Immunoglobulin-binding proteins with improved specificity
The present invention relates to modified immunoglobulin-binding proteins, e.g., Staphylococcus protein A, having improved binding specificity for immunoglobulins and methods of making and using the same.
1. A method of affinity purifying one or more immunoglobulins from a sample, the method comprising the steps of:
(a) providing a sample comprising one or more immunoglobulins;
contacting the sample with a chromatography matrix under conditions such that the one or more immunoglobulins bind to the matrix, wherein the chromatography matrix comprises an immunoglobulin-binding protein which binds an Fc portion of the one or more immunoglobulins but exhibits reduced binding to a Fab portion of the one or more immunoglobulins relative to wild-type protein A and comprises one or more isolated domains comprising the amino acid sequence set forth in SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and wherein the one or more isolated domains comprise: (i) at least the glycine residue at position 29 of SEQ ID NO: 10 replaced with an amino acid residue selected from the group consisting of a lysine residue, a leucine residue and an arginine residue, as shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; or (ii) at least the glycine residue at position 29 of SEQ ID NO: 11 replaced with an amino acid residue selected from the group consisting of a lysine residue, a leucine residue and an arginine residue, as shown in SEQ ID NO:26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively; or (iii) at least the alanine at position 29 of SEQ ID NO:12 replaced with an amino acid residue selected from the group consisting of a lysine residue, a leucine residue and an arginine residue, as shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively, and
(b) recovering the one or more bound immunoglobulins by eluting under suitable conditions.
2. The method of claim 1 , wherein the one or more immunoglobulins is selected from the group consisting of IgG, IgA or IgM, or a fragment thereof.
3. The method of claim 1 , wherein the one or more immunoglobulins is an IgG or a fragment thereof.
4. The method of claim 1 , wherein the Fc portion of an immunoglobulin is part of an Fc fusion protein.
5. The method of claim 1 , wherein the amino acid residue is lysine.
6. The method of claim 1 , wherein the immunoglobulin-binding protein is coupled to a solid support.
7. The method of claim 6 , wherein the solid support is selected from the group consisting of controlled pore glass, agarose, methacrylate, and polystyrene.
8. The method of claim 1 , wherein the immunoglobulin-binding protein further comprising at least a portion of the carboxy-terminal region of Staphylococcus protein A.