Support for affinity chromatography and method for isolating immunoglobulin
Provided are a support for affinity chromatography which has excellent alkali resistance, and a method for isolating immunoglobulin. A support for affinity chromatography, containing an immobilized protein ligand represented by the following formula (1): R—R 2 (1) wherein R represents a polypeptide consisting of 4 to 30 amino acid residues that contains an amino acid sequence represented by ATK or ASK; and R 2 represents a polypeptide consisting of 50 to 500 amino acid residues containing an immunoglobulin-binding domain consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, the partial sequence thereof, or an amino acid sequence having 70% or more identity to these sequences; with the proviso that a terminus at which R 2 binds to R is C-terminus or N-terminus of the immunoglobulin-binding domain.
1. A support immobilizing a protein ligand comprising:
an anchor peptide consisting of 4 to 30 amino acid residues containing an amino acid sequence represented by ATK or ASK; and
an immunoglobulin binding domain consisting of the amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or at least 90% identical to SEQ ID NO: 2 and that is no longer than 500 amino acid residues;
wherein the C- or N-terminus of said immunoglobulin binding domain is bound to said anchor peptide and the anchor peptide is bound to the support, optionally via a spacer; and wherein the anchor peptide comprises the sequence XX-UU-XX (SEQ ID NO:48), where X is K, S or T, and U is an optional amino acid residue which may be any amino acid residue,
wherein the anchor peptide comprises the sequence XXX-GA-XX (SEQ ID NO: 49), where X is A, K, S or T, and/or
wherein the anchor peptide comprises ASK, ASKATK (SEQ ID NO: 45), ATKASK (SEQ ID NO: 44), or ATKATK (SEQ ID NO: 47); and
wherein the support has a dynamic binding capacity for IgG that is higher than an otherwise identical support not comprising said anchor peptide after the support is treated with 0.5 M sodium hydroxide for 15 hrs.
2. The support according to claim 1 , wherein the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 is the amino acid sequence represented by SEQ ID NO: 3.
3. The support according to claim 1 , wherein the anchor peptide contains the amino acid sequence represented by a combination of ATK and ASK.
4. The support according to claim 1 , wherein the anchor peptide contains the amino acid sequence represented by ATKASK (SEQ ID NO: 44) or ASKATK (SEQ ID NO: 45).
5. The support according to claim 1 , wherein the anchor peptide contains the amino acid sequence represented by ASKASK (SEQ ID NO: 46).
6. The support according to claim 1 , wherein the anchor peptide contains the amino acid sequence represented by ATKATK (SEQ ID NO: 47).
7. The support according to claim 1 , wherein the protein ligand is immobilized on the support via epoxy group ring-opening reaction between an amino group or a thiol group in the ligand and an epoxy group in the support.
8. The support of claim 1 that is an affinity chromatography support.
9. The support of claim 8 that is in a particulate form.
10. The support of claim 8 that comprises synthetic polymer particles having a particle size ranging from 20 μm to 200 μm.
11. The support of claim 8 that comprises synthetic polymer particles having a particle size ranging from 100 nm to 400 nm.
12. The support of claim 8 that comprises porous particles having a specific surface area of 50 m 2 /g to 150 m 2 /g.
13. The support of claim 8 that has a dynamic binding capacity (“DBC”) for human polyclonal IgG ranging from 40 to 45 after contact with 0.5 M sodium hydroxide for 15 hrs.
14. The support of claim 8 that has a retention ratio of the immunoglobulin binding domain ranging from 89.0 to 96.8 after contact with 0.5 M sodium hydroxide for 15 hrs.
15. The support of claim 8 that comprises polysaccharide particles having a particle size ranging from 50 μm to 200 μm.
16. The support of claim 8 that comprises polysaccharide particles having a particle size ranging from 500 nm to 1,400 nm.
17. The support of claim 8 , wherein said protein ligand ranges in length from 70 to 1,000 amino acid residues.
18. The support of claim 8 , wherein said protein ligand ranges in length from 80 to 600 amino acid residues.
19. The support of claim 1 , wherein the immunoglobulin binding domain comprises G29A or an alanine substitution at a residue corresponding to G29.
20. A method for isolating an immunoglobulin comprising:
applying a sample containing immunoglobulin through the support for affinity chromatography according to claim 1 to adsorb the immunoglobulin to the support; and
eluting the immunoglobulin from the support.
21. A method for isolating an immunoglobulin comprising:
applying a sample containing immunoglobulin through the support according to claim 7 to adsorb the immunoglobulin to the support; and
eluting the adsorbed immunoglobulin from the support.