IMMUNOGLOBULIN-BINDING PROTEIN VARIANTS WITH INCREASED ALKALI-TOLERANCE AND USE THEREOF

20230356187

18/305522

April 24, 2023

Provided are immunoglobulin-binding protein variants with increased alkali tolerance, and uses thereof, and more particularly to immunoglobulin-binding protein variants with increased alkali tolerance relative to the wild type due to mutations in amino acids at specific positions in the B3 domain of the immunoglobulin-binding proteins

1. A polypeptide comprising an amino acid mutation of SEQ ID NO:4, wherein the amino acid mutation is substitution of at least one amino acid selected from the group consisting of: beginning from a proline (Pro, P) which is the second amino acid in the amino acid sequence of SEQ ID NO: 4, the 28th amino acid (glutamic acid (Glu, E)), the 43rd amino acid (lysine (Lys, K)), the 13th amino acid (tyrosine (Tyr, Y)), the 51st amino acid (aspartic acid (Asp, D)), and the 60th amino acid (asparagine (Asn, N) of the amino acid sequence of SEQ ID NO: 4, with an amino acid different from the original amino acid.

2. The polypeptide of claim 1, wherein the polypeptide comprises at least one substitution selected from the group consisting of the following: the 28th amino acid (E) is replaced with glycine (Gly, G), alanine (Ala, A), leucine (Leu, L), proline (Pro, P), or tryptophan (Trp, W); the 43rd amino acid (K) is replaced with proline (Pro, P) or glutamic acid (Glu, E); the 13th amino acid (Y) is replaced with phenylalanine (Phe, F); the 51st amino acid (D) is replaced with valine (Val, V) or threonine (Thr, T); and the 60th amino acid (N) is replaced with phenylalanine (Phe, F).

3. The polypeptide of claim 1, wherein the 28th amino acid (E) is replaced with glycine (Gly, G), alanine (Ala, A), leucine (Leu, L), proline (Pro, P), or tryptophan (Trp, W).

4. The polypeptide of claim 3, further comprising at least one substitution selected from the group consisting of the following: the 43rd amino acid (K) is replaced with proline (Pro, P) or glutamic acid (Glu, E); the 13th amino acid (Y) is replaced with phenylalanine (Phe, F); the 51st amino acid (D) is replaced with valine (Val, V) or threonine (Thr, T); and the 60th amino acid (N) is replaced with phenylalanine (Phe, F).

5. The polypeptide of claim 1, comprising the following amino acid mutation in the amino acid sequence of SEQ ID NO: 4: E28A, E28G, E28L, E28P, E28W, E28G+Y13F, E28G+K43P, E28G+D51V, E28G+N60E, E28L, E28P, E28W, E28G+K43E, E28G+D51T, E28G+N60F, or Y13F+E28G+K43P.

6. The polypeptide of claim 1, represented by the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 11, 12, 23, 24, 25, 26, 27, 28, or 47.

7. The polypeptide of claim 1, further comprising at least one selected from the group consisting of the A, B1, B2, B4, C, W, and M domain of protein L.

8. The polypeptide of claim 1, wherein the polypeptide has increased alkali tolerance relative to the polypeptide of SEQ ID NO: 4.

9. A multimer, comprising at least two repeating units of the polypeptide of claim 1.

10. The multimer of claim 9, represented by the amino acid sequence of SEQ ID NO: 49.

11. The multimer of claim 9, further comprising at least one selected from the group consisting of the A, B 1, B2, B4, C, W, and M domain of protein L.

12. A protein L variant, comprising the polypeptide of claim 1, or a multimer comprising at least two repeating units of the polypeptide, as a B3 domain

13. A nucleic acid molecule encoding the polypeptide of claim 1, a multimer comprising at least two repeating units of the polypeptide, or a protein L variant comprising the polypeptide or the multimer as a B3 domain.

14. A recombinant vector comprising the nucleic acid molecule of claim 13.

15. A recombinant cell comprising the nucleic acid molecule of claim 13 or a recombinant vector comprising the nucleic acid molecule.

16. A matrix for chromatography, wherein the polypeptide of claim 1, a multimer comprising at least two repeating units of the polypeptide, or a protein L variant comprising the polypeptide or the multimer as a B3 domain, is coupled to a solid support.

17. A composition for binding an immunoglobulin, comprising at least one selected from the group consisting of (1) the polypeptide of claim 1, (2) a multimer comprising two or more repeating units of the polypeptide, (3) a protein L variant comprising the polypeptide or the multimer as a B3 domain, (4) a nucleic acid molecule encoding the polypeptides, the multimer, or the protein L variant, (5) a recombinant vector comprising the nucleic acid molecule, (6) a recombinant cell comprising the nucleic acid molecule or a recombinant vector; and (7) a matrix for chromatography, wherein the polypeptide, the multimer, or the protein L variant is coupled to a solid support.

18. A composition for immunoglobulin isolation or purification, comprising (a) the polypeptide of claim 1, (b) a multimer comprising two or more repeating units of the polypeptide, (c) a protein L variant comprising the polypeptide or the multimer as a B3 domain; or (d) a matrix for chromatography, wherein a plurality of ligands comprising the polypeptides, multimer, or protein L variant are coupled to a solid support.

19. A method of isolating or purifying an immunoglobulin, comprising, adsorbing an immunoglobulin by contacting a sample comprising the immunoglobulin with at least one selected from the group consisting of the following: (a) the polypeptide of claim 1, (b) a multimer comprising two or more repeating units of the polypeptide, (c) a protein L variant comprising the polypeptide or the multimer as a B3 domain; and (d) a matrix for chromatography, wherein a plurality of ligands comprising the polypeptides, the multimer, or the protein L variant are coupled to a solid support.

20. A method of isolating or purifying at least one target compound from a liquid comprising adsorbing the target compound by contacting a sample comprising the target compound with at least one selected from the group consisting of the following: (a) the polypeptide of claim 1, (B) a multimer comprising two or more repeating units of the polypeptide, (c) a protein L variant comprising the polypeptide or the multimer as a B3 domain; and (d) a matrix for chromatography, wherein a plurality of ligands comprising the polypeptides, the multimer, or the protein L variant are coupled to a solid support.

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