Protein Refolding Method

Abstract

The present invention provides a method for producing a protein which has a restored native higher-order structure by bringing a protein which has lost its native higher-order structure into contact at pH 6.5 to 9.0 with a 1 to 3% aqueous solution of a specific surfactant, such as lauroylglutamic acid to obtain a solubilized solution of the protein; and then adding the solubilized solution to a buffer with pH 6.5 to 9.0 containing arginine or an arginine derivative at a concentration of 0.1 to 1.2 M to lower the concentration of the specific surfactant, such as lauroylglutamic acid, in the obtained mixture solution down to 0.02 to 0.275%. According to the present invention, it is possible to easily restore the native higher-order structure of a protein while smoothly removing the surfactant from the protein.

1 . A method for producing a protein having a restored native higher-order structure, the method comprising:
 (1) bringing a protein which has become insoluble or lost its native higher-order structure into contact at pH 6.5 to 9.0 with a 1 to 3% aqueous solution of a surfactant selected from the group consisting of dicarboxylic acids having C 8  to C 16  acyl groups and salts thereof, decanoylsarcosine and salts thereof, decanoylalanine and salts thereof, decanoic acid and salts thereof, lauryltrimethylammonium chloride, and combinations thereof, to obtain a solubilized solution of the protein;   (2) adding the solubilized solution to a buffer with pH 6.5 to 9.0 comprising an additive selected from the group consisting of arginine, an arginine derivative, and combinations thereof, wherein said additive is at a concentration of 0.05 to 1.2 M, to lower the concentration of the surfactant to 0.02 to 0.5%, to obtain a mixture; and   (3) recovering from the mixture the protein having a restored native higher-order structure.   
     
     
         2 . The method according to  claim 1 , wherein the surfactant is selected from the group consisting of dicarboxylic acids having C 8  to C 16  acyl groups and salts thereof, decanoylsarcosine and salts thereof, decanoylalanine and salts thereof and combinations thereof. 
     
     
         3 . The method according to  claim 1 , wherein the surfactant is a dicarboxylic acid having C 8  to C 16  acyl groups or salts thereof. 
     
     
         4 . The method according to  claim 1 , further comprising, between the step (1) and the step (2), a step (A) of:
 diluting the solubilized solution obtained in the step (1) to obtain a diluted solution having a surfactant concentration of 0.8 to 1.5% and pH of 6.5 to 9.0; and   incubating the diluted solution at 5° C. to 40° C. for at least 0.5 hours.   
     
     
         5 . The method according to  claim 1 , wherein, in the step (2), the concentration of the surfactant and the concentration of the additive are decreased stepwise or gradually, and maintaining the mixture at 5 to 48° C. for 1 hour to 5 days. 
     
     
         6 . The method according to  claim 1 , wherein the arginine derivative is selected from the group consisting of arginine with an acyl group having 1 to 6 carbon atoms, arginine butyl esters, agmatine, and arginine acid. 
     
     
         7 . The method according to  claim 1 , wherein the protein which has become insoluble or lost its native higher-order structure is a recombinant protein. 
     
     
         8 . The method according to  claim 1 , wherein the protein which has become insoluble or lost its native higher-order structure is a protein having an immunoglobulin structure in a domain thereof. 
     
     
         9 . The method according to  claim 8 , wherein the protein having an immunoglobulin structure in a domain thereof is selected from the group consisting of an antibody fragment having a part of an antibody domain, artificial antibodies, diabodies, minibodies, Fc fusion proteins and combinations thereof. 
     
     
         10 . The method according to  claim 9 , wherein the antibody fragment is selected from the group consisting of scFv, Fab, Fab′, (F(ab′) 2 ), and combinations thereof. 
     
     
         11 . The method according to  claim 9 , wherein the Fc fusion protein is obtained by fusing a cytokine, a receptor extracellular domain, or a peptide to an antibody Fc domain. 
     
     
         12 . The method according to  claim 1 , further comprising, when the protein having a restored native higher-order structure comprises a disulfide bond, a step (B) of causing a redox reaction of the protein which has become insoluble or lost its native higher-order structure to form a intramolecular disulfide bond and/or a intermolecular disulfide bond. 
     
     
         13 . The method according to  claim 12 , wherein the redox reaction comprises adding a redox material and/or a copper ion to the solubilized solution obtained in the step (1) and/or the buffer obtained in the step (2). 
     
     
         14 . A method for restoring a native higher-order structure of a protein which has become insoluble or lost its native higher-order structure, the method comprising:
 (1) bringing the protein which has become insoluble or lost its native higher-order structure into contact at pH 6.5 to 9.0 with a 1 to 3% aqueous solution of a surfactant selected from the group consisting of dicarboxylic acids having C 8  to C 16  acyl groups and salts thereof, decanoylsarcosine and salts thereof, decanoylalanine and salts thereof, decanoic acid and salts thereof, lauryl trimethyl ammonium chloride, and combinations thereof, to obtain a solubilized solution of the protein; and   (2) adding the solubilized solution to a buffer with pH 6.5 to 9.0 comprising an additive selected from the group consisting of arginine, an arginine derivative, and combinations thereof, wherein said additive is at a concentration of 0.05 to 1.2 M, to lower the concentration of the surfactant to 0.02 to 0.5%, to obtain a mixture.