Mutated immunoglobulin-binding protein
View Patent ↗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. An immunoglobulin-binding protein capable of binding to other regions of the immunoglobulin molecule than the complementarity determining regions (CDR), wherein at least the asparagine residue at position 6 or 11 of a parental immunoglobulin-binding protein defined by SEQ ID NO. 1 or 2, has been mutated to an amino acid other than glutamine, which mutation has conferred an increased chemical stability at alkaline pH-values compared to the parental molecule.
2. The protein of claim 1 , which is an Fc fragment-binding protein.
3. The protein of claim 1 , wherein the mutation(s) are selected from the group consisting of N6A; N11S; N11S and N23T; and N6A and N23T; and wherein the parental molecule comprises the sequence defined by SEQ ID NO. 2.
4. The protein of claim 1 , wherein the asparagine residue in position 23 has been mutated, preferably to a threonine residue.
5. The protein of claim 1 , wherein one or two asparagine residues are mutated.
6. The protein of claim 1 , further comprising an N23T mutation.
7. A multimer comprised of mutated protein units of claim 1 , which comprises two or more repetitive units.
8. The multimer of claim 7 , which further comprises a C-terminal cysteine.
9. The multimer of claim 7 , wherein the protein units are linked by elements including up to about 15 amino acids.
10. The multimer of claim 7 , further comprising one or more of the E, D, A, B, and C domains of Staphylococcus protein A.
11. The multimer of claim 7 , which is a tetramer.
12. The multimer of claim 11 , wherein each protein unit is the protein defined by SEQ ID NO. 2 and includes the mutation N23T.