Protein A B and Z domain mutants and their applications

Abstract

Protein A B and Z domain mutants and their applications. Specifically, there is provided an isolated polypeptide selected from: (1) a polypeptide having a substitution mutation at one or more positions selected from the 3rd, 6th, 9th, 15th, and 23rd relative to the native B domain of Protein A as set forth in SEQ ID NO:1 or the Z domain of Protein A as set forth in SEQ ID NO:2; and (2) A polypeptide having at least 85% sequence identity with the polypeptide of (1) above, and having substitution mutations at one or more of the 3rd, 6th, 9th, 15th and 23rd positions. Also provided are a fusion protein or protein A comprising the polypeptide, and an isolation matrix. The fusion protein or protein A comprising the polypeptide has significantly improved alkaline stability. The alkaline stability is significantly improved during an alkaline washing process.

Description

[Technical field]

[0001] The present invention relates to B domain and Z domain mutants of Protein A and their applications. [Background technology]

[0002] In recent years, antibody drugs have attracted attention in the global pharmaceutical market due to their high specificity. Among them, monoclonal antibodies are one of the most rapidly growing fields in the biopharmaceutical industry. The higher the purity of monoclonal antibodies, the better their effects and higher their efficiency. Research on the purification of various antibody drugs, including monoclonal antibodies, is currently an important research topic in the field. Protein A affinity chromatography media has become the most widely used method for purifying monoclonal antibodies due to its special adsorption ability for specific Fc fragments of antibodies. However, Protein A affinity chromatography media has problems such as ligand detachment and reduced binding loading during the antibody purification process. In addition, because 0.5-1.0M NaOH solution is used in the cleaning in place (CIP) process in the purification process, Protein A affinity chromatography media is destroyed by high concentration NaOH, which significantly reduces its service life. Therefore, scientific researchers have been working hard to improve the alkali resistance of Protein A. For example, Susanne Gilich et al. have proposed to improve the stability of domains of protein ligands under alkaline conditions by protein engineering, and those skilled in the art have studied the mutations of amino acids in each domain of protein ligands to find amino acids that can improve the alkaline resistance of protein ligands.

[0003] Protein A is composed of immunoglobulin-binding domains E, D, A, B, and C, which are highly homologous. Among them, domain B has excellent binding specificity with immunoglobulin. Domain Z was obtained by substituting valine for the 1st alanine in domain B of Protein A and alanine for the 29th glycine. This mutation confers better chemical stability to domain Z, and domain Z obtained by mutating the domain amino acids also has a certain stability under alkaline conditions. However, it is still unstable under higher pH conditions and does not meet the requirements of the CIP washing step in the antibody purification process. Currently, the known Protein A chromatography media with mutated B domain or Z domain has a certain alkaline stability compared to the natural Protein A chromatography media. For example, CN101522278A improves alkaline resistance by mutating glycine at the 29th position in the B domain to alanine. CN1642976A disclosed that at least one asparagine residue in the Z domain is mutated to an amino acid other than alanine, threonine, or aspartic acid. Also, CN105377881A disclosed that at least the 15th glutamic acid residue in the B domain or Z domain is mutated to an amino acid other than asparagine or glutamine. These mutants have higher alkali resistance under alkaline conditions than the natural ligand. The chromatography media prepared with the above ligands can withstand washing with 0.1-0.5 M sodium hydroxide in the antibody purification process, but there is still a certain difference from the standard CIP (0.5-1.0 M sodium hydroxide). Summary of the Invention

[0004] A first aspect of the invention is an isolated polypeptide, the polypeptide comprising: (1) having a substitution mutation at one or more positions selected from the 3rd, 6th, 9th, 15th, and 23rd relative to the native B domain of protein A as set forth in SEQ ID NO:1 or the Z domain of protein A as set forth in SEQ ID NO:2; and a polypeptide wherein the substitution mutation at position 3 is a substitution of asparagine to leucine, isoleucine, valine, or tyrosine, the substitution mutation at position 6 is a substitution of asparagine to glutamic acid, leucine, isoleucine, valine, serine, or threonine, the substitution mutation at position 9 is a substitution of glutamine to isoleucine, leucine, valine, phenylalanine, or methionine, the substitution mutation at position 15 is a substitution of glutamic acid to threonine, tryptophan, leucine, valine, isoleucine, phenylalanine, serine, tyrosine, or aspartic acid, and the substitution mutation at position 23 is a mutation of asparagine to valine, isoleucine, leucine, or tyrosine; (2) A polypeptide having at least 85% sequence identity to the polypeptide of (1) above, and having substitution mutations at one or more of the 3rd, 6th, 9th, 15th, and 23rd positions.

[0005] In one or more embodiments, the substitution mutation at position 3 is an asparagine to leucine, valine, or tyrosine.

[0006] In one or more embodiments, the substitution mutation at position 6 is an asparagine to glutamic acid, leucine, or serine.

[0007] In one or more embodiments, the substitution mutation at position 9 is a glutamine to isoleucine, phenylalanine, or methionine.

[0008] In one or more embodiments, the substitution mutation at position 15 is a glutamic acid to threonine, tryptophan, leucine, isoleucine, valine, phenylalanine, serine, or tyrosine substitution.

[0009] In one or more embodiments, the substitution mutation at position 23 is an asparagine to valine, isoleucine, or tyrosine mutation.

[0010] In one or more embodiments, the polypeptide has the substitution mutation at at least the third position, and at least one position selected from the fifteenth, sixth, ninth and twenty-third positions, at least two positions, at least three positions, or all four positions.

[0011] In one or more embodiments, the polypeptide has the substitution mutation at at least the 6th position, and at least one position selected from the 3rd, 15th, 9th and 23rd positions, at least two positions, at least three positions, or all four positions.

[0012] In one or more embodiments, the polypeptide has the substitution mutation at at least the 9th position, and at least one position selected from the 3rd, 6th, 15th, and 23rd positions, at least two positions, at least three positions, or all four positions.

[0013] In one or more embodiments, the polypeptide has the substitution mutation at at least the 15th position, and has the substitution mutation at at least one position selected from the 3rd, 6th, 9th and 23rd positions, at least two positions, at least three positions, or all four positions.

[0014] In one or more embodiments, the polypeptide has the substitution mutation at least at position 23, and at least one position selected from positions 3, 6, 9, and 15, at least two positions, at least three positions, or all four positions.

[0015] In one or more embodiments, the 15th substitution mutation is E15I, E15L or E15V, the 3rd substitution mutation is N3L or N3V, the 6th substitution mutation is N6E or N6L, the 9th substitution mutation is Q9I or Q9F, and the 23rd substitution mutation is N23I, N23Y or N23V.

[0016] In one or more embodiments, the polypeptide has the substitution mutations at the 3rd, 9th and 15th positions, and the substitution mutation at the 6th and / or 23rd positions, preferably wherein the 3rd substitution mutation is N3L or N3V, the 6th substitution mutation is N6L or N6E, the 9th substitution mutation is Q9I or Q9F, the 15th substitution mutation is E15L, E15I or E15V, and the 23rd substitution mutation is N23V, N23I or N23Y.

[0017] In one or more embodiments, the polypeptide has said substitution mutations at positions 3, 6, 9 and 15. Preferably, the third substitution mutation is N3L or N3V, preferably N3V, the sixth substitution mutation is N6L or N6E, preferably N6E, the ninth substitution mutation is Q9I or Q9F, preferably Q9I, and the fifteenth substitution mutation is E15L, E15I or E15V, preferably E15L.

[0018] In one or more embodiments, the amino acid sequence of the polypeptide is set forth in any one of SEQ ID NOs:3-71 and 118-124.

[0019] A second aspect of the present invention provides an isolated polypeptide comprising: (i) a polypeptide according to any one of the embodiments of the first aspect of the invention; (ii) one or more coupling elements at the C-terminus or N-terminus of the amino acid sequence of the polypeptide according to (i); and, optionally, (iii) residues derived from a truncated signalling sequence.

[0020] In one or more embodiments, the coupling element is selected from a cysteine ​​residue, a plurality of lysine residues, and a plurality of histidine residues, and the residues of the truncated signaling sequence are AQ.

[0021] A third aspect of the present invention provides a fusion protein comprising an amino acid sequence resulting from the fusion of 2-8 polypeptides according to any one of the embodiments of the first aspect of the present invention, wherein the 2-8 polypeptides are different, partially identical or completely identical, and wherein a linker sequence is optionally present between the polypeptides.

[0022] In one or more embodiments, of the 2 to 8 polypeptides contained in the fusion protein, at least one polypeptide has the substitution mutation at the 15th position, and optionally at least one position selected from the 3rd, 6th, 9th and 23rd positions, at least two positions, at least three positions, or all four positions, and preferably at least one polypeptide has the substitution mutation at the 3rd, 9th and 15th positions, and also has the substitution mutation at the 6th and / or 23rd positions.

[0023] In one or more embodiments, in the fusion protein, the polypeptide is one or more selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:71, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:68, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, and SEQ ID No:124.

[0024] In one or more embodiments, the C-terminus or N-terminus of the fusion protein further comprises one or more coupling elements and / or residues from the truncated signaling sequence, preferably the coupling elements are selected from a cysteine ​​residue, a plurality of lysine residues, and a plurality of histidine residues.

[0025] In one or more embodiments, the amino acid sequence of the fusion protein is set forth in any one of SEQ ID NOs: 73-109 and 111-117.

[0026] A fourth aspect of the present invention provides a recombinant Protein A, wherein the B domain of said recombinant Protein A is a polypeptide according to any one of the embodiments of the first aspect of the present invention.

[0027] A fifth aspect of the present invention provides an isolated nucleic acid molecule, the polynucleotide sequence of which is selected from (1) a polynucleotide sequence encoding a polypeptide, a fusion protein or a recombinant Protein A according to any one of the embodiments of the present invention, and (2) a complementary sequence of the polynucleotide sequence according to (1).

[0028] A sixth aspect of the present invention provides a nucleic acid construct, the nucleic acid construct comprising a nucleic acid molecule according to any one of the embodiments of the present invention, preferably the nucleic acid construct is an expression cassette, more preferably the nucleic acid construct is an expression vector or a cloning vector.

[0029] A seventh aspect of the present invention provides an expression system, the expression system comprising a nucleic acid construct according to any one of the embodiments of the present invention, preferably wherein the expression system is a host cell.

[0030] An eighth aspect of the present invention provides an isolation matrix comprising a polypeptide, a fusion protein and / or recombinant Protein A according to any one of the embodiments of the present invention coupled to a solid support.

[0031] In one or more embodiments, the polypeptide, fusion protein or recombinant Protein A is coupled to the solid support by a thioether bond.

[0032] In one or more embodiments, the solid support is selected from polyhydroxy-containing polymers, preferably polysaccharides, more preferably selected from dextran, starch, cellulose, pullulan, agar, agarose; synthetic polymers, preferably selected from polyvinyl alcohol, polystyrene, polystyrene divinylbenzene, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides; and supports of inorganic nature, preferably selected from silica, zirconia.

[0033] A ninth aspect of the present invention provides a chromatography column, said chromatography column comprising an isolation matrix according to any one of the embodiments of the present invention.

[0034] A tenth aspect of the present invention provides a method for isolating an Fc-containing protein, the method comprising the step of contacting a sample containing an immunoglobulin with a polypeptide, fusion protein, recombinant Protein A, isolation matrix or chromatography column according to any one of the embodiments of the present invention, preferably wherein the Fc-containing protein is an immunoglobulin.

[0035] In one or more embodiments, the method includes: (1) contacting a sample containing an Fc-containing protein with the isolation matrix; (2) washing the isolation matrix; (3) eluting the Fc-containing protein from the isolation matrix; and (4) washing the isolation matrix.

[0036] In one or more embodiments, the isolation matrix is ​​washed with a 0.1-2.0 M or 0.5-1.0 M NaOH or KOH solution.

[0037] An eleventh aspect of the present invention provides the use of a polypeptide, a fusion protein or recombinant Protein A according to any one of the embodiments of the present invention in the isolation of an Fc-containing protein or in the manufacture of an isolation matrix or a chromatography column for isolating an Fc-containing protein. [Brief description of the drawings]

[0038] [Figure 1] FIG. 1 is a gene result verification analysis diagram of (B domain) 4. [Diagram 2] Figure 2 is a genetic result verification analysis diagram of mutant B(N3V)4. [Diagram 3] FIG. 3 is a genetic result verification analysis diagram of mutant B(N6E)4. [Figure 4]FIG. 4 is a genetic result verification analysis diagram of mutant B(Q9I)4. [Diagram 5] FIG. 5 is a genetic result verification analysis diagram of mutant B(Q9F)4. [Figure 6] FIG. 6 is a genetic result verification analysis diagram of mutant B(E15L)4. [Figure 7] FIG. 7 is a genetic result verification analysis diagram of mutant B(E15I)4. [Figure 8] FIG. 8 is a genetic result verification analysis diagram of mutant B(N3V,N6E,Q9I,E15L)4. [Figure 9] FIG. 9 is a gene result verification analysis diagram of (Z domain) 4. [Figure 10] FIG. 10 is a genetic result verification analysis diagram of mutant Z(Q9I)4. [Figure 11] FIG. 11 is a genetic result verification analysis diagram of mutant Z(E15L)4. [Figure 12] FIG. 12 is a genetic result verification analysis diagram of mutant Z(E15I)4. [Figure 13] FIG. 13 is a genetic result verification analysis diagram of mutant Z(N3V,N6E,Q9I,E15L)4. [Figure 14] FIG. 14 is an alkaline stability graph of Z domain mutants shown in SEQ ID NOs:72-91. [Figure 15] FIG. 15 is an alkaline stability graph of the Z domains shown in SEQ ID NOs:72, 98-102, 104, 105 and 107-109. [Figure 16] FIG. 16 is an alkaline stability graph of B domain mutants shown in SEQ ID NOs:110-117. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form preferred embodiments.

[0040] As used herein, the terms "antibody" and "immunoglobulin" may be used interchangeably and have the meanings well known in the art. The antibody or immunoglobulin according to the present specification also includes an antibody fragment, and a fusion protein or complex containing an antibody fragment, as long as these fragments, fusion proteins or complexes contain the Fc portion of an antibody and can be isolated and purified by binding to Protein A. The antibody fragment may be a functionally active fragment thereof.

[0041] As used herein, amino acid residues may be described by the following abbreviations: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), valine (Val or V), and any amino acid residue (Xaa or X). Furthermore, herein, the amino acid sequences of peptides are conventionally written with the amino terminus (hereinafter referred to as the "N-terminus") on the left and the carboxy terminus (hereinafter referred to as the "C-terminus") on the right.

[0042] Protein A is widely used as an affinity media ligand for purifying immunoglobulins. Natural protein A has five domains, namely E domain, D domain, A domain, B domain and C domain, which bind to immunoglobulins, especially IgG, from N-terminus to C-terminus, in that order. Domain Z was obtained by substituting valine for alanine at position 1 and alanine for glycine at position 29 in domain B of protein A. The mutations conferred better chemical stability to domain Z. In the process of purifying immunoglobulins, immunoglobulins are bound to protein A and then eluted with alkali. During this process, protein A falls off from the affinity chromatography media, resulting in contamination of the immunoglobulin product.

[0043] According to the present invention, a mutant of the native B domain or Z domain of protein A obtained by performing a substitution mutation at least one of positions 3, 6, 9, 15, and 23 of the B domain or Z domain has significantly improved alkaline stability in an alkaline washing process compared to the native B domain or the Z domain before mutation. The concentration of the alkaline solution used may be increased from 0.1-0.5 M to 0.5-2.0 M, for example, to 0.5-1.0 M. This completes the present invention.

[0044] Thus, the present invention provides a polypeptide comprising a substitution mutation at at least one position selected from positions 3, 6, 9, 15 and 23, at least two positions, at least three positions, at least four positions, or all five positions relative to a native B domain as depicted in SEQ ID NO:1, or at least one position selected from positions 3, 6, 9, 15 and 23, at least two positions, at least three positions, at least four positions, or all five positions relative to a native Z domain as depicted in SEQ ID NO:2.

[0045] In one or more embodiments, the substitution mutation at position 3 may be a substitution of asparagine to leucine, isoleucine, valine, or tyrosine. In some embodiments, the substitution mutation at position 3 is a substitution of asparagine to leucine, valine, or tyrosine, preferably a substitution of leucine or valine. In some embodiments, the substitution mutation at position 3 is a substitution of asparagine to valine. In some embodiments, the polypeptides provided herein have a substitution mutation at position 3, preferably the amino acid sequence of the polypeptide is set forth in SEQ ID NO:3, 4, 5, or 118.

[0046] In one or more embodiments, the substitution mutation at position 6 may be a substitution of asparagine to glutamic acid, leucine, isoleucine, valine, serine, or threonine. In some embodiments, the substitution mutation at position 6 is a substitution of asparagine to glutamic acid, leucine, or serine. In some embodiments, the substitution mutation at position 6 is a substitution of asparagine to leucine. In some embodiments, the polypeptides provided herein have a substitution mutation at position 6, preferably the amino acid sequence of the polypeptide is set forth in SEQ ID NO:6, 7, 8, or 119.

[0047] In one or more embodiments, the substitution mutation at position 9 may be a substitution of glutamine to isoleucine, leucine, valine, phenylalanine, or methionine. In some embodiments, the substitution mutation at position 9 is a substitution of glutamine to isoleucine, phenylalanine, or methionine. In some embodiments, the polypeptides described herein have a substitution mutation at position 9, preferably the amino acid sequence of the polypeptide is set forth in SEQ ID NO:9, 10, 11, 120, or 121.

[0048] In one or more embodiments, the substitution mutation at position 15 may be a mutation or substitution of glutamic acid to threonine, tryptophan, leucine, valine, isoleucine, phenylalanine, serine, tyrosine, or aspartic acid. In some embodiments, the substitution mutation at position 15 may be a substitution of glutamic acid to threonine, tryptophan, leucine, valine, phenylalanine, serine, or tyrosine. In some embodiments, the substitution mutation at position 15 is a substitution of glutamic acid to leucine or serine. In some embodiments, the polypeptides described herein have a substitution mutation at position 15, preferably the amino acid sequence of the polypeptide is set forth in any one of SEQ ID NOs:12-20, 122, and 123.

[0049] In one or more embodiments, the substitution mutation at position 23 may be a substitution of asparagine to valine, isoleucine, leucine, or tyrosine. In some embodiments, the substitution mutation at position 23 is a substitution of asparagine to valine, isoleucine, or tyrosine. In some embodiments, the substitution mutation at position 23 is a substitution of asparagine to valine or isoleucine. In some embodiments, the polypeptides provided herein have a substitution mutation at position 23, preferably the amino acid sequence of the polypeptide is set forth in any one of SEQ ID NOs:21-23.

[0050] In one or more embodiments, the polypeptides of the present invention have a substitution mutation according to any one of the embodiments herein at at least the third position, and optionally at one or more positions selected from the 6th, 9th, 15th and 23rd positions, preferably at one or more positions selected from the 6th, 9th and 15th positions. In some other embodiments, the polypeptides of the present invention have a substitution mutation according to any one of the embodiments herein at at least the 6th position, and optionally at one or more positions selected from the 3rd, 9th, 15th and 23rd positions, preferably at one or more positions selected from the 3rd, 9th and 15th positions. In some other embodiments, the polypeptides of the present invention have a substitution mutation according to any one of the embodiments herein at at least the 9th position, and optionally at one or more positions selected from the 3rd, 6th, 15th and 23rd positions, preferably at one or more positions selected from the 3rd, 6th and 15th positions. In some further embodiments, the polypeptide of the present invention has a substitution mutation according to any one of the embodiments herein at at least position 15 and optionally at one or more positions selected from positions 3, 6, 9 and 23, preferably at positions 3, 6 and 9. In some further embodiments, the polypeptide of the present invention has a substitution mutation according to any one of the embodiments herein at at least position 23 and optionally at one or more positions selected from positions 3, 6, 9 and 15. In some embodiments, the substitution mutation at position 3 is N3L or N3V, the substitution mutation at position 6 is N6L or N6E, the substitution mutation at position 9 is Q9I or Q9F, the substitution mutation at position 15 is E15L, E15I or E15V, and the substitution mutation at position 23 is N23V, N23I or N23Y.

[0051] In some embodiments, a polypeptide of the invention comprises a substitution mutation according to any one of the embodiments herein at at least position 15, and a substitution mutation according to any one of the embodiments herein at at least one position, at least two positions, at least three positions, or all four positions selected from positions 3, 6, 9, and 23. In some embodiments, the substitution mutation at position 15 is E15T, E15W, E15L, E15V, E15I, E15F, E15S, E15Y, or E15T, the substitution mutation at position 3 is N3L, N3V, or N3Y, the substitution mutation at position 6 is N6S, N6L, or N6E, the substitution mutation at position 9 is Q9I or Q9F, and the substitution mutation at position 23 is N23V, N23I, or N23Y. Preferably, the substitution mutation at position 15 is E15I or E15V, the substitution mutation at position 3 is N3L or N3V, the substitution mutation at position 6 is N6E or N6L, the substitution mutation at position 9 is Q9I or Q9F, and the substitution mutation at position 23 is N23I, N23Y, or N23V.

[0052] In some embodiments, a polypeptide of the present invention has a substitution mutation according to any one of the embodiments herein at positions 3, 9 and 15, and a substitution mutation according to any one of the embodiments herein at position 6 and / or 23. In some embodiments, the substitution mutation at position 15 is E15T, E15W, E15L, E15V, E15I, E15F, E15S, E15Y or E15T, the substitution mutation at position 3 is N3L, N3V or N3Y, the substitution mutation at position 6 is N6S, N6L or N6E, the substitution mutation at position 9 is Q9I or Q9F, and the substitution mutation at position 23 is N23V, N23I or N23Y. Preferably, the substitution mutation at position 3 is N3L or N3V, the substitution mutation at position 6 is N6L or N6E, the substitution mutation at position 9 is Q9I or Q9F, the substitution mutation at position 15 is E15L, E15I or E15V, and the substitution mutation at position 23 is N23V, N23I or N23Y.

[0053] In some embodiments, the amino acid sequence of a polypeptide of the present invention has an N3V mutation at position 3, or an N6E mutation at position 6, or a Q9I or Q9F mutation at position 9, or an E15L or E15I mutation at position 15, or an N3V, N6E, Q9I or Q9F and E15L or E15I mutations at positions 3, 6, 9 and 15 relative to SEQ ID NO:1 or 2.

[0054] In some embodiments, the amino acid sequence of a polypeptide having substitution mutations at at least two positions selected from positions 3, 6, 9, 15, and 23 of the present disclosure is set forth in any one of SEQ ID NOs:24-71.

[0055] The polypeptide of the present invention can be used as it is as a ligand for binding and isolating immunoglobulins. Therefore, in some embodiments, the polypeptide further comprises one or more coupling elements for coupling the polypeptide to a solid support at its C-terminus or N-terminus. Suitable coupling elements are well known in the art and include, but are not limited to, a cysteine ​​residue, a plurality of lysine residues (e.g., 3-15 or 5-10), and a plurality of histidine residues (e.g., 3-15 or 5-10). The coupling element may be a cysteine ​​residue and located at the C-terminus of the polypeptide, and the thiol group of the cysteine ​​residue reacts with an electrophilic group on the solid support to couple the polypeptide to the solid support. The coupling element may be directly linked to the polypeptide, or may be linked to the N- or C-terminus of the polypeptide via a linker. The linker may be one commonly used in the art, and its presence does not affect the binding activity of the polypeptide. An exemplary linker is a linker sequence that includes or consists of G and S, and is typically 2-20 amino acid residues in length. An exemplary linker sequence is (GS) n , (G.S.S.) n , (GSSS) n and (GSSSS) n where n is an integer from 2 to 10, but the total length of the linker is typically 20 amino acid residues or less. The polypeptide may further comprise a number of amino acid residues (e.g., 15 or less, 10 or less, or 5 or less) at the N-terminus that are derived from the cloning process or from a signaling sequence. As a specific example, the polypeptide of the invention may comprise AQ at the N-terminus.

[0056] The present invention further provides a fusion protein, which is formed by fusing 2-8 polypeptides according to the present invention. Each of the polypeptides forming the fusion protein may be completely different, partially identical, or completely identical. The polypeptides may be directly linked to each other via peptide bonds at the C-terminus and N-terminus, respectively, or any two polypeptides may be linked by a linker. The linker may be one commonly used in the art, and its presence does not affect the binding activity of the polypeptide. An exemplary linker is a linker sequence containing or consisting of G and S, and is usually 2-20 amino acid residues in length. An exemplary linker sequence is (GS) n and (GSS) n etc., where n is an integer from 2 to 10, but the total length of the linker is typically 20 amino acid residues or less.

[0057] The fusion protein can be used as a ligand to bind and isolate immunoglobulins. Therefore, in some embodiments, the fusion protein further comprises one or more coupling elements for coupling the fusion protein to a solid support at its C-terminus or N-terminus. Suitable coupling elements are well known in the art and include, but are not limited to, a cysteine ​​residue, multiple lysine residues (e.g., 3-15 or 5-10), and multiple histidine residues (e.g., 3-15 or 5-10). The coupling element may be a cysteine ​​residue and located at the C-terminus of the fusion protein, and the thiol group in the cysteine ​​residue reacts with an electrophilic group in the solid support to couple the fusion protein to the solid support. The coupling element may be directly linked to the polypeptide, or may be linked to the N- or C-terminus of the polypeptide via a linker. The linker may be one commonly used in the art, and its presence does not affect the binding activity of the polypeptide. An exemplary linker is a linker sequence that includes or consists of G and S, and is typically 2-20 amino acid residues in length. An exemplary linker sequence is (GS) n and (GSS)n etc., where n is an integer from 2-10, but the total length of the linker is typically 20 amino acid residues or less. The fusion protein may further comprise a number of amino acid residues (e.g., 15 or less, 10 or less, or 5 or less) at the N-terminus that are derived from the cloning process or from a signaling sequence. As a specific example, the fusion protein may comprise AQ at its N-terminus.

[0058] In some embodiments, at least one of the 2-8 polypeptides contained in the fusion protein of the present invention has a substitution mutation according to any one of embodiments herein at position 15, and optionally has a substitution mutation according to any one of embodiments herein at at least one position selected from positions 3, 6, 9, and 23, at least two positions, at least three positions, or all four positions. Preferably, at least one of the polypeptides contained in the fusion protein has a substitution mutation according to any one of embodiments herein at positions 3, 9, and 15, and has a substitution mutation according to any one of embodiments herein at positions 6 and / or 23. Preferably, the substitution mutation at position 3 is N3L or N3V, the substitution mutation at position 6 is N6L or N6E, the substitution mutation at position 9 is Q9I or Q9F, the substitution mutation at position 15 is E15L, E15I or E15V, and the substitution mutation at position 23 is N23V, N23I or N23Y.

[0059] In some embodiments, the polypeptides of the invention forming the fusion proteins of the invention comprise one or more of the following polypeptides having substitution mutations: SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:71, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:68, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, and SEQ ID NO:124. The amino acid sequences of exemplary fusion proteins according to the invention are shown in any one of SEQ ID NOs:73-109 and 111-117.

[0060] The present invention further provides a recombinant protein A, the B domain of which is a Z domain mutant or a B domain mutant according to any embodiment of the present invention. The other domains of the recombinant protein A may be E domain, D domain, A domain and C domain well known in the art, including natural E domain, D domain, A domain and B domain, and known E domain, D domain, A domain and / or C domain that have been mutated but retain their biological activity. In addition to using the polypeptide of the present invention for the B domain, mutations or modifications made to other parts of protein A known in the art can be used as the recombinant protein A of the present invention.

[0061] The present specification also includes a polypeptide, a fusion protein, and a recombinant protein A having at least 85% sequence identity with a polypeptide, a fusion protein, or a recombinant protein A according to any one of the embodiments of the present specification, and having a substitution mutation according to any one of the embodiments of the present specification at one or more amino acid positions selected from 3, 6, 9, 15, and 23 corresponding to SEQ ID NO: 1 or 2. As used herein, "at least 85% identity" with respect to amino acid sequences and nucleotide sequences means 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity, more preferably 97% or more identity, more preferably 98% or more identity, and even more preferably 99% or more identity. The identity of two sequences can be calculated and aligned by tools well known in the art, including BLASTP, which is used for aligning amino acid sequences. In a preferred embodiment, the present invention includes variants of the amino acid sequences set forth in any one of SEQ ID NOs:3-71, 118-124 and 73-109, 111-117, each of which has at least 85%, preferably at least 90%, more preferably at least 97%, more preferably at least 99% sequence identity to the corresponding parent sequence, and in which the amino acid residues at positions 3, 6, 9, 15 and 23 of the corresponding parent remain the same.

[0062] The present invention also includes nucleic acid molecules encoding the above polypeptides, fusion proteins and recombinant Protein A, as well as complementary sequences thereof. The present invention includes all forms of the nucleic acid molecules of the present invention, including RNA and DNA. A complementary sequence in accordance with the present invention should be understood as a complementary sequence that is approximately the same length as the nucleic acid molecule.

[0063] The present invention also includes a nucleic acid construct comprising the nucleic acid molecule or a complementary sequence thereof. The nucleic acid construct may be an expression cassette comprising a promoter, the nucleic acid molecule, and a transcription termination sequence. Promoters and transcription termination sequences are well known in the art, and a person skilled in the art can appropriately select the promoter and transcription termination sequence depending on the host cell for expressing the polypeptide, fusion protein, or recombinant protein A.

[0064] In some embodiments, the nucleic acid construct is a vector, including expression vectors and cloning vectors. Expression vectors are vectors suitable for expressing foreign genes, such as nucleic acid molecules encoding the polypeptides, fusion proteins, or recombinant protein A of the present invention, in host cells, and include prokaryotic and eukaryotic expression vectors. Eukaryotic expression systems include yeast expression systems, mammalian cell expression systems, and insect cell expression systems. Cloning vectors are for amplifying target genes (e.g., nucleic acid molecules encoding the polypeptides, fusion proteins, or recombinant protein A of the present invention) in host cells. Cloning vectors include plasmid vectors, phage vectors, viral vectors, and any combination thereof, or vectors in combination with other genomic DNA. The most common host cell for molecular cloning is E. coli.

[0065] The present invention further provides an expression system comprising the nucleic acid molecule, nucleic acid construct or vector disclosed herein. The expression system is, for example, a Gram-positive or Gram-negative prokaryotic host cell system, such as E. coli or Bacillus, modified to express the polypeptide, fusion protein or recombinant Protein A of the present invention. In some embodiments, the expression system is a eukaryotic host cell system of yeast, such as Pichia or Saccharomyces.

[0066] The present invention further provides an isolation matrix for isolating immunoglobulins or Fc-containing proteins. The isolation matrix of the present invention comprises a polypeptide, a fusion protein and / or recombinant Protein A according to any one of the embodiments of the present invention coupled to a solid support. Due to the improved alkaline stability of the polypeptide, the fusion protein and the recombinant Protein A of the present invention, the isolation matrix of the present invention can withstand strong alkaline conditions (0.5-2.0M NaOH) during washing.

[0067] The solid supports in the isolation matrix of the present invention are solid supports known in the art and used for the isolation of immunoglobulins or Fc-containing proteins.

[0068] In the present specification, the shape of the solid support may be any shape, such as particles, membranes, plates, tubes, needles, fibers, etc. The solid support of the present invention may be a porous or non-porous material. In some embodiments, the solid support is a bead or particle type, porous or non-porous. The bead or particle type matrix may be used as a packed bed or in a suspension type, which includes an expanded bed, but the particles or beads are free to move.

[0069] When the solid support is a particle, the preferred particle size is 20-200 μm. For example, when the solid support is a synthetic polymer, the preferred particle size is 20-100 μm, preferably 30-80 μm. When the solid support is a polysaccharide, the preferred particle size is 50-200 μm, more preferably 60-150 μm. In the present specification, the "particle size" is the volume average diameter measured by the laser diffraction method of ISO 13320 and JIS Z 8825-1.

[0070] Examples of solid supports include, but are not limited to, polyhydroxy-containing polymers such as polysaccharides, including dextran, starch, cellulose, pullulan, agar, agarose, etc. In a preferred embodiment, the solid support comprises agar or agarose.

[0071] In some embodiments, the solid support of the present invention is a synthetic polymer having a hydrophilic surface. For example, these polymers may be polymers having hydroxyl groups, carboxyl groups, aminocarbonyl groups, amino groups, or oligoethyleneoxy groups or polyethyleneoxy groups on the outer surface (and also on the inner surface, if present) by hydrophilization treatment, and are preferably synthetic polymers of copolymers formed by crosslinking polyfunctional monomers such as polyfunctional (meth)acrylates and divinylbenzene. Exemplary synthetic polymers include polyvinyl alcohol, polystyrene, polystyrene divinylbenzene, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, and polymethacrylamides. When the polymer surface is hydrophobic, the surface may be hydrophilized so that the hydrophilic groups are exposed to the surrounding aqueous liquid.

[0072] In some embodiments, solid supports of the present invention include supports that are inorganic in nature, including, but not limited to, silica and zirconia.

[0073] In some embodiments, a solid support of the invention is in the form of a surface, chip, capillary, or filter, such as a membrane.

[0074] In the present invention, the polypeptide, fusion protein and recombinant protein A of the present invention are coupled to the corresponding solid support by conventional coupling techniques through the thiol, amino and / or carboxyl groups contained therein. Conventional coupling reagents include, but are not limited to, bicyclic oxides, epichlorohydrin, CNBr and N-hydroxysuccinimide. The introduction of a spacer group between the solid support and the polypeptide, fusion protein and recombinant protein A of the present invention can facilitate chemical coupling between them and the solid support.

[0075] In some embodiments, the polypeptide, fusion protein, or recombinant protein A of the present invention is coupled to a solid support via a thioether bond. In some embodiments, the polypeptide, fusion protein, or recombinant protein A of the present invention is coupled via a cysteine ​​at its C-terminus. In particular, the thiol group of the cysteine ​​is effectively coupled to electrophilic groups, such as epoxy groups, halohydrin groups, on the solid support to form a thioether bridge coupling.

[0076] In the isolation matrix of the invention, the concentration of the binding ligand (e.g. the polypeptide, fusion protein and / or recombinant Protein A according to the invention) coupled to the solid support is usually 5-20 mg / ml, e.g. 5-15 mg / ml. The amount of said binding ligand coupled is controlled by adjusting the concentration of the polypeptide, fusion protein and / or recombinant Protein A used in the coupling process, the coupling conditions used and / or the pore structure of the solid support used.

[0077] The present invention further provides a chromatography column comprising an isolation matrix of the present invention. Typically, the isolation matrix of the present invention is packed into a chromatography column.

[0078] The present invention further provides a method for isolating an Fc-containing protein (e.g., an immunoglobulin), the method comprising contacting a sample containing the Fc-containing protein with a polypeptide, fusion protein and / or recombinant Protein A according to any one of the embodiments herein. In some embodiments, the method comprises contacting the sample with an isolation matrix or chromatography column according to any one of the embodiments herein.

[0079] Specifically, in some embodiments, the method comprises: (1) contacting a sample containing an Fc-containing protein with an isolation matrix according to any one of the embodiments herein; (2) washing the isolation matrix; (3) eluting the Fc-containing protein from the isolation matrix; and (4) washing the isolation matrix.

[0080] As used herein, the sample may be any sample that contains an Fc-containing protein, in particular an immunoglobulin. Preferably, the immunoglobulin in accordance with the present invention is an IgG.

[0081] The detergent for washing the isolation matrix, the elution solution for eluting the protein, and the washing solution for washing the isolation matrix used in the method are the usual detergent, elution solution, and washing solution in the field (particularly in the field of protein A chromatography). For example, the detergent may be a PBS buffer. The elution solution may be a solution or buffer with a pH≦5, preferably the pH of the elution solution is 2.5-5 or 3-5. In some embodiments, the pH of the elution solution is 11 or higher, for example, the pH of the elution solution is 11-14 or 11-13. In some embodiments, elution is performed with a citrate solution, for example, a sodium citrate solution. The washing solution is usually alkaline and may have a pH of 13-14. In some embodiments, the washing solution is a sodium hydroxide solution or a potassium hydroxide solution, and the concentration is 0.1-2.0M, for example, 0.5-2.0M or 0.5-1.0M.

[0082] In some embodiments, the methods herein further comprise the step of collecting the eluate and isolating and purifying the protein, which may be further isolated and purified by anion or cation exchange chromatography, hybrid ion exchange chromatography, and / or hydrophobic interaction chromatography, depending on the isolated protein.

[0083] The present invention further provides an application of the polypeptide, fusion protein, and Protein A according to any one of the embodiments herein in the isolation and purification of Fc-containing proteins, or in the manufacture of an isolation matrix for isolating and purifying Fc-containing proteins.

[0084] The sequences relevant to this specification are as follows: SEQ ID NO:1 (native B domain) A DNKFNKEQQNAFYEILHLPNLNEEQRN G FIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:2 (Z domain) V DNKFNKEQQNAFYEILHLPNLNEEQRN A FIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:3,Z(N3L) VDLKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:4,Z(N3V) VDVKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:5,Z(N3Y) VDYKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:6,Z(N6S) VDNKFSKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:7,Z(N6L) VDNKFLKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:8,Z(N6E) <h2 style=";text-align:left;direction:ltr">VDNKFEKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:9,Z(Q9I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:10,Z(Q9F)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEFQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0085] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:11,Z(Q9M)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEMQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:12,Z(E15T)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYTILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:13,Z(E15W)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYWILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:14,Z(E15L)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:15,Z(E15V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:16,Z(E15I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0086] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:17,Z(E15F)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">VDNKFNKEQQNAFYFILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:18,Z(E15S)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYSILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:19,Z(E15Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYYILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:20,Z(E15D)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYDILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0087] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:21,Z(N23V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYEILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:22,Z(N23I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYEILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:23,Z(N23Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYEILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:24,Z(Q9I,E15T)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYTILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:25,Z(Q9I,E15W)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYWILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:26,Z(Q9I,E15L)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">VDNKFNKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:27,Z(Q9I,E15V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:28,Z(Q9I,E15I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:29,Z(Q9I,E15F)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYFILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:30,Z(Q9I,E15S)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYSILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0088] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:31,Z(Q9I,E15Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYYILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:32,Z(Q9I,E15D)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYDILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:33,Z(Q9F,E15T)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEFQNAFYTILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:34,Z(Q9F,E15W)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEFQNAFYWILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:35,Z(Q9F,E15L)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">VDNKFNKEFQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:36,Z(Q9F,E15V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEFQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:37,Z(Q9F,E15I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEFQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:38,Z(Q9F,E15F)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEFQNAFYFILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:39,Z(Q9F,E15S)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEFQNAFYSILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:40,Z(Q9F,E15Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEFQNAFYYILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0089] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:41,Z(Q9F,E15D)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEFQNAFYDILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:42,Z(N3L,Q9I,E15L)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDLKFNKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:43,Z(N3Y,Q9I,E15V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDYKFNKEIQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:44,Z(N3V,Q9I,E15I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFNKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:45,Z(N6E,Q9I,E15L)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFEKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:46,Z(N6E,Q9F,E15V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFEKEFQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:47,Z(N6E, Q9I,E15I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFEKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:48,Z(Q9I,E15L,N23V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFNKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:49,Z(Q9I,E15V,N23V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFNKEIQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:50,Z(Q9I,E15I,N23V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFNKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0090] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:51,Z(N3L,N6L,Q9I,E15L)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDLKFLKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:52,Z(N3Y,N6E,Q9I,E15I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">VDYKFEKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:53,Z(N3V,N6E,Q9I,E15L)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFEKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:54,Z(N3L,N6L,Q9F,E15I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDLKFLKEFQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:55,Z(N3Y,N6E,Q9I,E15L)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDYKFEKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:56,Z(N3V,N6E,Q9I,E15I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFEKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:57,Z(N3L,Q9I,E15L,N23V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDLKFNKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:58,Z(N3Y,Q9I,E15I,N23I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDYKFNKEIQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:59,Z(N3V,Q9I,E15L,N23Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFNKEIQNAFYLILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:60,Z(N6L,Q9F,E15I,N23V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0091] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:61,Z(N6E,Q9I,E15L,N23I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFEKEIQNAFYLILHLPNLIEEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:62,Z(N6E,Q9I,E15L,N23Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFEKEIQNAFYLILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:63,Z(N3L,N6L,Q9I,E15L,N23V)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDLKFLKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:64,Z(N3L,N6E,Q9I,E15I,N23I)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:65,Z(N3V,N6E,Q9I,E15L,N23Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFEKEIQNAFYLILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:66,Z(N3V,N6E,Q9I,E15I,N23Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFEKEIQNAFYIILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:67,Z(N3Y,N6E,Q9I,E15L,N23Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDYKFEKEIQNAFYLILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:68,Z(N3V,N6L,Q9I,E15V,N23Y)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDVKFLKEIQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> SEQ ID NO:69,Z(N3L,N6L,Q9F,E15I,N23V) VDLKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:70,Z(N3L,N6E,Q9F,E15L,N23I) VDLKFEKEFQNAFYLILHLPNLIEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0092] SEQ ID NO:71,Z(N3V,N6L,Q9F,E15V,N23Y) VDVKFLKEFQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:72,Z(Zドメイン)4 VDNKFNKEQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:73,Z(N3L)4 VDLKFNKEQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFNKEQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDLKFNKEQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFNKEQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:74,Z(N3B)4 VDVKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:75,Z(N6L)4 VDNKFLKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFLKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFLKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFLKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:76,Z(N6E)4 VDNKFEKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFEKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFEKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFEKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:77,Z(Q9I)4 VDNKFNKEIQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:78,Z(Q9F)4 VDNKFNKEFQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEFQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEFQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEFQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:79,Z(Q9M)4 VDNKFNKEMQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEMQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEMQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEMQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:80,Z(E15T)4 <h2 style=";text-align:left;direction:ltr">VDNKFNKEQQNAFYTILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYTILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQQNAFYTILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYTILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0093] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:81,Z(E15W)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYWILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYWILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQQNAFYWILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYWILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:82,Z(E15L)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:83,Z(E15V)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">VDNKFNKEQQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:84,Z(E15I)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:85,Z(E15F)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYFILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYFILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQQNAFYFILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYFILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:86,Z(E15S)4<h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYSILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYSILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYSILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYSILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:87,Z(E15Y)4 VDNKFNKEQQNAFYYILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYYILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYYILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYYILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:88,Z(E15D)4 VDNKFNKEQQNAFYDILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYDILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYDILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYDILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:89,Z(N23V)4 <h2 style=";text-align:left;direction:ltr">VDNKFNKEQQNAFYEILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQQNAFYEILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:90,Z(N23I)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYEILHLPNLIEEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLIEEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQQNAFYEILHLPNLIEEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLIEEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:91,Z(N23Y)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQQNAFYEILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFNKEQQNAFYEILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:92,Z(Q9I,E15L)4<h2 style=";text-align:left;direction:ltr"> VDNKFNKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:93,Z(Q9I,E15V)4 VDNKFNKEIQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYVILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:94,Z(Q9I,E15I)4 VDNKFNKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:95,Z(N3L,N6L,Q9I,E15L)4 VDLKFLKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAP SEQ ID NO:96,Z(N3Y,N6E,Q9I,E15I)4 VDYKFEKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDYKFEKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDYKFEKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDYKFEKEIQNAFYIILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAP SEQ ID NO:97,Z(N3V,N6E,Q9I,E15L)4 VDVKFEKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFEKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFEKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFEKEIQNAFYLILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAP SEQ ID NO:98,Z(N3L,Q9I,E15L,N23V)4 <h2 style=";text-align:left;direction:ltr">VDLKFNKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFNKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDLKFNKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFNKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:99,Z(N6L,Q9F,E15I,N23V)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDNKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDNKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:100,Z(N3L,N6L,Q9I,E15L,N23V)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDLKFLKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDLKFLKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEIQNAFYLILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:101,Z(N3L,N6E,Q9I,E15I,N23I)4<h2 style=";text-align:left;direction:ltr"> VDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:102,Z(N3V,N6L,Q9F,E15V,N23Y)4 VDVKFLKEFQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFLKEFQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFLKEFQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFLKEFQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:103,Z(N3V,N6E,Q9I,E15I,N23Y)4 VDVKFEKEIQNAFYIILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFEKEIQNAFYIILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFEKEIQNAFYIILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFEKEIQNAFYIILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:104,Z(N3L,N6L,Q9F,E15I,N23V)4 <h2 style=";text-align:left;direction:ltr">VDLKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDLKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:105,Z(N3L,N6E,Q9F,E15L,N23I)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDLKFEKEFQNAFYLILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEFQNAFYLILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDLKFEKEFQNAFYLILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEFQNAFYLILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:106,Z(N3Y,N6E,Q9I,E15L,N23Y)4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDYKFEKEIQNAFYLILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDYKFEKEIQNAFYLILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDYKFEKEIQNAFYLILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDYKFEKEIQNAFYLILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:107,Z(N3L,N6E,Q9I,E15I,N23I)6<h2 style=";text-align:left;direction:ltr"> VDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFEKEIQNAFYIILHLPNLIEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:108,Z(N3V,N6L,Q9I,E15V,N23Y)6 VDVKFLKEIQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFLKEIQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFLKEIQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFLKEIQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFLKEIQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDVKFLKEIQNAFYVILHLPNLYEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:109,Z(N3L,N6L,Q9F,E15I,N23V)6 VDLKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEFQNAFYIILHLPNLVEEQRNA FIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDLKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEFQNAFYIILHLPNLVEEQRNA FIQSLKDDPSQSANLLAEAKKLNDAQAPKVDLKFLKEFQNAFYIILHLPNLVEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:110,(Bドメイン)4 ADNKFNKEQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQKADNKFNKEQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK ADNKFNKEQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQKADNKFNKEQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0096] SEQ ID NO:111,B(N3V)4 ADVKFNKEQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADVKFNKEQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK ADVKFNKEQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADVKFNKEQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:112,B(N6E)4 ADNKFEKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFEKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFEKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFEKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:113,B(Q9I)4 ADNKFNKEIQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEIQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEIQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEIQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:114,B(Q9F)4 ADNKFNKEFQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEFQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEFQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEFQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:115,B(E15L)4 ADNKFNKEQQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:116,B(E15I)4 ADNKFNKEQQNAFYIILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYIILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYIILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYIILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:117,B(N3V,N6E,Q9I,E15L)4 ADVKFEKEIQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADVKFEKEIQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADVKFEKEIQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADVKFEKEIQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:118,B(N3V) ADVKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:119,B(N6E) ADNKFEKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:120,B(Q9I) ADNKFNKEIQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0097] SEQ ID NO:121,B(Q9F) ADNKFNKEFQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO: 122,B(E15L) ADNKFNKEQQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:123,B(E15I) ADNKFNKEQQNAFYIILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK SEQ ID NO:124,B(N3V,N6E,Q9I,E15L) ADVKFEKEIQNAFYLILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK EXAMPLES

[0098] The present invention will be further described below with reference to specific examples. It should be noted that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In the following examples, the experimental methods for which no specific conditions are specified generally follow conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0099] Production Example 1: Production of Z domain mutants and B domain mutants After designing the mutation sites, the entire gene was synthesized, expressed and purified by Sangon Biotech.

[0100] For expression and purification, a single recombinant plasmid is transformed into E. coli and then fermented and cultured in LB liquid medium for inducible expression. After fermentation, the cells are harvested, the cell walls are destroyed by pyrolysis to release the expression product, and then centrifuged. The isolated liquid is purified by IgG affinity medium, the isolated liquid is sample-loaded through the affinity medium, the medium is washed with 10 mM phosphate buffer, the target protein is collected in pH 3.8 buffer, adjusted to neutral, and stored for use.

[0101] Genetic result verification analysis diagrams of some of the synthesized mutants are shown in Figure 1-13.

[0102] Preparation Example 2: Preparation of affinity chromatography media using Z domain mutants and B domain mutants The obtained Z domain mutant and B domain mutant are used to prepare affinity chromatography media by conventional methods. An exemplary preparation process specifically includes the following steps:

[0103] a. Activation 10 g of high-rigidity agarose was taken, washed with purified water, and dried by suction. 10 g of the above agarose was weighed, and 20 mL of purified water was added. 0.2 g of sodium hydroxide and 10 mL of epoxychloropropane were added to a 100 mL flask, and the mixture was reacted at a constant temperature of 27°C for 2 hours. The activated gel was washed with 1 L of water to obtain activated agarose microspheres.

[0104] b. Crosslinking The activated agarose microspheres were placed in a 100 mL flask, and 150 mg NaHCO3, 10 mg Na2CO3, 150 mg NaCl, and 10 mg EDTA were added. After uniform stirring, 50 mL of the mutant solution was added and reacted at a constant temperature of 34°C for 8 hours to complete the crosslinking.

[0105] c. Blocking The crosslinked product was added to 100 mL of blocking buffer (ethanolamine solution), the pH was adjusted to 8.6 with sodium hydroxide, and the reaction was carried out at a constant temperature of 26°C for 2 hours to complete the blocking of the remaining epoxy groups and reduce their effects.

[0106] Example 1 Kinetic analysis was performed on the Z domain mutants and B domain mutants in Table 1 by the following method.

[0107] The experimental procedure is as follows. 1) IgG was cross-linked to a Series S CM5 chip. a. IgG was diluted to 5 μg / ml, 10 μg / ml, 15 μg / ml, and 20 μg / ml with 10 mM acetic acid-NaOH (pH 5.0). b. Prepare working buffer (10 mM acetic acid-NaOH, pH 5.0), stripping solution 1 (0.5% SDS), stripping solution 2 (50 mM glycine-NaOH, pH 9.5), blocking buffer (1 M ethanolamine pH 8.5), and activating agent (0.4 M EDC, 0.1 M NHS). c. Immobilization was performed on Biacore-4000. d. The cross-linking results are shown in the table below. JPEG2025503793000001.jpg75148

[0108] 2)Dynamic analysis a. The mutant samples were diluted with 1XPBS (pH 7.6) to 0 μg / ml, 1.615 μg / ml, 2.4375 μg / ml, 3.25 μg / ml, 4.875 μg / ml, 6.5 μg / ml, 9.75 μg / ml, 13 μg / ml, 19.5 μg / ml, and 26 μg / ml. b. Gel buffer: 1X PBS (pH 7.6) and regeneration solution (50 mM citric acid-NaOH pH 3.0) were prepared. c. Kinetics and affinity were performed on a Biacore-4000. JPEG2025503793000002.jpg14150

[0109] The experimental results are shown in Table 1 below. Table 1 shows the kinetic analysis results of the Z domain mutants and the B domain mutants by Biacore.

[0110] [Table 1]

[0111] The results show that the KD values ​​of each mutant for the corresponding Z domain or B domain are almost the same, and the dissociation constants do not show any significant changes, indicating that the mutants obtained by mutating the Z domain and B domain of the present invention have no effect on the binding ability to IgG.

[0112] Example 2 The IgG binding ability and alkaline stability of the Z domain mutant and B domain mutant affinity chromatography media in Table 2 were evaluated by the following methods.

[0113] The experimental procedure is as follows. 1) 4.4 mL of affinity chromatography media of Z domain mutant or B domain mutant was taken and loaded onto a chromatography column with 20% ethanol at a flow rate of 5 ml / min. Buffers: Buffer A (PBS, pH 7.6), Buffer B (0.1 M Sodium Citrate, pH 3.0), Buffer C (1 M NaOH). Sample: A 1 mg / ml purified IgG sample was prepared (the purified product was diluted with buffer A).

[0114] 2) Measurement of 10% dynamic loading: Equipment: AKTA Pure. Measurement process: a. Buffer A balanced chromatography column, flow rate: 1.8 ml / min. b. If the IGg sample does not flow through the column, detect the UV absorption peak and measure λ max This results in 10% λ max was calculated. c. Sample load: Flow rate: 1.8 ml / min. The UV absorption peak of the basal outflow was recorded. d. The two were summed to obtain the UV absorption peak corresponding to 10% dynamic loading. e. Continue loading the sample until the UV absorption peak reaches the UV absorption peak in step d, the corresponding volume is the 10% dynamic loading volume.

[0115] 3) Resistance to alkaline cycles: Room temperature control: 23±0.5℃. According to the decrease in the loading amount, the sample loading amount of the program is updated, and other programs remain unchanged. Measurement process: a. PBS balance: flow rate: 1.8ml / min, volume: 1cv. b. Sample load: Flow rate: 1.8 ml / min, 200 ml (subsequent program will update sample load volume according to decreasing loading volume). c. PBS wash: flow rate: 1.8ml / min, volume: 5cv. d. 0.1M Sodium Citrate Elution: Flow rate: 1.8ml / min, Volume: 3cv. e. Treated with 1M NaOH for 2 hours. First, 1 cv was run at a flow rate of 1.8 ml / min to fill the chromatography column with 1M NaOH, and then the flow rate was changed to 0.5 ml / min, volume: 60 ml, and time: 2 hours. f. PBS balance: flow rate: 1.8ml / min, volume: 10cv.

[0116] 4) The above alkali treatment process was carried out 13 times, and the corresponding loading amounts were recorded and graphed. The results are shown in Table 2 and Figures 14, 15 (Z(N3V,N6E,Q9I,E15L)4) and 16. Table 2 shows the IgG capacity (calculated as dynamic binding loading amount, mg / ml) at 10% penetration (Qb10%) of each sample.

[0117] [Table 2]

[0118] The results show that the initial IgG binding strength of the mutants is not significantly different from that of the Z domain, and the initial IgG binding strength of the mutants in the table is all above 130 mg / ml, indicating that they have excellent affinity. After 24 hours of alkaline treatment with 1.0 M NaOH, a very small number of mutants show IgG binding strengths slightly inferior to that of the Z domain, while the IgG binding strength of many mutants is superior to that of the Z domain and B domain. Among them, mutant Z(E15S)4 has 43.57% excess IgG capacity after 24 hours of alkaline treatment. These data show that the mutation sites selected by the present invention can improve the alkaline stability of the Z domain and B domain.

[0119] Example 3 Example 2 was carried out using affinity chromatography media prepared with the Z domain mutants in Table 3, and the results are shown in Table 3 and Figure 15. Table 3 shows the IgG capacity (calculated as dynamic binding loading, mg / ml) at 10% penetration (Qb10%) for each sample.

[0120] [Table 3]

[0121] Table 3 shows the alkaline resistance of mutants mutated at multiple sites in the Z domain. After 24 hours of treatment with 1.0 M NaOH, the excess IgG binding capacity of the mutants was improved to some extent to the Z domain, indicating that the above mutations improved the alkaline resistance. Among them, mutant Z(N3L,N6L,Q9F,E15I,N23V)6 still had 63.93% IgG capacity after 24 hours of alkali treatment.

Claims

1. 1. An isolated polypeptide, the polypeptide comprising: (1) having substitution mutations at one or more positions selected from the 3rd, 6th, 9th, 15th, and 23rd positions relative to the native B domain of protein A as set forth in SEQ ID NO:1 or the Z domain of protein A as set forth in SEQ ID NO:2; and a polypeptide wherein the substitution mutation at position 3 is a substitution of asparagine to leucine, isoleucine, valine, or tyrosine, the substitution mutation at position 6 is a substitution of asparagine to glutamic acid, leucine, isoleucine, valine, serine, or threonine, the substitution mutation at position 9 is a substitution of glutamine to isoleucine, leucine, valine, phenylalanine, or methionine, the substitution mutation at position 15 is a substitution of glutamic acid to threonine, tryptophan, leucine, valine, isoleucine, phenylalanine, serine, tyrosine, or aspartic acid, and the substitution mutation at position 23 is a mutation of asparagine to valine, isoleucine, leucine, or tyrosine; (2) A polypeptide having at least 85% sequence identity with the polypeptide of (1) and having substitution mutations at one or more of the 3rd, 6th, 9th, 15th and 23rd positions.

2. 2. The polypeptide of claim 1, the substitution mutation at position 3 is an asparagine to leucine, valine, or tyrosine; the substitution mutation at position 6 is an asparagine to glutamic acid, leucine, or serine substitution; the substitution mutation at position 9 is a substitution of glutamine to isoleucine, phenylalanine, or methionine; the substitution mutation at position 15 is a substitution of glutamic acid with threonine, tryptophan, leucine, isoleucine, valine, phenylalanine, serine, or tyrosine; A polypeptide, characterized in that the substitution mutation at position 23 is an asparagine to valine, isoleucine, or tyrosine mutation.

3. 3. The polypeptide according to claim 1 or 2, having said substitution mutation at at least the 3rd position and having said substitution mutation at at least one position selected from the 15th, 6th, 9th and 23rd positions, at least two positions, at least three positions or all four positions; having said substitution mutation at at least the 6th position, and having said substitution mutation at at least one position, at least two positions, at least three positions, or all four positions selected from the 3rd, 15th, 9th, and 23rd positions; having said substitution mutation at at least the 9th position, and having said substitution mutation at at least one position selected from the 3rd, 6th, 15th and 23rd positions, at least two positions, at least three positions, or all four positions; having said substitution mutation at at least the 15th position, and having said substitution mutation at at least one position selected from the 3rd, 6th, 9th and 23rd positions, at least two positions, at least three positions, or all four positions; or having said substitution mutation at at least position 23, and having said substitution mutation at at least one position, at least two positions, at least three positions, or all four positions selected from said positions 3, 6, 9, and 15; Preferably, the polypeptide is characterized in that the 15th substitution mutation is E15I, E15L or E15V, the 3rd substitution mutation is N3L or N3V, the 6th substitution mutation is N6E or N6L, the 9th substitution mutation is Q9I or Q9F, and the 23rd substitution mutation is N23I, N23Y or N23V.

4. 3. The polypeptide of claim 1 or 2, wherein the polypeptide has the substitution mutations at positions 3, 9 and 15, and the substitution mutations at positions 6 and / or 23; Preferably, the polypeptide, wherein the third substitution mutation is N3L or N3V, the sixth substitution mutation is N6L or N6E, the ninth substitution mutation is Q9I or Q9F, the fifteenth substitution mutation is E15L, E15I or E15V, and the twenty-third substitution mutation is N23V, N23I or N23Y.

5. 2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide is set forth in any one of SEQ ID NOs: 3-71 and 118-124.

6. 1. An isolated polypeptide, characterized in that it is composed of the following (i), (ii) and (iii): (i) a polypeptide according to any one of claims 1 to 5, (ii) one or more coupling elements at the C-terminus or N-terminus of the amino acid sequence of the polypeptide according to (i), and optionally (iii) residues derived from a truncated signaling sequence.

7. 7. The polypeptide of claim 6, wherein the coupling element is selected from a cysteine ​​residue, a plurality of lysine residues and a plurality of histidine residues, and the residue of the truncated signaling sequence is AQ.

8. A fusion protein comprising an amino acid sequence formed by fusing 2 to 8 polypeptides according to any one of claims 1 to 5, wherein the 2 to 8 polypeptides are different from each other, partially identical or completely identical, and wherein a linker sequence is optionally present between the polypeptides.

9. A fusion protein as described in claim 8, characterized in that, among 2 to 8 polypeptides contained in the fusion protein, at least one polypeptide has the substitution mutation at the 15th position, and optionally at least one position selected from the 3rd, 6th, 9th and 23rd positions, at least two positions, at least three positions, or all four positions, preferably, at least one polypeptide has the substitution mutation at the 3rd, 9th and 15th positions, and also has the substitution mutation at the 6th and / or 23rd positions.

10. 9. The fusion protein of claim 8, wherein the polypeptide is one or more selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:71, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:68, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123 and SEQ ID NONo:

124.

11. A fusion protein according to any one of claims 8 to 10, characterized in that the C-terminus or N-terminus of the fusion protein further comprises one or more coupling elements and / or residues derived from a truncated signalling sequence, preferably the coupling elements being selected from a cysteine ​​residue, a plurality of lysine residues and a plurality of histidine residues.

12. The fusion protein according to claim 8, characterized in that the amino acid sequence of the fusion protein is set forth in any one of SEQ ID NOs: 73-109 and 110-117.

13. A recombinant protein A, wherein the B domain of said recombinant protein A is a polypeptide according to any one of claims 1 to 5.

14. An isolated nucleic acid molecule, the polynucleotide sequence of which is (1) a polynucleotide sequence encoding a polypeptide according to any one of claims 1 to 7, a fusion protein according to any one of claims 8 to 12, or a recombinant protein A according to claim 13; (2) A nucleic acid molecule selected from the complementary sequence of the polynucleotide sequence of (1).

15. A nucleic acid construct, comprising a nucleic acid molecule according to claim 14, preferably wherein the nucleic acid construct is an expression cassette, more preferably wherein the nucleic acid construct is an expression vector or a cloning vector.

16. An expression system comprising the nucleic acid construct of claim 15, preferably said expression system being a host cell.

17. 14. An isolation matrix, characterized in that said isolation matrix comprises a polypeptide according to any one of claims 1-7, a fusion protein according to any one of claims 8-12 and / or a recombinant protein A according to claim 13 coupled to a solid support.

18. 18. The isolation matrix according to claim 17, characterized in that the polypeptide, fusion protein or recombinant Protein A is coupled to the solid support by a thioether bond.

19. 19. The isolation matrix of claim 18, wherein the solid support is a polyhydroxy-containing polymer, preferably a polysaccharide, more preferably a polymer selected from dextran, starch, cellulose, pullulan, agar, and agarose; a synthesized polymer, preferably selected from polyvinyl alcohol, polystyrene, polystyrene divinylbenzene, polyhydroxyalkyl acrylate, polyhydroxyalkyl methacrylate, polyacrylamide, and polymethacrylamide; An isolation matrix, characterized in that it is chosen from supports of inorganic nature, preferably chosen from silica, zirconia.

20. A chromatography column, characterized in that the chromatography column comprises an isolation matrix according to any one of claims 17 to 19.

21. 21. A method for isolating an Fc-containing protein, the method comprising the step of contacting a sample containing an immunoglobulin with a polypeptide according to any one of claims 1-7, a fusion protein according to any one of claims 8-12, recombinant Protein A according to claim 13, an isolation matrix according to any one of claims 17-19 or a chromatography column according to claim 20, characterized in that the Fc-containing protein is an immunoglobulin.

22. 22. The method of claim 21, comprising: (1) contacting a sample containing an Fc-containing protein with the isolation matrix; (2) washing the isolation matrix; and (3) eluting the Fc-containing protein from the isolation matrix; and (4) washing the isolation matrix; Preferably, the method is characterized by washing the isolation matrix with a 0.1-2.0M or 0.5-1.0M NaOH or KOH solution.

23. The use of a polypeptide according to any one of claims 1 to 7, a fusion protein according to any one of claims 8 to 12 or a recombinant protein A according to claim 13 in the isolation of an Fc-containing protein or in the manufacture of an isolation matrix or a chromatography column for the isolation of an Fc-containing protein.

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