Method of affinity separation and ligands for use therein

Abstract

Methods of affinity separation wherein the affinity ligand is immobilized proteinaceous ligand wherein one or more of its asparagine (Asn) residues has been modified. Methods of making a stabilized combinatorial protein by a) modification of Asn residues within a protein molecule to increase stability of the protein in alkaline conditions, and b) randomization of the protein molecule to modify its binding characteristics, and combinatorial proteins wherein in a step separate from the randomization step, the stability of the protein in alkaline conditions has been increased by modifying one or more of its Asn residues.

1 . (canceled)  
     
     
         2 . A method of stabilizing an affinity ligand by modifying one or more of its Asn residues.  
     
     
         3 . A method of preparing a combinatorial library of protein molecules wherein the protein has been rendered less sensitive to alkaline pH by modification of one or more of its Asn residues before it is randomized.  
     
     
         4 . A method of phage display wherein a protein expressed on the phage surface has had one or more of its Asn residues modified in a step separate to any modifications introduced in order to modify binding characteristics of the protein.  
     
     
         5 . A method of making a stabilized combinatorial protein comprising the steps of: 
 a) modification of Asn residues within a protein molecule to increase stability of the protein in alkaline conditions; and    b) randomization of the protein molecule to modify its binding characteristics.    
     
     
         6 . A combinatorial protein wherein in a step separate from the randomization step, the stability of the protein in alkaline conditions has been increased by modifying one or more of its Asn residues.  
     
     
         7 . A fusion protein comprising a first part wherein one or more naturally occurring Asn residues have been modified and a second part being a randomized protein molecule selected for its specific binding properties.  
     
     
         8 . (canceled)  
     
     
         9 . A method as claimed in  claim 2 , wherein one or more Asn residues in said ligand or said protein are replaced with a less alkaline-sensitive amino acid.  
     
     
         10 . A method as claimed in  claim 2 , wherein two or more Asn residues are modified.  
     
     
         11 . A method as claimed in  claim 2 , wherein all the Asn residues are modified.  
     
     
         12 . A method as claimed in  claim 2 , wherein Asn residues on the surface of the three-dimensional structure of the ligand are modified.  
     
     
         13 . The protein of  claim 6 , wherein said Asn residues are replaced with an amino acid selected from lysine, aspartic acid and leucine.  
     
     
         14 . A method as claimed in  claim 2 , wherein said affinity ligand is a combinational protein.  
     
     
         15 . A method as claimed in  claim 5  which further comprises the step of: 
 c) selecting a randomized affinity ligand by expression in a surface display library.    
     
     
         16 . A method as claimed in  claim 14 , wherein said affinity ligand is a randomised protein selected by expression in a surface display library.  
     
     
         17 . A method as claimed in  claim 14 , wherein said combinatorial protein is derived from an immunoglobulin molecule or a fragment or derivative thereof, staphylococcal protein A (SPA) or a fragment, domain or derivative thereof, or a DNA binding protein, or fragment or domain thereof.  
     
     
         18 . A method as claimed in  claim 17 , wherein said combinatorial protein comprises domain Z (a derivative of the B domain of SPA), or a derivative thereof.  
     
     
         19 . A method as claimed in  claim 2 , wherein said affinity ligand comprises Albumin-Binding Protein (ABD) or a fragment or derivative thereof.  
     
     
         20 . Albumin Binding Protein (ABD) or fragments or derivatives thereof wherein one or more native Asn residues have been replaced by a less alkaline sensitive amino acid, said protein, fragment or derivative being suitable for use in a method or affinity separation.  
     
     
         21 . A fusion protein as claimed in  claim 7  wherein the first part is ABD and the second part is domain Z (a derivative of the B domain of SPA) or a derivative thereof, said fusion protein being suitable for use in a method of affinity separation.  
     
     
         22 . A protein as claimed in  claim 6  wherein said combinatorial protein is derived from an immunoglobulin molecule or a fragment or derivative thereof, staphylococcal protein A (SPA) or a fragment, domain or derivative thereof, or a DNA binding protein, or fragment or domain thereof.  
     
     
         23 . A protein as claimed in  claim 22  wherein said combinatorial protein comprises domain Z (a derivative of the B domain of SPA), or a derivative thereof.  
     
     
         24 . A protein as claimed in  claim 6  wherein said affinity ligand comprises Albumin-Binding Protein (ABD) or a fragment or derivative thereof.  
     
     
         25 . A nucleic acid molecule encoding a protein as defined in  claim 6 .  
     
     
         26 . A host cell expressing a protein as defined in  claim 6.