Interaction trap systems for detecting protein interactions

Abstract

Disclosed herein is a method of determining whether a first protein is capable of physically interacting with a second protein, involving: (a) providing a host cell which contains (i) a reporter gene operably linked to a protein binding site; (ii) a first fusion gene which expresses a first fusion protein, the first fusion protein including the first protein covalently bonded to a binding moiety which is capable of specifically binding to the protein binding site; and (iii) a second fusion gene which expresses a second fusion protein, the second fusion protein including the second protein covalently bonded to a gene activating moiety and being conformationally-constrained; and (b) measuring expression of the reporter gene as a measure of an interaction between the first and the second proteins. Also disclosed are methods for assaying protein interactions, and identifying antagonists and agonists of protein interactions. Proteins isolated by these methods are also discussed. Finally, populations of eukaryotic cells are disclosed, each cell having a recombinant DNA molecule encoding a conformationally-constrained intracellular peptide.

1 . A method for identifying an antagonist or agonist protein, said method comprising:
 (a) providing a host cell which contains a pair of interacting proteins, and a reporter gene whose expression is mediated by the pair of interacting proteins;   (b) introducing into said host cell a DNA coding for a candidate agonist or antagonist protein, wherein said candidate agonist or antagonist protein has reduced structural flexibility due to covalent bonding of the amino and carboxy termini of said agonist or antagonist protein to a conformation-constraining protein or disulfide bonding between cysteine residues at the amino and carboxy termini of said agonist or antagonist protein; and   (c) measuring expression of said reporter gene, wherein an increase in expression of said reporter gene identifies said candidate protein as an agonist protein and a decrease in expression of said reporter gene identifies said candidate protein as an antagonist protein.   
     
     
         2 . The method according to  claim 1 , wherein a change in expression of the reporter gene affects cell viability or is detectable in a color assay leading to the presence or absence of color. 
     
     
         3 . The method of  claim 1 , wherein said method further comprises the step of isolating said agonist or antagonist protein. 
     
     
         4 . A method for identifying an antagonist or agonist protein, said method comprising:
 (a) providing a pair of interacting proteins, and a reporter gene whose expression is mediated by the pair of interacting proteins;   (b) adding to said pair of interacting proteins a candidate agonist or antagonist protein, wherein said candidate agonist or antagonist protein has reduced structural flexibility due to covalent bonding of the amino and carboxy termini of said agonist or antagonist protein to a conformation-constraining protein or disulfide bonding between cysteine residues at the amino and carboxy termini of said agonist or antagonist protein; and   (c) assaying for an increase or decrease in reporter gene expression, wherein an increase in reporter gene expression identifies said candidate protein as an agonist protein and a decrease in reporter gene expression identifies said candidate protein as an antagonist protein.   
     
     
         5 . The method of  claim 4 , wherein said method further comprises the step of isolating said agonist or antagonist protein. 
     
     
         6 . A method for the identification, in an intracellular screen, of peptides that affect an identifiable characteristic of a cell, said method comprising:
 (a) transforming, into a population of eukaryotic host cells, a DNA library encoding peptides having reduced structural flexibility due to covalent bonding of the amino and carboxy termini of said peptide to a conformation-constraining protein or disulfide bonding between cysteine residues at the amino and carboxy termini of said peptide, there being at least 100 different recombinant molecules encoding different peptides in said population, each molecule being in at least one cell of said population; and   (b) detecting the identifiable characteristic, wherein the identifiable characteristic is cell viability or a perturbation of cell cycle progression.   
     
     
         7 . The method according to  claim 6 , wherein the DNA encoding the peptides is random.