Detection of Molecular Interactions Using a Reduced Affinity Enzyme Complementation Reporter System

Abstract

Methods and compositions for detecting molecular interactions are provided. Aspects of the invention include the use of a reduced affinity enzyme complementation reporter system. Also provided are systems and kits for use in practicing embodiments of the methods.

What is claimed is: 
     
         1 . A method of determining whether a first and second protein interact, said method comprising:
 (a) providing a cell comprising:
 (i) a first fusion protein of said first protein and a first β-galactosidase fragment, wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide; and 
 (ii) a second fusion protein of said second protein and a second β-galactosidase fragment; 
 wherein said first and second β-galactosidase fragments have an affinity for each other which provides a known level of β-galactosidase activity in the absence of an interaction between said first and second proteins that is lower than the activity observed in the presence of an interaction between said first and second proteins; and 
   (b) evaluating said cell for β-galactosidase activity to determine whether said first and second proteins interact.   
     
     
         2 . The method according to  claim 1 , wherein said providing comprises introducing nucleic acids encoding said first and second fusion proteins into said cell. 
     
     
         3 . The method according to  claim 2 , wherein said nucleic acids are introduced into said cell sequentially. 
     
     
         4 . The method according to  claim 2 , wherein said nucleic acids are introduced into said cell simultaneously. 
     
     
         5 . The method according to  claim 1 , wherein said method further comprises contacting said cell with a candidate interaction modulatory agent prior to said evaluating. 
     
     
         6 . The method according to  claim 1 , wherein said evaluating comprises comparing observed β-galactosidase activity to said known level of β-galactosidase activity. 
     
     
         7 . The method according to  claim 1 , wherein said first β-galactosidase fragment has a binding affinity for said second β-galactosidase fragment that is lower than a β-galactosidase fragment consisting of amino acids 3 to 92 of  E. coli  wild-type β-galactosidase. 
     
     
         8 . The method according to  claim 7 , wherein said first β-galactosidase fragment comprises at least one amino acid variation as compared to a β-galactosidase fragment consisting of amino acids 3 to 92 of  E. coil  wild-type β-galactosidase. 
     
     
         9 . The method according to  claim 8 , wherein said at least one amino acid variation is a substitution. 
     
     
         10 . The method according to  claim 8 , wherein said at least one amino acid variation is a deletion. 
     
     
         11 . The method according to  claim 8 , wherein said variation occurs between residues 31 and 41. 
     
     
         12 . The method according to  claim 1 , wherein said cell is a mammalian cell. 
     
     
         13 . The method according to  claim 1 , wherein said cell is a yeast cell. 
     
     
         14 . The method according to  claim 1 , wherein said interaction occurs at an intracellular location. 
     
     
         15 . The method according to  claim 1 , wherein said interaction occurs at a plasma-membrane location. 
     
     
         16 . A cell comprising:
 (a) a first fusion protein of a first protein and a first β-galactosidase fragment, wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide; and   (b) a second fusion protein of a second protein and a second β-galactosidase fragment;   wherein said first and second β-galactosidase fragments have a low affinity for each other that provides a known level of β-galactosidase activity in the absence of an interaction between said first and second proteins that is lower than the activity observed in the presence of an interaction between said first and second proteins.   
     
     
         17 . The cell according to  claim 16 , wherein said first and second fusion proteins are intracellular proteins. 
     
     
         18 . The cell according to  claim 16 , wherein at least one of said first and second fusion proteins is a membrane bound protein. 
     
     
         19 . The cell according to  claim 15 , wherein both of said first and second fusion proteins are membrane bound proteins. 
     
     
         20 . A kit comprising:
 (a) cell comprising:
 (i) a first fusion protein of a first protein and a first β-galactosidase fragment, wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide; and 
 (ii) a second fusion protein of said second protein and a second β-galactosidase fragment; 
 wherein said first and second β-galactosidase fragments have a low affinity for each other which provides a known level of β-galactosidase activity in the absence of an interaction between said first and second proteins that is lower than the activity observed in the presence of an interaction between said first and second proteins; and 
   (b) a β-galactosidase substrate.   
     
     
         21 . A kit comprising:
 (a) a first nucleic acid encoding a first β-galactosidase fragment; and   (b) a second nucleic acid encoding a second β-galactosidase fragment;   wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide and has a binding affinity for said second β-galactosidase fragment that is lower than a β-galactosidase fragment consisting of amino acids 3 to 92 of  E. coli  wild-type β-galactosidase.   
     
     
         22 . The kit according to  claim 21 , wherein said first and second nucleic acids are present on vectors. 
     
     
         23 . The kit according to  claim 22 , wherein said vectors comprise a restriction site positioned on said vector such that when a protein coding sequence is inserted into said vector using said restriction site, said vector encodes a fusion protein of said protein and a β-galactosidase fragment. 
     
     
         24 . The kit according to  claim 22 , wherein said kit further comprises a cell. 
     
     
         25 . The kit according to  claim 22 , wherein said kit further comprises a β-galactosidase substrate.