Mutated immunoglobulin-binding polypeptides
View Patent ↗The invention discloses a polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a B or C domain of Staphylococcus Protein A (SpA), as specified by SEQ ID NO 1 or SEQ ID NO 2, or of Protein Z, as specified by SEQ ID NO 3, wherein at least the glutamic acid residue at position 15 has been mutated to an amino acid other than asparagine. The invention also discloses multimers of said polypeptide, as well as separation matrices comprising the multimers or polypeptides.
1. A separation matrix, comprising a plurality of polypeptides or multimers of the polypeptides coupled to a solid support, wherein the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10, 12, 13, or a combination thereof.
2. The separation matrix of claim 1 , wherein the polypeptides or multimers thereof have been coupled to the solid support via thioether bonds.
3. The separation matrix of claim 1 , wherein the solid support is a polysaccharide.
4. A method of isolating an immunoglobulin using the separation matrix of claim 1 , the method comprising the steps of: a) contacting a liquid sample comprising an immunoglobulin with the separation matrix of claim 1 , b) washing said separation matrix with a washing liquid, c) eluting the immunoglobulin from the separation matrix with an elution liquid, and d) cleaning the separation matrix with a cleaning liquid.
5. The method of claim 4 , wherein the cleaning liquid is alkaline having a pH of 13-14.
6. The method of claim 4 , wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.
7. The method of claim 4 , wherein steps a)-d) are repeated at least 10 times.
8. The separation matrix of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 6.
9. The separation matrix of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 7.
10. The separation matrix of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 8.
11. The separation matrix of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 9.
12. The separation matrix of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 10.
13. The separation matrix of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 12.
14. The separation matrix of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 13.
15. The separation matrix of claim 1 , wherein the polypeptide is the amino acid multimer with each monomer of the multimer comprising a sequence of any one of SEQ ID NOS: 6-10, 12, or 13.
16. The separation matrix of claim 1 , wherein the polypeptide is the amino acid multimer with each monomer comprising a sequence of SEQ ID NO: 6.
17. The separation matrix of claim 1 , wherein polypeptide is the amino acid multimer with each monomer comprising a sequence of SEQ ID NO: 7.
18. The separation matrix of claim 1 , wherein the polypeptide is the amino acid multimer with each monomer comprising a sequence of SEQ ID NO: 8.
19. The separation matrix of claim 1 , wherein the polypeptide is the amino acid multimer with each monomer comprising a sequence of SEQ ID NO: 9.
20. The separation matrix of claim 1 , wherein the polypeptide is the amino acid multimer with each monomer comprising a sequence of SEQ ID NO: 10.
21. The separation matrix of claim 1 , wherein the polypeptide is a multimer with each monomer comprising a sequence of SEQ ID NO: 12.
22. The separation matrix of claim 1 , wherein the polypeptide is a multimer with each monomer comprising a sequence of SEQ ID NO: 13.
23. The separation matrix of claim 1 , wherein the multimer comprises a plurality of the polypeptide units linked by elements comprising up to 15 amino acids.
24. The separation matrix of claim 1 , wherein the multimer is a dimer, trimer, tetramer, pentamer or hexamer.
25. The separation matrix of claim 1 , further comprising at the C-terminal or N-terminal of the polypeptide or multimer of the polypeptide one or more coupling element to couple the polypeptide to the support, wherein the coupling element is selected from the group consisting of a cysteine residue, a plurality of lysine residues and a plurality of histidine residues.