Gag binding protein

Abstract

A method is provided for introducing a GAG binding site into a protein comprising the steps: identifying a region in a protein which is not essential for structure maintenance introducing at least one basic amino acid into said site and/or deleting at least one bulky and/or acidic amino acid in said site, whereby said GAG binding site has a GAG binding affinity of Kd≦10 μM, preferably ≦1 μM, still preferred ≦0.1 μM, as well as modified GAG binding proteins.

1 . A method of making a modified GAG binding protein by modifying a GAG binding site of the GAG binding protein, wherein the GAG binding site is modified by a method comprising the steps of:
 a) identifying a region in the protein which is not essential for structure maintenance; and   b) introducing at least one basic amino acid into the site and/or deleting at least one bulky and/or acidic amino acid in the site;   whereby the GAG binding site has a GAG binding affinity of Kd≦10 μM.   
     
     
         2 . A method according to  claim 1 , wherein the GAG binding site has a GAG binding affinity of ≦1 μM. 
     
     
         3 . A method according to  claim 1 , wherein the GAG binding site has a GAG binding affinity of ≦0.1 μM. 
     
     
         4 . A method according to  claim 1 , wherein the GAG binding affinity is higher by a factor of minimum 5 compared with wild-type GAG binding protein. 
     
     
         5 . A method according to  claim 1 , wherein at least one basic amino acid selected from the group consisting of Arg, Lys, and His is inserted into the GAG binding region. 
     
     
         6 . A method according to  claim 1 , wherein the protein is a chemokine, preferably IL-8, RANTES or MCP-1. 
     
     
         7 . A method according to  claim 6 , wherein the GAG binding region is a C terminal a-helix. 
     
     
         8 . A method according to  claim 7 , wherein positions 17, 21, 70, and/or 71 are substituted by Arg, Lys, His, Asn, and/or Gln. 
     
     
         9 . A method according to  claim 1 , wherein the increased GAG binding affinity is an increased binding affinity to heparan sulfate and/or heparin. 
     
     
         10 . A method according to  claim 1 , wherein a further biologically active region is modified thereby inhibiting or down-regulating a further biological activity of the protein. 
     
     
         11 . A method according to  claim 10 , wherein the further biologically active region is modified by deletion, insertion, and/or substitution, preferably with alanine, a sterically and/or electrostatically similar residue. 
     
     
         12 . A method according to  claim 10 , wherein the protein is a chemokine and the further biological activity is leukocyte activation. 
     
     
         13 . A method according to  claim 12 , wherein the protein is IL-8 and the further biologically active region is located within the first 10 N-terminal amino acids. 
     
     
         14 . A method according to  claim 13 , wherein the protein is a mutant with the first 6 N-terminal amino acids deleted. 
     
     
         15 . A method according to  claim 14 , wherein the protein is a mutant, and wherein the protein is modified such that
 Arg is at position 17, Lys is at position 70, and Arg is at position 71;   Arg is at position 17, Arg is at position 70, and Lys is at position 71;   
       or
 Lys is at positions 70 and 71.