Mutated immunoglobulin-binding protein
The present invention relates to an immunoglobulin-binding protein, wherein at least one asparagine residue has been mutated to an amino acid other than glutamine or aspartic acid, which mutation confers an increased chemical stability at pH-values of up to about 13-14 compared to the parental molecule. The protein can for example be derived from a protein capable of binding to other regions of the immunoglobulin molecule than the complementarity determining regions (CDR), such as protein A, and preferably the B-domain of Staphylococcal protein A. The invention also relates to a matrix for affinity separation, which comprises an immunoglobulin-binding protein as ligand coupled to a solid support, in which protein ligand at least one asparagine residue has been mutated to an amino acid other than glutamine.
1. A matrix for affinity chromatography, comprising a plurality of ligands coupled to a solid support,
wherein the ligands comprise an immunoglobulin-binding protein, or multimer thereof that binds to other regions of the immunoglobulin molecule than the complementarity determining regions (CDR), and
wherein at least one of N23 and N43 of a parental immunoglobulin-binding protein defined by SEQ ID NOS:1 or 2 have been mutated to amino acid residues selected from A, I, S, T, E, or H,
wherein the N23 and N43 asparagine residues can be mutated to amino acid residues that are the same or different from each other, and
wherein the mutations have conferred an increased chemical stability at alkaline pH-values compared to the parental immunoglobulin-binding protein.
2. The matrix of claim 1 , wherein the mutations are N23T and N43E and the ligands have been coupled to the support by thioether bonding.
3. The matrix of claim 1 , wherein the support is a polysaccharide.
4. The matrix of claim 1 , which selectively binds an immunoglobulin selected from the group consisting of IgG, IgA, and IgM.
5. The matrix of claim 4 , which selectively binds an IgG immunoglobulin.
6. The matrix of claim 1 , wherein the ligands exhibit an increased binding capacity, during two or more separations with intermittent alkaline cleaning, compared to the parental protein molecule.
7. The matrix of claim 6 , wherein the cleaning is performed with NaOH and the concentration thereof is up to about 1 M.
8. The matrix of claim 7 , wherein the cleaning is performed with NaOH and the concentration thereof is about 0.5 M.
9. The matrix of claim 1 , wherein the protein is an Fc fragment-binding protein.
10. The matrix of claim 1 , wherein each ligand includes a multimer comprising two or more repetitive protein units.
11. The matrix of claim 10 , wherein the protein units of the multimer are linked by elements including up to about 15 amino acids.
12. The matrix of claim 10 , wherein the multimer further comprises one or more of the E, D, A, B, and C domains of Staphylococcus protein A.
13. The matrix of claim 10 , wherein the multimer is a tetramer having a sequence of SEQ ID NO:22 or SEQ ID NO:23.