Designed proteins for pH switchable antibody purification
Modified Fc-binding domain polypeptides or polypeptides including one or more modified Fc-binding domains are provided. In some examples, a modified Fc-binding domain includes or consists of the amino acid sequence of any one of SEQ ID NOs: 4, 6, or 8. Nucleic acids encoding the modified Fc-binding domains or polypeptides including one or more of the modified Fc-binding domains are also provided. In some embodiments, the nucleic acids are included in a vector and may be operably linked to a promoter. Methods for purifying a polypeptide including one or more Fc regions are also provided. Kits and reagents including the modified Fc-binding domain polypeptides or polypeptides including one or more modified Fc-binding domains are provided. In some examples, the modified Fc-binding domain polypeptides or polypeptides including one or more modified Fc-binding domains are linked to a solid support.
1. A modified Fc-binding domain polypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
2. The modified Fc-binding domain polypeptide of claim 1 , wherein the modified Fc-binding domain consists of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
3. A nucleic acid encoding the modified Fc-binding domain polypeptide of claim 1 .
4. The nucleic acid of claim 3 , comprising the nucleic acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.
5. An immunoglobulin-binding protein comprising one or more modified Fc-binding domain polypeptides of claim 1 .
6. The immunoglobulin-binding protein of claim 5 , comprising the amino acid sequence of any one of SEQ ID NOs: 10-12.
7. A nucleic acid encoding the immunoglobulin-binding protein of claim 5 .
8. The nucleic acid of claim 7 , comprising the nucleic acid sequence of any one of SEQ ID NOs: 13-15.
9. A vector comprising the nucleic acid of claim 3 , operably linked to a promoter.
10. The modified Fc-binding domain polypeptide of claim 1 or an immunoglobulin-binding protein comprising one or more of the modified Fc-binding domain polypeptides, linked to a solid support.
11. The modified Fc-binding domain polypeptide or the immunoglobulin-binding protein linked to a solid support of claim 10 , wherein the solid support comprises agarose, sepharose, or polyacrylamide.
12. A kit comprising the modified Fc-binding domain polypeptide or the immunoglobulin-binding protein linked to a solid support of claim 10 and one or more buffers.
13. The kit of claim 12 , wherein the one or more buffers comprises an elution buffer with a pH of about 7-8.2.
14. A method of purifying a polypeptide including one or more Fc regions, comprising:
contacting the modified Fc-binding domain polypeptide of claim 1 or an immunoglobulin-binding protein comprising one or more of the modified Fc-binding domain polypeptides with a polypeptide including one or more Fc regions at a pH of about 6 or less to form a complex of the modified Fc-binding domain or the immunoglobulin-binding protein and the polypeptide including one or more Fc regions; and
contacting the complex with a buffer above about pH 7 to dissociate the polypeptide including one or more Fc regions.
15. The method of claim 14 , wherein the modified Fc-binding domain polypeptide or the immunoglobulin-binding protein is linked to a solid support.
16. The method of claim 14 , wherein the modified Fc-binding domain polypeptide or the immunoglobulin-binding protein is contacted with the polypeptide including one or more Fc regions at a pH of about 5.6.
17. The method of claim 14 , wherein the buffer above about 6 pH 7 to dissociate the polypeptide including one or more Fc regions comprises a buffer with pH about 8.2.
18. The nucleic acid of claim 4 , consisting of the nucleic acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.
19. The immunoglobulin-binding protein of claim 6 , consisting of the amino acid sequence of any one of SEQ ID NOs: 10-12.
20. The nucleic acid of claim 8 , consisting of the nucleic acid sequence of any one of SEQ ID NOs: 13-15.