Alkali-resistant variants of protein A and their use in affinity chromatography
The present invention relates to immunoglobulin (Ig)-binding proteins with alkali-resistance properties. In one embodiment, the present invention provides for a variant of an Ig-binding protein, the variant comprising the Ig-binding protein having at least one asparagine residue substituted with a histidine, a serine, an aspartic acid or a threonine residue. The at least one substitution may confer to the variant Ig-binding protein an increased stability in alkaline solutions when compared to the wild-type Ig-binding. The present invention relates also to matrices for affinity separation of immunoglobulins comprising the Ig-binding proteins of the present invention, and to methods of using the Ig-binding proteins of the present invention to separate immunoglobulins from mixture compositions.
1. An Ig-binding protein comprising the peptide sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 14.
2. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 2.
3. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 3.
4. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 4.
5. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 5.
6. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 6.
7. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 7.
8. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 8.
9. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 9.
10. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 10.
11. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 11.
12. The Ig-binding protein of claim 1 , comprising the peptide sequence of SEQ ID NO: 14.
13. A matrix, comprising:
the Ig-binding protein of claim 1 coupled to a solid support.
14. The matrix of claim 13 ,
wherein the Ig-binding protein is coupled to the solid support through a linker peptide.
15. The matrix of claim 14 ,
wherein the linker peptide comprises the amino acid sequence of SEQ ID NO: 15.
16. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 2.
17. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 3.
18. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 4.
19. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 5.
20. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 6.
21. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 7.
22. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 8.
23. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 9.
24. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 10.
25. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 11.
26. The matrix of claim 13 , wherein the Ig-binding protein comprises the peptide sequence of SEQ ID NO: 14.
27. A method of separating an immunoglobulin from a composition, the method comprising:
contacting the composition with the matrix of claim 13 , thereby separating the immunoglobulin from another substance,
wherein the composition comprises the immunoglobulin and the other substance.