Method for producing protein composition, and protein composition

Abstract

The present invention aims to provide a method for producing a protein composition, the method being capable of suppressing changes such as degradation and denaturation of a protein which occur when a highly hydrophilic protein or a protein that undergoes denaturation by complete removal of water is irradiated with radiation. The present invention provides a method for producing a protein composition containing a protein (A), a radical scavenger (RS), and at least one hydrogen-bond-formable compound (HC) selected from the group consisting of amino acids, peptides, and proteins other than the protein (A), the method including a sterilization step of radiosterilizing an unsterilized protein composition, wherein the unsterilized protein composition contains the protein (A), the radical scavenger (RS), and the hydrogen-bond-formable compound (HC), the protein (A) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups, the hydrogen-bond-formable compound (HC) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups, the at least one functional group in the protein (A) is capable of binding to the at least one functional group in the hydrogen-bond-formable compound (HC) via a hydrogen bond, the unsterilized protein composition has a water content of 0 to 30% by weight based on the weight of the unsterilized protein composition, a weight ratio of the radical scavenger (RS) to the protein (A) in the unsterilized protein composition [radical scavenger (RS)/protein (A)] is 0.01 to 1.0, and a molar ratio of a total molar number of functional groups in the hydrogen-bond-formable compound (HC) to a total molar number of functional groups in the protein (A) in the unsterilized protein composition [the total molar number of functional groups in the hydrogen-bond-formable compound (HC)/the total molar number of functional groups in the protein (A)] is 0.01 to 0.50.

1 . A method for producing a protein composition containing a protein (A), a radical scavenger (RS), and at least one hydrogen-bond-formable compound (HC) selected from the group consisting of amino acids, peptides, and proteins other than the protein (A),
 the method comprising:   a sterilization step of radiosterilizing an unsterilized protein composition,   wherein the unsterilized protein composition contains the protein (A), the radical scavenger (RS), and the hydrogen-bond-formable compound (HC),   the protein (A) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups,   the hydrogen-bond-formable compound (HC) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups,   the at least one functional group in the protein (A) is capable of binding to the at least one functional group in the hydrogen-bond-formable compound (HC) via a hydrogen bond,   the unsterilized protein composition has a water content of 0 to 30% by weight based on the weight of the unsterilized protein composition,   a weight ratio of the radical scavenger (RS) to the protein (A) in the unsterilized protein composition [radical scavenger (RS)/protein (A)] is 0.01 to 1.0, and   a molar ratio of a total molar number of functional groups in the hydrogen-bond-formable compound (HC) to a total molar number of functional groups in the protein (A) in the unsterilized protein composition [the total molar number of functional groups in the hydrogen-bond-formable compound (HC)/the total molar number of functional groups in the protein (A)] is 0.01 to 0.50.   
     
     
         2 . The method for producing a protein composition according to  claim 1 ,
 wherein in the unsterilized protein composition, the at least one functional group of the protein (A) is on a side chain of an amino acid,   the at least one functional group on a side chain of an amino acid in the protein (A) is capable of binding to the at least one functional group in the hydrogen-bond-formable compound (HC) via a first hydrogen bond, and   the first hydrogen bond has a distance of 1.3 to 1.9 Å.   
     
     
         3 . The method for producing a protein composition according to  claim 1 ,
 wherein in the unsterilized protein composition, the at least one functional group in the protein (A) is an amide group in a peptide bond in the protein (A),   the amide group in a peptide bond in the protein (A) is bound to the hydrogen-bond-formable compound (HC) via a second hydrogen bond, and   the second hydrogen bond has a distance of 1.3 to 1.9 Å.   
     
     
         4 . The method for producing a protein composition according to  claim 1 ,
 wherein the amino acid composition of the protein (A) is such that the number of proline (P) residues accounts for 1 to 50%, the number of serine (S) residues accounts for 1 to 50%, and the number of valine (V) residues accounts for 1 to 50%, based on the total number of amino acids in the protein (A).   
     
     
         5 . The method for producing a protein composition according to  claim 1 ,
 wherein the protein (A) contains a repetitive sequence (X).   
     
     
         6 . The method for producing a protein composition according to  claim 5 ,
 wherein the repetitive sequence (X) includes any one amino acid sequence (a1) selected from the group consisting of GAGAGS (1), RGD, YIGSR (2), GVGVP (3), PGVGV (4), VPGVG (5), GVPGV (6), VGVPG (7), GPP, GAP, GAHGPAGPK (8), GAA, VAAGY (9), GAGAGAS (10), LGPLGP (11), GAHGPAGPK (12), GAPGPAGPPGSRGDPGPP (13), GAQGPAGPG (14), GAPGAPGSQGAPGLQ (15), GAPGTPGPQGLPGSP (16), GAAVTGRGDSPASAAGY (17), and GAAPGASIKVAVSAGPSAGY (18).   
     
     
         7 . The method for producing a protein composition according to  claim 5 ,
 wherein the repetitive sequence (X) includes an amino acid sequence consisting of 2 to 200 repeats of GAGAGS (1).   
     
     
         8 . The method for producing a protein composition according to  claim 5 ,
 wherein the repetitive sequence (X) includes a sequence (Y) consisting of 2 to 200 repeats of an amino acid sequence (a2) that is one of GVGVP (3), PGVGV (4), VPGVG (5), GVPGV (6), VGVPG (7), GPP, GAP, or GAHGPAGPK (8) and/or a sequence (Y1) in which 1 to 100 amino acids in the sequence (Y) are replaced by lysine (K) or arginine (R).   
     
     
         9 . The method for producing a protein composition according to  claim 8 ,
 wherein a ratio of a number of units of GAGAGS (1) to a total number of the amino acid sequences (a2) and amino acid sequences (a2′) described below in one molecule of the protein (A) [(the number of units of GAGAGS (1)):(the total number of the amino acid sequences (a2) and (a2′))] is [1:2] to [1:20],   
       amino acid sequence (a2′): an amino acid sequence in which one to five amino acids in the amino acid sequence (a2) are replaced by lysine (K) or arginine (R). 
     
     
         10 . The method for producing a protein composition according to  claim 5 ,
 wherein the repetitive sequence (X) includes a sequence (Y2) consisting of 1 to 50 repeats of an amino acid sequence (a3) that is one of GAAVTGRGDSPASAAGY (17) or GAAPGASIKVAVSAGPSAGY (18).   
     
     
         11 . The method for producing a protein composition according to  claim 1 ,
 wherein the protein (A) has a molecular mass of 15 to 200 kDa as determined by SDS-polyacrylamide gel electrophoresis.   
     
     
         12 . The method for producing a protein composition according to  claim 1 ,
 wherein the radical scavenger (RS) has a structure that is at least one selected from the group consisting of oxygen-containing conjugated structures and nitrogen-containing conjugated structures.   
     
     
         13 . The method for producing a protein composition according to  claim 1 ,
 wherein the radical scavenger (RS) has a radical scavenging ability against diphenylpicrylhydrazyl radicals of 0.01 to 90 mg Trolox eq/mg.   
     
     
         14 . A protein composition, comprising:
 a protein (A),   wherein the protein composition further contains a radical scavenger (RS), and at least one hydrogen-bond-formable compound (HC) selected from the group consisting of amino acids, peptides, and proteins other than the protein (A),   the protein (A) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups,   the hydrogen-bond-formable compound (HC) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups,   the at least one functional group in the protein (A) is capable of binding to the at least one functional group in the hydrogen-bond-formable compound (HC) via a hydrogen bond,   a weight ratio of the radical scavenger (RS) to the protein (A) in the protein composition [radical scavenger (RS)/protein (A)] is 0.01 to 1.0,   a molar ratio of a total molar number of functional groups in the hydrogen-bond-formable compound (HC) to a total molar number of functional groups in the protein (A) in the protein composition [the total molar number of functional groups in the hydrogen-bond-formable compound (HC)/the total molar number of functional groups in the protein (A)] is 0.01 to 0.50, and   the protein composition has been radiosterilized.