to the exchange of the proteins is measured to indicate a molecular process.

1 . A composition for detecting a molecular process, the composition comprising a first protein, a second protein, and an exchange protein, the exchange protein for interacting with either the first protein to form a first complex, or with the second protein to form a second complex, wherein when the exchange protein dissociates from the first protein to associate with the second protein, a change in signal is produced, thereby indicating the molecular process. 
     
     
         2 . The composition of  claim 1 , wherein the composition is for detecting a molecular process in a cell. 
     
     
         3 . The composition of  claim 1 , wherein the exchange protein, first protein or second protein are dimerization dependent fluorescent proteins (ddFPs), and one of the first signalling protein and second signalling protein is a red ddFP and the other is a green ddFP. 
     
     
         4 . The composition of  claim 1 , wherein the molecular process is a loss of protein-protein interaction or physical connection. 
     
     
         5 . The composition of  claim 1 , wherein the change in signal is a change in fluorescence intensity, a change in fluorescence color, or a change in subcellular localization. 
     
     
         6 . The composition of  claim 1 , wherein the change in signal generated fluorescence is a green-to-red or red-to-green change in fluorescence color. 
     
     
         7 . The composition of  claim 1 , wherein the exchange, first or second proteins are conjugated with one or more further proteins. 
     
     
         8 . The composition of  claim 1 , wherein the one or more further proteins interact with each other, or dissociate from each other, thereby changing the signal. 
     
     
         9 . A reporter system for detecting a molecular process comprising a live cell which co-expresses a first protein, a second protein, and an exchange protein, the exchange protein for interacting with either the first protein to form a first complex, or with the second protein to form a second complex, wherein when the exchange protein dissociates from the first protein to associate with the second protein, a change in signal is produced, thereby indicating the molecular process. 
     
     
         10 . The system of  claim 9 , wherein the at least two of the first protein, second protein and exchange protein are combined into a single polypeptide chain. 
     
     
         11 . The system of  claim 10 , wherein all three of the first protein, second protein and exchange protein are combined into a single polypeptide chain. 
     
     
         12 . The system of  claim 9 , wherein the live cell is transfected with at least one expression plasmid expresses one, two or all three of the first protein, second protein and exchange protein.