Assays and kits for detecting protein binding

Abstract

The invention provides methods for determining the interactions between phage-displayed proteins and test molecules. The phage-displayed proteins are contacted with a reference moiety in the presence and absence of a test molecule; the behavior of the phage-displayed proteins as a function of concentration of the test molecule permits calculation of the binding affinity of the phage-displayed protein for the test molecule.

1 . A method for assessing interactions between a test molecule and polypeptide members of a set of polypeptides comprising: 
 contacting a first polypeptide member of a set of polypeptides with a first reference moiety and a test molecule, said first polypeptide member being a first phage-displayed polypeptide and said first reference moiety being capable of binding at least one polypeptide member of said set of polypeptides;    contacting a second polypeptide member of said set of polypeptides with a second reference moiety and said test molecule, said second polypeptide member being a second phage-displayed polypeptide and said second reference moiety being capable of binding at least one polypeptide member of said set of polypeptides; and    evaluating an interaction of said phage-displayed polypeptides to said reference moieties, said interaction being indicative of an interaction between polypeptide members of said set of polypeptides and said test molecule.    
     
     
         2 . A method for assessing interactions between a set of test molecules and polypeptide members of a set of polypeptides comprising: 
 contacting a first polypeptide member of a set of polypeptides with a first reference moiety and a set of test molecules, said first polypeptide member being a first phage-displayed polypeptide and said first reference moiety being capable of binding at least one polypeptide member of said set of polypeptides;    contacting a second polypeptide member of said set of polypeptides with a second reference moiety and said set of test molecules, said second polypeptide member being a second phage-displayed polypeptide and said second reference moiety being capable of binding at least one polypeptide member of said set of polypeptides; and    evaluating an interaction of said phage-displayed polypeptides to said reference moieties, said interaction being indicative of an interaction between polypeptide members of said set of polypeptides and said test molecules.    
     
     
         3 . The method of  claim 2  further comprising contacting individual test molecule members of said set of test molecules to said polypeptide members of said set of polypeptides and said reference moieties.  
     
     
         4 . The method of  claim 1  or  2  further comprising categorizing said member polypeptides from said set of polypeptides into subsets, said subsets being created based on the interaction between phage-displayed polypeptides and reference moieties.  
     
     
         5 . The method of  claim 1  or  2  wherein said first and second reference moieties are the same.  
     
     
         6 . The method of  claim 1  or  2  wherein said members of said set of polypeptides are contacted one at a time with said reference moieties and said test molecules.  
     
     
         7 . The method of  claim 1  or  2  wherein multiple members of said set of polypeptides are contacted with said reference moieties and said test molecules at the same time.  
     
     
         8 . The method of  claim 1  or  2  wherein said interaction is a binding interaction.  
     
     
         9 . The method of  claim 1  or  2  wherein said reference moiety is immobilized on a solid support.  
     
     
         10 . The method of  claim 1  or  2  wherein said evaluating of said binding between said phage-displayed polypeptides and said reference moieties is performed using a phage plaque assay, fluorescence polarization, or a quantitative polymerase chain reaction.  
     
     
         11 . The method of  claim 1  or  2  wherein said phage-displayed polypeptides are phage-displayed kinases.  
     
     
         12 . The method of  claim 11  wherein said reference moiety is a kinase modulator.  
     
     
         13 . The method of  claim 12  wherein said kinase modulator binds to the ATP site of a kinase.  
     
     
         14 . The method of  claim 13  wherein said kinase modulator is at least one of a modulator selected from staurosporine, purvalanol B, SU5402, imatinib mesylate, SU6668, Iressa, PD-173955, and SB202190.  
     
     
         15 . The method of  claim 1  or  2  wherein said polypeptide is displayed on a T7 bacteriophage.  
     
     
         16 . The method of  claim 1  or  2  wherein said evaluating of said binding of said phage-displayed polypeptide to said reference moiety comprises: 
 quantifying the amount of said phage-displayed polypeptide bound to said reference moiety.    
     
     
         17 . The method of  claim 16  wherein said quantifying comprises: 
 determining the concentration of said test molecule at which about 50% of the phage-displayed polypeptide is bound to said reference moiety relative to the amount bound in the absence of said test molecule, whereby said concentration at which 50% of the phage-displayed polypeptide is bound to said reference moiety is identified as the value of the dissociation constant.    
     
     
         18 . The method of  claim 1  or  2  wherein said reference moiety is attached to a streptavidin-coated magnetic bead via a biotin moiety.  
     
     
         19 . A kit for assessing a potential interaction between a polypeptide and a test molecule comprising: 
 said polypeptide, wherein said polypeptide is a phage-displayed polypeptide; and    a reference moiety, wherein said reference moiety is capable of binding said phage-displayed polypeptide.    
     
     
         20 . The kit of  claim 19  wherein said polypeptide is a kinase.  
     
     
         21 . The kit of  claim 20  wherein said reference moiety binds to a ATP site of a kinase.  
     
     
         22 . The kit of  claim 21  wherein said reference moiety is a kinase modulator selected from staurosporine, purvalanol B, SU5402, imatinib mesylate, SU6668, Iressa, PD-173955, and SB202190.  
     
     
         23 . The kit of  claim 19  wherein said polypeptide is displayed on a T7 bacteriophage.