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. In an immunoglobulin-binding protein having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 capable of binding to regions of the immunoglobulin molecule other than complementarity determining regions (CDR), the improvement comprising mutating at least one asparagine residue of a parental, or a functional variant thereof, with an amino acid other than glutamine, wherein the mutation(s) are selected from the group consisting of N23T and N3A; N23T and N3A and N6D; and N23T and N43E.
2. The protein of claim 1 , which is an Fc fragment-binding protein.
3. A multimer comprised of two or more mutated protein units of claim 1 .
4. The multimer of claim 3 , wherein the protein units are linked by elements including up to about 15 amino acids.
5. The multimer of claim 3 , further comprising one or more of the E, D, A, B, and C domains of Staphylococcus protein A.
6. The multimer of claim 3 , comprising three mutated protein units.
7. The multimer of claim 6 , wherein the protein unit is the protein defined by SEQ ID NO. 2 and includes the mutation N23T.