Protein binding miniature proteins and uses thereof

Abstract

In certain aspects, the present invention provides miniature proteins resulted from a protein scaffold such as an avian pancreatic polypeptide that can be modified by substitution of at least one amino acid residue. In other aspects, the present invention provides diagnostic and therapeutic uses of these miniature proteins.

1 . An avian pancreatic polypeptide (aPP) modified by substitution of at least one amino acid residue, wherein said at least one residue is located within a type II polyproline (PPII) helix of the polypeptide when the polypeptide is in a tertiary form. 
     
     
         2 . The modified polypeptide of  claim 1 , wherein at least two or three amino acid residues on the PPII helix are substituted. 
     
     
         3 . The modified polypeptide of  claim 1 , further modified by substitution of at least one amino acid residue in the linker region between the PPII helix and the alpha helix domain of the polypeptide. 
     
     
         4 . The modified polypeptide of  claim 3 , wherein at least two amino acid residues on the linker region are substituted. 
     
     
         5 . The modified polypeptide of  claim 1 , wherein said at least one substituted residue of the PPII helix is selected from a site on a first protein through which the first protein interacts with a second protein. 
     
     
         6 . The modified polypeptide of  claim 5 , wherein the first protein is selected from the group consisting of: zyxin, vinculin, and the ActA protein of  Listeria monocytogenes.    
     
     
         7 . The modified polypeptide of  claim 5 , wherein the second protein is selected from the group consisting of:  Drosophila  Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP). 
     
     
         8 . The modified polypeptide of  claim 5 , wherein the site is a protein binding site. 
     
     
         9 . The modified polypeptide of  claim 1 , having an amino acid sequence selected from any of SEQ ID NOs: 1-5. 
     
     
         10 . The modified polypeptide of  claim 1 , wherein the modified polypeptide binds to a protein selected from the group consisting of:  Drosophila  Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP). 
     
     
         11 . The modified polypeptide of  claim 10 , wherein the modified polypeptide preferentially binds to one protein selected from the group consisting of:  Drosophila  Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP), but doe not bind to the other proteins of the group. 
     
     
         12 . The modified polypeptide of  claim 1 , wherein the modified polypeptide inhibits interaction between the known protein and the second protein. 
     
     
         13 - 17 . (canceled) 
     
     
         18 . An isolated polypeptide selected from the group consisting of:
 (a) an isolated polypeptide comprising any of the amino acid sequences as set forth in SEQ ID NOs: 1-5;   (b) an isolated polypeptide comprising a fragment of at least twelve contiguous amino acids of any of SEQ ID NOs: 1-5;   (c) an isolated polypeptide comprising one or more amino acid substitutions in any of the amino acid sequences as set forth in SEQ ID NOs: 1-5; and   (d) an isolated polypeptide at least 95 percent identical to any of SEQ ID NOs: 1-5.   
     
     
         19 - 20 . (canceled) 
     
     
         21 . A method of identifying an avian pancreatic polypeptide (aPP) miniature protein that modulates the interaction between a known protein and a second protein, comprising a step of isolating at least one recombinant phage clone from a phage display library that displays a protein scaffold that modulates the association between a known protein and a second protein, the phage display library comprising a plurality of recombinant phase that express a protein scaffold modified by substitution of at least one amino acid residue, said at least one residue being exposed on a type II polyproline (PPII) helix of the polypeptide when the polypeptide is in a tertiary form. 
     
     
         22 - 38 . (canceled)