GLYCOPROTEIN PRODUCTION METHOD AND SCREENING METHOD
A method for producing a glycoprotein, which is uniform in terms of functions derived from a sugar chain (e.g., a blood half-life) and physiological activities, i.e., a glycoprotein, which is uniform in terms of the amino acid sequence, the sugar chain structure and the higher-order structure; specifically, a method for producing a glycoprotein, which is uniform in terms of the amino acid sequence, the sugar chain structure, and the higher-order structure, includes the following steps (a) to (c): (a) folding a glycoprotein, which is uniform in terms of the amino acid sequence and the sugar chain structure; (b) fractionating the folded glycoprotein by column chromatography; and (c) collecting a fraction having a specified activity.
1 . A method for producing a glycoprotein having uniform amino acid sequence, sugar chain structure, and higher order structure, comprising the following steps (a) to (c):
(a) folding a glycoprotein having uniform amino acid sequence and sugar chain;
(b) fractionating the folded glycoprotein by column chromatography; and
(c) collecting a fraction having a predetermined activity.
2 . The method according to claim 1 , further comprising, after the step (c), the steps of:
(d) unfolding a glycoprotein contained in a fraction not collected in the step (c);
(e) refolding the unfolded glycoprotein;
(f) fractionating the refolded glycoprotein by column chromatography and collecting a fraction having a predetermined activity; and
(g) repeating the steps (d) to (f) as needed.
3 . A method for screening for a glycoprotein having a predetermined physiological activity, comprising the following steps (i) to (iii):
(i) folding a glycoprotein having uniform amino acid sequence and sugar chain;
(ii) fractionating the folded glycoprotein by column chromatography; and
(iii) measuring an activity of each of the fractions to determine whether or not it has a predetermined activity.
4 . A method for obtaining a glycoprotein mixture having a desired physiological activity, comprising the following steps (A) to (D):
(A) folding a glycoprotein having uniform amino acid sequence and sugar chain;
(B) fractionating the folded glycoprotein by column chromatography;
(C) measuring an activity of each of the fractions; and
(D) determining a mixing ratio of the fractions to obtain a desired activity and mixing the fractions according to the ratio thus obtained.
5 . The method according to claim 1 , wherein at least a part of the glycoprotein having uniform amino acid sequence and sugar chain are produced by a method comprising the following steps (1) to (6):
(1) esterifying a hydroxyl group of a resin having a hydroxyl group and a carboxyl group of an amino acid having an amino group protected with a fat-soluble protecting group or a carboxyl group of a glycosylated amino acid having an amino group protected with a fat-soluble protecting group;
(2) removing the fat-soluble protecting group to generate a free amino group;
(3) amidating the free amino group and a carboxyl group of an amino acid having an amino group protected with a fat-soluble protecting group or a carboxyl group of a glycosylated amino acid having an amino group protected with a fat-soluble protecting group;
(4) after the step (3), removing the fat-soluble protecting group to generate a free amino group;
(5) repeating the steps (3) and (4) once or more; and
(6) cleaving an ester bond formed in the step (1) by an acid.
6 . The method according to claim 5 , wherein a part of the glycoprotein having uniform amino acid sequence and sugar chain are produced by the steps (1) to (6), and wherein the glycoprotein is produced by a method further comprising the following step (7):
(7) linking a part of the glycoprotein obtained in the step (6) with other peptides or glycopeptides by a ligation method.
7 . The method according to claim 2 , wherein at least a part of the glycoprotein having uniform amino acid sequence and sugar chain are produced by a method comprising the following steps (1) to (6):
(1) esterifying a hydroxyl group of a resin having a hydroxyl group and a carboxyl group of an amino acid having an amino group protected with a fat-soluble protecting group or a carboxyl group of a glycosylated amino acid having an amino group protected with a fat-soluble protecting group;
(2) removing the fat-soluble protecting group to generate a free amino group;
(3) amidating the free amino group and a carboxyl group of an amino acid having an amino group protected with a fat-soluble protecting group or a carboxyl group of a glycosylated amino acid having an amino group protected with a fat-soluble protecting group;
(4) after the step (3), removing the fat-soluble protecting group to generate a free amino group;
(5) repeating the steps (3) and (4) once or more; and
(6) cleaving an ester bond formed in the step (1) by an acid.
8 . The method according to claim 3 , wherein at least a part of the glycoprotein having uniform amino acid sequence and sugar chain are produced by a method comprising the following steps (1) to (6):
(1) esterifying a hydroxyl group of a resin having a hydroxyl group and a carboxyl group of an amino acid having an amino group protected with a fat-soluble protecting group or a carboxyl group of a glycosylated amino acid having an amino group protected with a fat-soluble protecting group;
(2) removing the fat-soluble protecting group to generate a free amino group;
(3) amidating the free amino group and a carboxyl group of an amino acid having an amino group protected with a fat-soluble protecting group or a carboxyl group of a glycosylated amino acid having an amino group protected with a fat-soluble protecting group;
(4) after the step (3), removing the fat-soluble protecting group to generate a free amino group;
(5) repeating the steps (3) and (4) once or more; and
(6) cleaving an ester bond formed in the step (1) by an acid.
9 . The method according to claim 4 , wherein at least a part of the glycoprotein having uniform amino acid sequence and sugar chain are produced by a method comprising the following steps (1) to (6):
(1) esterifying a hydroxyl group of a resin having a hydroxyl group and a carboxyl group of an amino acid having an amino group protected with a fat-soluble protecting group or a carboxyl group of a glycosylated amino acid having an amino group protected with a fat-soluble protecting group;
(2) removing the fat-soluble protecting group to generate a free amino group;
(3) amidating the free amino group and a carboxyl group of an amino acid having an amino group protected with a fat-soluble protecting group or a carboxyl group of a glycosylated amino acid having an amino group protected with a fat-soluble protecting group;
(4) after the step (3), removing the fat-soluble protecting group to generate a free amino group;
(5) repeating the steps (3) and (4) once or more; and
(6) cleaving an ester bond formed in the step (1) by an acid.
10 . The method according to claim 7 , wherein a part of the glycoprotein having uniform amino acid sequence and sugar chain are produced by the steps (1) to (6), and wherein the glycoprotein is produced by a method further comprising the following step (7):
(7) linking a part of the glycoprotein obtained in the step (6) with other peptides or glycopeptides by a ligation method.
11 . The method according to claim 8 , wherein a part of the glycoprotein having uniform amino acid sequence and sugar chain are produced by the steps (1) to (6), and wherein the glycoprotein is produced by a method further comprising the following step (7):
(7) linking a part of the glycoprotein obtained in the step (6) with other peptides or glycopeptides by a ligation method.
12 . The method according to claim 9 , wherein a part of the glycoprotein having uniform amino acid sequence and sugar chain are produced by the steps (1) to (6), and wherein the glycoprotein is produced by a method further comprising the following step (7):
(7) linking a part of the glycoprotein obtained in the step (6) with other peptides or glycopeptides by a ligation method.