Ligand and use thereof
The present invention is within the field of protein engineering and purification. The invention relates to a target-binding polypeptide mutant of an IgG binding polypeptide, such as Protein A, Protein G, Protein L or Protein M, comprising a metal binding motif. More closely the invention relates to an Fc binding ligand comprising an engineered protein based on the Protein A derived Z domain, to which a calcium binding EF-loop has been introduced.
1. A Fc-binding polypeptide mutant of an IgG-binding polypeptide comprising the amino acid sequence selected from SEQ ID NOs: 1-9 (Zmat1-Zmat9),wherein the Fc-binding polypeptide mutant is a mutant of domain Z derived from Staphylococcus Protein A (SpA) domain B and comprises a metal binding motif inserted between helix 2 and 3 of the mutant of domain Z, wherein Fc binding occurs in the presence of a metal ion and wherein the Fc-binding polypeptide mutant optionally comprises the following Z domain mutation: N23T.
2. The Fc-binding polypeptide mutant according to claim 1 , wherein the metal binding motif is calcium binding and the Fc-binding is in the presence of calcium ions.
3. The Fc-binding polypeptide mutant according to claim 1 , comprising a linker before the metal binding motif.
4. The Fc-binding polypeptide mutant according to claim 1 , comprising the amino acid sequence of SEQ ID NO: 8 (Zmat8).
5. The Fc-binding polypeptide mutant according to claim 1 , comprising the following domain Z mutations: F5L, N28K, K35I.
6. A ligand comprising the Fc-binding polypeptide mutant according to claim 1 .
7. The ligand according to claim 6 , comprising the Fc-binding polypeptide mutant comprising the amino acid sequence of SEQ ID NO: 8 (Zmat8).
8. The ligand according to claim 6 , wherein the ligand is a multimer selected from a dimer, trimer, tetramer, pentamer or hexamer.
9. A chromatography matrix comprising the ligand according to claim 6 or multimer thereof.
10. A polypeptide comprising the amino acid sequence selected from SEQ ID Nos:1-9 (Zmat1-Zmat9).
11. The polypeptide of claim 10 , wherein the amino acid sequence is SEQ ID NO:8.
12. A method for purification of target or sample proteins, comprising binding the target or sample proteins to a the ligand according to claim 6 or multimer thereof in the presence of a metal ion; and eluting bound proteins by addition of a metal binding molecule.
13. The method according to claim 12 , wherein the ligand or multimer ligand is immobilized to a solid phase.
14. The method according to claim 12 , wherein the bound proteins are eluted at a pH above 4.5.
15. The method according to claim 14 , where the bound proteins are eluted at pH 5-7.
16. The method according to claim 15 , where the bound proteins are eluted at pH 5.4-5.6.
17. The method according to claim 12 , wherein the target or sample proteins are antibodies or portions thereof.
18. The method according to claim 17 , wherein the target or sample proteins are IgG.
19. The method according to claim 17 , wherein the target or sample proteins are Fc-fusion proteins.
20. The method according to claim 12 , wherein the elution with metal binding molecule is with a chelating agent.
21. A nucleic acid or vector encoding a polypeptide comprising the amino acid sequence selected from SEQ ID NOs: 1-9.
22. An expression vector comprising the nucleic acid or vector of claim 21 .