DNA & protein binding miniature proteins

Abstract

The present invention provides a protein scaffold, such as an avian pancreatic polypeptide, that can be modified by substitution of two or more amino acid residues that are exposed on the alpha helix domain of the polypeptide when the polypeptide is in a tertiary form.

1 . A composition, comprising: 
 an avian pancreatic polypeptide modified by substitution of at least one amino acid residue, the at least one residue comprising a basic region leucine zipper structure and being exposed on the alpha helix domain of the polypeptide when the polypeptide is in a tertiary form.    
     
     
         2 . The composition of  claim 1 , wherein the avian pancreatic polypeptide is modified by substitution of at least six residues.  
     
     
         3 . The composition of  claim 1 , wherein the avian pancreatic polypeptide is modified by substitution of at least eight residues.  
     
     
         4 . The composition of  claim 1 , wherein the avian pancreatic polypeptide is modified by substitution of at least ten residues.  
     
     
         5 . The composition of  claim 1 , wherein the avian pancreatic polypeptide is modified by substitution of at least twelve residues.  
     
     
         6 . The composition of  claim 1 , wherein the at least one residue is selected from a site on a known protein through which interaction of the known protein with another molecule occurs.  
     
     
         7 . The composition of  claim 6 , wherein the known protein is selected from the group consisting of GCN4, CEBP, Max, Myc, and MyoD.  
     
     
         8 . The composition of  claim 6 , wherein the site is a binding site.  
     
     
         9 . The composition of  claim 8 , wherein the binding site is a DNA binding site.  
     
     
         10 . The composition of  claim 9 , wherein the DNA binding site is selected from the group consisting of the CRE half site, the CEBP site, the MyoD half site, and the Q50 engrailed variant site.  
     
     
         11 . A composition, comprising: 
 an avian pancreatic polypeptide modified by substitution of at least one amino acid residue, the at least one residue comprising a basic-helix-loop-helix structure and being exposed on the alpha helix domain of the polypeptide when the polypeptide is in a tertiary form.    
     
     
         12 . The composition of  claim 11 , wherein the avian pancreatic polypeptide is modified by substitution of at least six residues.  
     
     
         13 . The composition of  claim 11 , wherein the avian pancreatic polypeptide is modified by substitution of at least eight residues.  
     
     
         14 . The composition of  claim 11 , wherein the avian pancreatic polypeptide is modified by substitution of at least ten residues.  
     
     
         15 . The composition of  claim 11 , wherein the avian pancreatic polypeptide is modified by substitution of at least twelve residues.  
     
     
         16 . The composition of  claim 11 , wherein the at least one residue is selected from a site on a known protein through which interaction of the known protein with another molecule occurs.  
     
     
         17 . The composition of  claim 16 , wherein the known protein is selected from the group consisting of double minute two, Bcl-2, protein kinase A, Jun, and Fos.  
     
     
         18 . The composition of  claim 6 , wherein the site is a binding site.  
     
     
         19 . The composition of  claim 18 , wherein the binding site is a DNA binding site.  
     
     
         20 . The composition of  claim 19 , wherein the DNA binding site is selected from the group consisting of the CRE half site, the CEBP site, the MyoD half site, and the Q50 engrailed variant site.  
     
     
         21 . A composition, comprising: 
 an isolated polypeptide selected from the group consisting of:    (a) an isolated polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 34, 35, 36, 37, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71 or 72;    (b) an isolated polypeptide comprising a fragment of at least twelve amino acids of any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 34, 35, 36, 37, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71 or 72;    (c) an isolated polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 34, 35, 36, 37, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71 or 72; comprising one or more conservative amino acid substitutions;    (d) an isolated polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 34, 35, 36, 37, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71 or 72; comprising one or more naturally occurring amino acid sequence substitutions; and    (e) an isolated polypeptide with at least 95% amino acid homology to any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 34, 35, 36, 37, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71 or 72.    
     
     
         22 . A composition, comprising: 
 an avian pancreatic polypeptide modified by substitution of at least one amino acid residue, the at least one residue being a DNA binding site and being exposed on the alpha helix domain of the polypeptide when the polypeptide is in a tertiary form.    
     
     
         23 . The composition of  claim 22 , wherein the at least one residue is selected from a site on a known protein through which interaction of the known protein with DNA occurs.  
     
     
         24 . A composition, comprising: 
 an avian pancreatic polypeptide modified by substitution of at least one amino acid residue, the at least one residue being a protein binding site and being exposed on the alpha helix domain of the polypeptide when the polypeptide is in a tertiary form.    
     
     
         25 . The composition of  claim 24 , wherein the at least one residue is selected from a site on a known protein through which interaction of the known protein with a protein occurs.