Fc binding proteins with cysteine in the c-terminal helical region
The present invention relates to Fc binding proteins comprising one or more domains with Cysteine in the C-terminal helical region. The invention further relates to affinity matrices comprising the Fc binding proteins of the invention. The invention also relates to a use of these Fc binding proteins or affinity matrices for affinity purification of immunoglobulins and to methods of affinity purification using the Fc binding proteins of the invention.
1. An affinity separation matrix comprising an Fc binding protein comprising one or more domains, wherein at least one domain comprises an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence with at least 91% identity thereto, and wherein at least one amino acid a position corresponding to position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 of SEQ ID NO: 3 is cysteine.
2. The affinity separation matrix of claim 1 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NO: 3.
3. The affinity separation matrix of claim 1 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence with at least 93% identity to SEQ ID NO: 3.
4. The affinity separation matrix of claim 1 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NOs: 17-20, 40-52, 64, 65, or 74-76.
5. The affinity separation matrix of claim 1 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence with at least 91% identity thereto, wherein at least one amino acid in a position corresponding to position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 of SEQ ID NO: 4 is cysteine.
6. The affinity separation matrix of claim 5 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence with at least 93% identity to SEQ ID NO: 4.
7. The affinity separation matrix of claim 5 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NOs: 21-25.
8. The affinity separation matrix of claim 1 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NO: 5, or an amino acid sequence with at least 91% identity thereto, wherein at least one amino acid in in a position corresponding to position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 of SEQ ID NO: 5 is cysteine.
9. The affinity separation matrix of claim 8 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence with at least 93% identity to SEQ ID NO: 5.
10. The affinity separation matrix of claim 8 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NOs: 53-57.
11. The affinity separation matrix of claim 1 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence with at least 91% identity thereto, wherein at least one amino acid in in a position corresponding to position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 of SEQ ID NO: 6 is cysteine.
12. The affinity separation matrix of claim 11 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence with at least 93% identity to SEQ ID NO: 6.
13. The affinity separation matrix of claim 11 , wherein the at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NOs: 58-63.
14. The affinity separation matrix of claim 1 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or an amino acid sequence with at least 91% identity thereto, wherein at least one amino acid in a position corresponding to position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 of SEQ ID NO: 7 is cysteine.
15. The affinity separation matrix of claim 14 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8.
16. The affinity separation matrix of claim 14 , wherein at least one domain of the Fc binding protein comprises an amino acid sequence of SEQ ID NOs: 26-39 or 90-95.
17. The affinity separation matrix of claim 1 , wherein the at least one domain of the Fc binding protein has a deletion of 1, 2, or 3 amino acids within the first 4 amino acids of its N-terminus and/or a deletion of 1 or 2 amino acids within the first 2 amino acids of its C-terminus.
18. The affinity separation matrix of claim 1 , wherein the Fc binding protein comprises 2, 3, 4, 5, 6, 7, or 8 domains linked to each other.
19. The affinity separation matrix of claim 18 , wherein the Fc binding protein is a homo-multimer or a hetero-multimer.
20. The affinity separation matrix of claim 9 , wherein one or more domains of the Fc binding protein are linked to each other directly or with one or more linkers, optionally wherein the one or more linkers is a peptide linker.
21. The affinity separation matrix of claim 1 , wherein the Fc binding protein is conjugated to the matrix via a cysteine in a position corresponding to position 40, 42, 43, 46, 47, 50, 51, 53, or 54 of SEQ ID NO: 3.
22. The affinity separation matrix of claim 1 , wherein at least one amino acid of the Fc binding protein in a position corresponding to position 43, 46, or 47 of SEQ ID NO: 3 is cysteine.