Protein capable of binding specifically to immunoglobulin, and immunoglobulin-binding affinity ligand

Abstract

An object of the present invention is to create a novel engineered Protein A ligand having better antibody dissociation properties in the acidic condition compared with known engineered Protein A ligands. The present invention provides a protein having an affinity for an immunoglobulin, including an amino acid sequence obtained by introducing, into an amino acid sequence derived from any of E, D, A, B and C domains of Protein A, at least one amino acid substitution at any one or more of amino acid residues corresponding to positions 31 to 37 of the A, B and C domains (positions 29 to 35 of the E domain, positions 34 to 40 of the D domain), which are conserved in all the domains, the protein having a lower affinity for an Fab region of an immunoglobulin than a protein having the amino acid sequence before introduction of the substitution.

1 - 25 . (canceled) 
     
     
         26 . A method for separating and purifying an antibody, the method comprising:
 adsorbing an immunoglobulin comprising a Fab region onto an affinity separation matrix; and   eluting the antibody adsorbed onto the affinity separation matrix with an acidic solution,   wherein the affinity separation matrix comprises a carrier and a ligand immobilized on the carrier,   wherein the ligand is a protein comprising an amino acid having a sequence identity of not less than 90% to the amino acid sequence of SEQ ID NO:5, and   wherein the ligand comprises at least one substitution selected from the group consisting of:
 a substitution for Ser at position 33 of SEQ ID NO:5; 
 a substitution for Lys at position 35 of SEQ ID NO:5; 
 a substitution for Asp at position 36 of SEQ ID NO:5; and 
 a substitution for Asp at position 37 of SEQ ID NO:5, and 
   wherein the ligand has an affinity for the Fab region lower than an affinity for the Fab region that a protein comprising the amino acid sequence of SEQ ID NO:5 has.   
     
     
         27 . The method according to  claim 26 ,
 wherein the substitution for Ser at position 33 of SEQ ID NO: 5 is a substitution of Glu, Leu, or Thr,   wherein the substitution for Lys at position 35 of SEQ ID NO: 5 is a substitution of Arg,   wherein the substitution for Asp at position 36 of SEQ ID NO: 5 is a substitution of Arg or Ile, and   wherein the substitution for Asp at position 37 of SEQ ID NO: 5 is a substitution of Glu.   
     
     
         28 . The method according to  claim 26 , wherein the pH of the acidic solution is 3.0-5.0. 
     
     
         29 . The method according to  claim 26 , wherein the carrier comprises a water-insoluble base material. 
     
     
         30 . The method according to  claim 29 , wherein the water-insoluble base material comprises a synthetic polymer or a polysaccharide. 
     
     
         31 . The method according to  claim 30 , wherein the water-insoluble base material comprises the polysaccharide, and the polysaccharide is cellulose. 
     
     
         32 . The method according to  claim 31 , wherein the immunoglobulin is immunoglobulin G or an immunoglobulin G derivative, and wherein the immunoglobulin G derivative is selected from the group consisting of: chimeric immunoglobulin G; humanized immunoglobulin G; and immunoglobulin G comprising an Fc region with a modified sugar chain. 
     
     
         33 . The method according to  claim 26 , wherein the ligand further comprises a substitution of Ala for Gly at position 29 of SEQ ID NO:5. 
     
     
         34 . The method according to  claim 33 , wherein the ligand comprises the substitution of Glu, Leu, or Thr for Ser at position 33 of SEQ ID NO:5. 
     
     
         35 . The method according to  claim 33 , wherein the ligand comprises the substitution of Arg or Ile for Asp at position 36 of SEQ ID NO:5. 
     
     
         36 . The method according to  claim 33 , wherein the ligand comprises the substitution of Arg for Lys at position 35 of SEQ ID NO:5 and the substitution of Glu for Asp at position 37 of SEQ ID NO:5.