Method for producing protein by precipitation

Abstract

The present invention provides a method for producing a target protein in the form of a fusion protein at a high recovery ratio. The present invention relates to a method for producing a fusion protein made of a protein having a self-assembly capability and a target protein comprising the following steps (1) to (4): (1) preparing a solution containing the fusion protein; (2) adjusting a pH of the solution obtained in step (1) to such a pH that a recovery ratio calculated according to the following equation is 10% or more, where the recovery ratio (%)=[an amount of the fusion protein in a solution obtained in step (4)/{the amount of the fusion protein in the solution obtained in step (4)+an amount of the fusion protein in a solution after solid separation in step (3)}]×100; (3) separating a solid from the solution obtained in step (2); and (4) dissolving the solid separated in step (3) into a solution having a pH of 12 or below but higher than the pH of the solution obtained in step (2) by 0.1 or more.

1 . A method for producing a fusion protein made of a protein having a self-assembly capability and a target protein, comprising the following steps (1) to (4):
 (1) preparing a solution containing the fusion protein;   (2) adjusting a pH of the solution obtained in step (1) to such a pH that a recovery ratio calculated according to the following equation is 10% or more, wherein
   the recovery ratio (%)=[an amount of the fusion protein in a solution obtained in step (4)/{the amount of the fusion protein in the solution obtained in step (4)+an amount of the fusion protein in a solution after solid separation in step (3)}]×100;
 
   (3) separating a solid from the solution obtained in step (2); and   (4) dissolving the solid separated in step (3) into a solution having a pH of 12 or below but higher than the pH of the solution obtained in step (2) by 0.1 or more.   
     
     
         2 . The method according to  claim 1 , wherein the protein having a self-assembly capability is a cell surface protein. 
     
     
         3 . The method according to  claim 2 , wherein the cell surface protein is a CspB mature protein or a portion thereof. 
     
     
         4 . The method according to  claim 3 , wherein the CspB mature protein or the portion thereof is any one of the following (a) and (b):
 (a) a protein consisting of an amino acid sequence of SEQ ID NO: 3; and   (b) a protein having a homology of 95% or more with the amino acid sequence of SEQ ID NO: 3.   
     
     
         5 . The method according to  claim 3 , wherein the portion of the CspB mature protein is a sequence consisting of 6 to 250 amino acid residues from the N-terminus of the CspB mature protein. 
     
     
         6 . The method according to  claim 5 , wherein the portion of the CspB mature protein is a sequence consisting of 6, 17, 50, or 250 amino acid residues from the N-terminus of the CspB mature protein. 
     
     
         7 . The method according to  claim 1 , wherein the number of amino acid residues in the target protein is 10 to 1000. 
     
     
         8 . The method according to  claim 1 , wherein an amino acid sequence used for an enzymatic cleavage or a chemical cleavage is further incorporated between the protein having a self-assembly capability and the target protein. 
     
     
         9 . The method according to  claim 8 , wherein the amino acid sequence used for the enzymatic cleavage between the protein having a self-assembly capability and the target protein is a ProTEV protease recognition sequence, a trypsin recognition sequence, or a Factor Xa protease recognition sequence. 
     
     
         10 . The method according to  claim 1 , wherein the pH in step (2) is 9 or below. 
     
     
         11 . The method according to  claim 1 , wherein the pH is adjusted in step (2) using an acid selected from the group consisting of sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, and trifluoroacetic acid. 
     
     
         12 . The method according to  claim 1 , wherein the separation in step (3) is performed by centrifugation and/or membrane filtration. 
     
     
         13 . The method according to  claim 1 , wherein the solution obtained in step (1) is a supernatant of a culture solution of a coryneform bacterium having a gene construct capable of expressing the fusion protein. 
     
     
         14 . The method according to  claim 1 , wherein the recovery ratio specified in step (2) is 30% or more. 
     
     
         15 . A method for producing a target protein, comprising the following steps (1) to (5):
 (1) preparing a solution containing a fusion protein made of a protein having a self-assembly capability and the target protein, the fusion protein containing an amino acid sequence used for an enzymatic cleavage or a chemical cleavage between the protein having a self-assembly capability and the target protein;   (2) adjusting a pH of the solution obtained in step (1) to such a pH that a recovery ratio calculated according to the following equation is 10% or more, wherein
   the recovery ratio (%)=[an amount of the fusion protein in a solution obtained in step (4)/{the amount of the fusion protein in the solution obtained in step (4)+an amount of the fusion protein in a solution after solid separation in step (3)}]×100;
 
   (3) separating a solid from the solution obtained in step (2);   (4) dissolving the solid separated in step (3) into a solution having a pH of 12 or below but higher than the pH of the solution obtained in step (2) by 0.1 or more; and   (5) enzymatically or chemically cleaving the fusion protein at a site of the amino acid sequence between the protein having a self-assembly capability and the target protein simultaneously with step (4), during step (4), or after step (4).   
     
     
         16 . The method according to  claim 15 , wherein the step of cleaving the fusion protein is an enzymatically cleaving step. 
     
     
         17 . The method according to  claim 15 , wherein the protein having a self-assembly capability is a cell surface protein. 
     
     
         18 . The method according to  claim 17 , wherein the cell surface protein is a CspB mature protein or a portion thereof. 
     
     
         19 . The method according to  claim 18 , wherein the CspB mature protein or the portion thereof is any one of the following (a) and (b):
 (a) a protein consisting of an amino acid sequence of SEQ ID NO: 3; and   (b) a protein having a homology of 95%, or more with the amino acid sequence of SEQ ID NO: 3.   
     
     
         20 . The method according to  claim 18 , wherein the portion of the CspB mature protein is a sequence consisting of 6 to 250 amino acid residues from the N-terminus of the CspB mature protein. 
     
     
         21 . The method according to  claim 20 , wherein the portion of the CspB mature protein is a sequence consisting of 6, 17, 50, or 250 amino acid residues from the N-terminus of the CspB mature protein. 
     
     
         22 . The method according to  claim 15 , wherein the number of amino acid residues in the target protein is 10 to 1000. 
     
     
         23 . The method according to  claim 15 , wherein the target protein is teriparatide. 
     
     
         24 . The method according to  claim 15 , wherein the target protein is a bivalirudin intermediate represented by SEQ ID NO: 93. 
     
     
         25 . The method according to  claim 15 , wherein the amino acid sequence used for the enzymatic cleavage between the protein having a self-assembly capability and the target protein is a ProTEV protease recognition sequence, a trypsin recognition sequence, or a Factor Xa protease recognition sequence. 
     
     
         26 . The method according to  claim 15 , wherein the pH in step (2) is 9 or below. 
     
     
         27 . The method according to  claim 15 , wherein the pH is adjusted in step (2) using an acid selected from the group consisting of sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, and trifluoroacetic acid. 
     
     
         28 . The method according to  claim 15 , wherein the separation in step (3) is performed by centrifugation and/or membrane filtration. 
     
     
         29 . The method according to  claim 15 , wherein the solution obtained in step (1) is a supernatant of a culture solution of a coryneform bacterium having a gene construct capable of expressing the fusion protein. 
     
     
         30 . The method according to  claim 15 , comprising a step of (6) purifying the fusion protein or the target protein after step (4) and/or step (5). 
     
     
         31 . The method according to  claim 30 , wherein step (6) is performed by column chromatography. 
     
     
         32 . The method according to  claim 15 , wherein the recovery ratio specified in step (2) is 300 or more. 
     
     
         33 . A method for forming and separating a solid of a fusion protein made of a protein having a self-assembly capability and a target protein, the method comprising:
 forming the solid by adjusting a pH of a solution containing the fusion protein to 9 or below; and   then separating the solid.