Immunoglobulin-binding proteins and affinity supports using the same

Abstract

The present invention provides a carrier for affinity chromatography that maintains high immunoglobulin binding capacity and alkali resistance. An immunoglobulin-binding protein comprising at least one modified immunoglobulin-binding domain that is a polypeptide consisting of an amino acid sequence that is an amino acid sequence of an immunoglobulin-binding domain selected from the group consisting of a B domain, a Z domain, a C domain, and variants thereof of staphylococcal protein A, with at least one amino acid residue inserted between positions corresponding to positions 3 and 4 of the amino acid sequence of the B domain, the Z domain, or the C domain.

Description

Technical Field

[0001] This invention relates to immunoglobulin-binding proteins, affinity carriers using the same, methods for isolating immunoglobulins using the affinity carrier, and methods for manufacturing antibody pharmaceuticals. Background Technology

[0002] Affinity chromatography is a chromatographic method that uses a chromatographic column packed with a ligand immobilization carrier, which is obtained by immobilizing a substance (ligand) that specifically binds to a substance intended for separation or purification onto an insoluble carrier. Affinity chromatography is used for the separation and purification of biological substances such as proteins and nucleic acids (Patent Document 1). Examples of carriers used in affinity chromatography include cross-linked sugar particles, such as those represented by agarose gel, and particles primarily composed of synthetic polymers.

[0003] In the fabrication of carriers for affinity chromatography, ligands, substances that specifically bind to the target substance, need to be immobilized on the carrier. Staphylococcus aureus protein A (SpA) and its variants are known as representative affinity chromatography ligands with the ability to specifically bind to immunoglobulins. Because SpA can bind to the Fc region of immunoglobulins without significantly affecting the high selectivity of immunoglobulins for antigens, it can efficiently capture and purify immunoglobulins and proteins containing the Fc region.

[0004] In the natural form of SpA, the domains that bind to immunoglobulins consist of five domains, E, D, A, B, and C, sequentially from the N-terminus. These domains, along with the Z domain (a modified version of the B domain), have been used as affinity chromatography ligands. Furthermore, to enhance immunoglobulin binding affinity, substances combining two or more of these domains are generally used as ligands.

[0005] The aforementioned SpA immunoglobulin-binding domains each contain three α-helical structures, and it is known that the two α-helical sites on the N-terminal side facilitate binding to immunoglobulins. Furthermore, it has been reported that in the B and C domains of the aforementioned domains, a turn structure is formed by the Asn at position 3 and the Lys at position 4 of the N-terminus (Non-Patent Literature 1). Therefore, in ligands formed by linking multiple B, C, or Z domains belonging to a modified B domain, it is presumed that due to the aforementioned turn structure, the domains are connected in a bent configuration, resulting in a steric hindrance. This steric hindrance is a significant problem in the fabrication of affinity chromatography carriers using repeating structures of the B, C, or Z domains as ligands. That is, due to the aforementioned steric hindrance, the immunoglobulin binding capacity of the ligand is low, resulting in a large amount of carrier required to purify a certain amount of immunoglobulin. Affinity chromatography carriers using SpA as a ligand are generally very expensive, and requiring a large amount of carrier is not preferable from a manufacturing cost perspective.

[0006] Affinity chromatography carriers are often reused in bioseparation applications. Therefore, a cleaning process known as in-situ washing (CIP) is typically performed during reuse to remove impurities and return the carrier to its original state. Alkaline solutions such as sodium hydroxide are used as reagents for CIP. However, such alkaline conditions are harsh for affinity chromatography carriers using proteins as ligands, sometimes resulting in loss of binding ability to target molecules due to ligand inactivation or cleavage. Deamidation of Asn and Gln residues is widely known as a major factor in ligand inactivation under these alkaline conditions. In particular, it has been reported that Asn is highly sensitive to alkaline conditions, and its deamidation is structure-dependent, often occurring at amino acid sequence sites represented by Asn-Gly or Asn-Ser (Non-Patent Literature 2).

[0007] As a prior art for avoiding ligand inactivation under alkaline conditions, examples include ligands with reduced alkali sensitivity obtained by deleting or modifying Asn residues. By using these ligands, affinity chromatography carriers that maintain their immunoglobulin binding ability even after repeated CIPs using alkaline solutions are provided. For example, Patent Document 2 provides a ligand for an affinity chromatography carrier containing the B, C, or Z domains of SpA, wherein the ligand contains the deletion of at least three consecutive amino acids from the N-terminus starting at position 1 or 2 of at least one domain. Patent Document 3 provides a ligand for an affinity chromatography carrier containing the C domain of SpA with consecutive amino acid deletions from positions 3 to 6 on the N-terminus. Patent Document 4 provides a ligand for an affinity chromatography carrier modified with Asn residues in the Z and B domains of SpA. However, the above-mentioned prior art primarily aims to improve the alkali resistance of the carrier and does not address improving the immunoglobulin binding ability of the carrier.

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: Japanese Patent Application Publication No. 6-281638

[0011] Patent Document 2: Japanese Patent Application Publication No. 2012-254981

[0012] Patent Document 3: Japanese Patent No. 5345539

[0013] Patent Document 4: Japanese Patent Publication No. 2002-527107

[0014] Non-patent literature

[0015] Non-patent literature 1: Structure, 2014, 22: 1467-1477

[0016] Non-patent literature 2: Journal of Biotechnology, 2000, 80: 169-178 Summary of the Invention

[0017] There is a need for an affinity vector that maintains high immunoglobulin binding capacity and alkali resistance, and is highly efficient and cost-effective. One aspect of the invention is to provide a novel ligand for the affinity vector that enhances immunoglobulin binding capacity. Another aspect of the invention is to provide an affinity vector that maintains the aforementioned high immunoglobulin binding capacity even when repeatedly subjected to CIP using alkaline solutions.

[0018] Therefore, in one embodiment, the present invention provides an immunoglobulin-binding protein. This immunoglobulin-binding protein comprises at least one modified immunoglobulin-binding domain, which is a polypeptide consisting of an amino acid sequence selected from the B domain, Z domain, C domain, and variants thereof of Staphylococcus aureus protein A (SpA), wherein at least one amino acid residue is inserted between positions 3 and 4 of the amino acid sequence corresponding to the B domain, Z domain, or C domain.

[0019] In one embodiment of the immunoglobulin-binding protein of the present invention, the immunoglobulin-binding domain selected from the above-mentioned B domain, Z domain, C domain and their variants is an immunoglobulin-binding domain composed of any one of the amino acid sequences represented by serial numbers 1 to 3 or an amino acid sequence having at least 70% homology with them.

[0020] In one embodiment of the immunoglobulin-binding protein of the present invention, at least one of the modified immunoglobulin-binding domains is selected from the following polypeptides:

[0021] A polypeptide consisting of an amino acid sequence in which at least one amino acid residue is inserted between positions 3 and 4 of the amino acid sequence represented by Serial No. 1, which is an immunoglobulin binding domain consisting of an amino acid sequence represented by Serial No. 1 or an amino acid sequence having at least 70% homology with it.

[0022] A polypeptide comprising an amino acid sequence having at least one amino acid residue inserted between positions 3 and 4 of the amino acid sequence represented by Serial Number 2, relating to an immunoglobulin-binding domain of the amino acid sequence represented by Serial Number 2 or an amino acid sequence having at least 70% homology with it; and

[0023] A polypeptide consisting of an amino acid sequence in which at least one amino acid residue is inserted between positions 3 and 4 of the amino acid sequence represented by Serial No. 3, which is an immunoglobulin binding domain consisting of an amino acid sequence represented by Serial No. 3 or an amino acid sequence having at least 70% homology with it.

[0024] In one embodiment of the immunoglobulin-binding protein of the present invention, at least one amino acid residue is selected from at least one of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp and Tyr.

[0025] In one embodiment of the immunoglobulin-binding protein of the present invention, the immunoglobulin-binding domain composed of an amino acid sequence having at least 70% homology with the amino acid sequence represented by any one of the above sequence numbers 1 to 3 is a Val1 / Ala29 variant.

[0026] In one embodiment, the immunoglobulin-binding protein of the present invention comprises 2 to 12 of the above-described modified immunoglobulin-binding domains.

[0027] In another embodiment, the present invention provides a polynucleotide encoding the aforementioned variant immunoglobulin-binding protein.

[0028] In another embodiment, the present invention provides a vector comprising the aforementioned polynucleotides.

[0029] In another embodiment, the present invention provides a recombinant comprising the above-described carrier.

[0030] In another embodiment, the present invention provides a method for manufacturing an immunoglobulin-binding protein, comprising: expressing the polynucleotide using a cell-free protein synthesis system or expressing the polynucleotide in the recombinant.

[0031] Furthermore, in another embodiment, the present invention provides a method for manufacturing an immunoglobulin-binding protein. This method comprises inserting at least one amino acid residue between positions 3 and 4 of an amino acid sequence of an immunoglobulin-binding domain selected from the B, Z, C domains, and their variants, of a protein A from Staphylococcus aureus, corresponding to the amino acid sequence of the B, Z, or C domain.

[0032] Furthermore, in another embodiment, the present invention provides a method for enhancing the immunoglobulin-binding capacity of immunoglobulin-binding proteins. This method involves inserting at least one amino acid residue between positions 3 and 4 of the amino acid sequence corresponding to the immunoglobulin-binding domain of Staphylococcus aureus protein A, selected from the B, Z, C domains and their variants.

[0033] In one embodiment of the method for manufacturing immunoglobulin-binding proteins and the method for improving immunoglobulin binding capacity of the present invention, the immunoglobulin-binding domain selected from the B domain, Z domain, C domain and their variants is an immunoglobulin-binding domain composed of an amino acid sequence represented by any one of the sequence numbers 1 to 3 or an amino acid sequence having at least 70% homology with them.

[0034] In one embodiment, the method for manufacturing the immunoglobulin-binding protein and the method for improving immunoglobulin binding capacity of the present invention include the following:

[0035] For the immunoglobulin binding domain consisting of the amino acid sequence represented by Serial No. 1 or an amino acid sequence having at least 70% homology with it, at least one amino acid residue is inserted between positions 3 and 4 of the amino acid sequence represented by Serial No. 1.

[0036] For the immunoglobulin-binding domain consisting of the amino acid sequence represented by Serial Number 2 or an amino acid sequence having at least 70% homology with it, at least one amino acid residue is inserted between positions 3 and 4 of the amino acid sequence represented by Serial Number 2; or

[0037] For the immunoglobulin binding domain consisting of the amino acid sequence represented by sequence number 3 or an amino acid sequence having at least 70% homology with it, at least one amino acid residue is inserted between positions 3 and 4 of the amino acid sequence represented by sequence number 3.

[0038] In one embodiment of the method for manufacturing immunoglobulin-binding protein and the method for improving immunoglobulin binding capacity of the present invention, the above-mentioned at least one amino acid residue is selected from at least one of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp and Tyr.

[0039] In one embodiment of the method for manufacturing the immunoglobulin-binding protein and the method for improving immunoglobulin binding capacity of the present invention, the immunoglobulin-binding domain composed of an amino acid sequence having at least 70% homology with the amino acid sequence represented by any one of the above sequence numbers 1 to 3 is a Val1 / Ala29 variant.

[0040] In one embodiment, the method for manufacturing the immunoglobulin-binding protein and the method for improving immunoglobulin binding capacity of the present invention further include: connecting 2 to 12 of the above-mentioned immunoglobulin-binding domains with at least one amino acid residue inserted.

[0041] Furthermore, in another embodiment, the present invention provides an affinity carrier formed by immobilizing the aforementioned immunoglobulin-binding protein on a water-insoluble substrate.

[0042] Furthermore, in another embodiment, the present invention provides a method for isolating immunoglobulins using the aforementioned affinity carrier.

[0043] Furthermore, in another embodiment, the present invention provides a method for manufacturing an antibody pharmaceutical product using the aforementioned affinity carrier.

[0044] The variant immunoglobulin-binding protein of the present invention is useful as a ligand for affinity chromatography carriers. This variant immunoglobulin-binding protein is immobilized on the carrier by chemical bonding of reactive side chains such as amino, thiol, or carboxyl groups present on the surface of an insoluble carrier. This variant immunoglobulin-binding protein exhibits high immunoglobulin binding capacity, and this high capacity is maintained even after repeated CIP using alkaline solutions. Therefore, when using this variant immunoglobulin-binding protein as a ligand for affinity chromatography carriers, for example, in the purification of immunoglobulins, a greater amount of immunoglobulin can be purified with a given amount of carrier, and the dynamic binding capacity of immunoglobulins does not easily decrease even with repeated use. As a result, immunoglobulin purification processes can be implemented at low cost. Detailed Implementation

[0045] All patent documents, non-patent documents and other publications cited in this specification are incorporated herein by reference in their entirety.

[0046] In this specification, sequence homology of amino acid and nucleotide sequences was calculated using the Lipman-Pearson method (Science, 227, 1435-41, 1985). Specifically, the homology analysis program of the genetic information processing software Genetyx-Win (Ver. 5.1.1; software developer) was used, and the unit size to compare (ktup) was interpreted as 2 for calculation.

[0047] In this specification, "at least 70% homology" of amino acid sequences and nucleotide sequences means 70% or more homology, preferably 80% or more homology, more preferably 85% or more homology, even more preferably 90% or more homology, even more preferably 95% or more homology, even more preferably 98% or more homology, and even more preferably 99% or more homology.

[0048] In this specification, "corresponding positions" on amino acid and nucleotide sequences can be determined by aligning the target sequence and reference sequence (e.g., the amino acid sequence represented by sequence number 3) in a manner that maximizes the homology of conserved amino acid residues or nucleotides present in each amino acid or nucleotide sequence. Alignment can be performed using well-known algorithms in an order known to those skilled in the art. For example, alignment can also be performed manually based on the Lippmann-Pearson method described above, but can be performed using the Clustal W multiple sequence alignment program (Thompson, J. Det al., 1994, Nucleic Acids Res., 22: 4673–4680) by default. Alternatively, Clustal W2 or Clustal omega, as revisions of Clustal W, can also be used. Clustal W, Clustal W2, and Clustal omega can be accessed, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the Japanese DNA database (DDBJ [www.ddbj.nig.ac.jp / Welcome-j.html]) operated by the National Institute of Genetics.

[0049] In this specification, "immunoglobulin-binding protein" refers to a protein capable of binding immunoglobulins. "Immunoglobulin-binding domain" refers to the domains contained within immunoglobulin-binding proteins that are associated with immunoglobulin binding. Examples include the A, B, C, D, and E domains of Staphylococcus aureus protein A (SpA), as well as the Z domain, a modified version of the B domain.

[0050] 1. Carriers for affinity chromatography

[0051] 1.1. Mutant immunoglobulin-binding proteins

[0052] The variant immunoglobulin-binding protein of the present invention comprises at least one modified immunoglobulin-binding domain derived from the B, C, or Z domain of SpA. This modified immunoglobulin-binding domain is a polypeptide consisting of an amino acid sequence in which at least one amino acid residue is inserted between an Asn residue at position 3 corresponding to the B, Z, or C domain and a Lys residue at position 4. The variant immunoglobulin-binding protein of the present invention can be used as a ligand for an affinity vector.

[0053] As the parent domain of the modified immunoglobulin-binding domain contained in the mutant immunoglobulin-binding protein of the present invention, examples of the parent domain include the B domain, Z domain, C domain of SpA having immunoglobulin-binding ability, and variants thereof. Among them, the B domain, Z domain, and C domain are preferred. The B domain, Z domain, and C domain of SpA are polypeptides composed of the amino acid sequences represented by SEQ ID NO: 1, 2, and 3, respectively. These domains have an Asn residue at the 3rd position and a Lys residue at the 4th position in their amino acid sequences, and have a turn structure composed of these amino acid residues.

[0054] Examples of variants of the above B domain, Z domain, or C domain that can be used as the above parent domain include polypeptides composed of an amino acid sequence having at least 70% homology with the amino acid sequences represented by SEQ ID NO: 1, 2, or 3 and having immunoglobulin-binding ability and functioning as an immunoglobulin-binding domain. Moreover, this variant has consecutive Asn and Lys residues at positions corresponding to the 3rd to 4th positions of the amino acid sequences represented by SEQ ID NO: 1, 2, or 3 in its amino acid sequence, and has a turn structure composed of these amino acid residues. An example of a variant of the above B domain, Z domain, or C domain is the Val1 / Ala29 variant of the immunoglobulin-binding domain composed of the amino acid sequence represented by SEQ ID NO: 1, 2, or 3.

[0055] The above variants can be prepared by modifying the B domain, Z domain, or C domain of SpA by addition, deletion, substitution, or deletion of amino acid residues, chemical modification of amino acid residues, etc. As methods for addition, deletion, substitution, or deletion of amino acid residues, known methods such as site-specific mutation of polynucleotides encoding the above domains can be cited.

[0056] The modified immunoglobulin-binding domain contained in the variant immunoglobulin-binding protein of the present invention can be obtained by inserting at least one amino acid residue between the Asn residue at position 3 and the Lys residue at position 4 of the amino acid sequence corresponding to the parent domain. For example, the modified immunoglobulin-binding domain is a polypeptide composed of an amino acid sequence in which at least one amino acid residue is inserted between the Asn residue at position 3 and the Lys residue at position 4 of the amino acid sequence of the immunoglobulin-binding domain represented by sequence number 1, 2, or 3. Alternatively, the modified immunoglobulin-binding domain is a polypeptide composed of an amino acid sequence in which at least 70% homology with the amino acid sequence represented by sequence number 1, 2, or 3 is expressed, and at least one amino acid residue is inserted between the Asn residue and the Lys residue of the amino acid sequence in which the variant immunoglobulin-binding domain has consecutive Asn and Lys residues at positions 3 to 4 of the amino acid sequence represented by sequence number 1, 2, or 3. These peptides have immunoglobulin binding capacity and function as immunoglobulin binding domains.

[0057] The insertion of at least one amino acid residue into the aforementioned parent domain is not particularly limited. For example, at least one, preferably one to four, and more preferably one to two amino acid residues selected from Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr can be included. Among these, Phe, Leu, Ile, and Pro are more preferred, followed by His, Tyr, and Trp, then Arg, Gln, Glu, Asp, Val, and Met, and finally Thr and Ala. Therefore, the amino acid residues inserted into the aforementioned parent domain are preferably at least one, 1 to 4, and more preferably 1 to 2, selected from Arg, Asp, Gln, Glu, His, Met, Val, Phe, Leu, Ile, Pro, Trp, and Tyr. More preferably, they are at least one, 1 to 4, and more preferably 1 to 2, selected from His, Phe, Leu, Ile, Pro, Trp, and Tyr. Even more preferably, they are at least one, 1 to 4, and more preferably 1 to 2, selected from Phe, Leu, Ile, and Pro. The reason for preferring these amino acid residues is presumably because it can alter the turn structure formed by the Asn residue at position 3 and the Lys residue at position 4, and can avoid the highly base-sensitive amino acid sequences represented by Asn-Gly and Asn-Ser. When the number of inserted amino acid residues is two or more, they can be completely different types of amino acid residues or can contain multiple amino acid residues of the same type. Further preferably, the amino acid residues inserted into the above-mentioned B, Z or C domains or their variants are selected from any one of Phe, Leu, Ile and Pro.

[0058] As a method for inserting amino acid residues into the aforementioned parent domain, one example is inserting a nucleotide sequence encoding the amino acid residue to be inserted into the nucleotide sequence encoding the parent domain. Specific methods for inserting nucleotide sequences include site-specific mutation, homologous recombination, and SOE (splicing by overlap extension)-PCR (Gene, 1989, 77: 61-68), the detailed order of which is well known to those skilled in the art.

[0059] The variant immunoglobulin-binding protein of the present invention comprises one or more modified immunoglobulin-binding domains in which amino acid residues are inserted between Asn and Lys, but preferably two or more such modified immunoglobulin-binding domains, more preferably 2 to 12, and even more preferably 3 to 8. The domains are interconnected. More specifically, the C-terminus of a domain is connected to or in reverse order of the N-terminus of an adjacent domain. Because the variant immunoglobulin-binding protein of the present invention has a region in which multiple modified immunoglobulin-binding domains are arranged linearly, it can prevent steric hindrance and immunoglobulin binding obstruction caused by the curved arrangement of the domains. Therefore, the variant immunoglobulin-binding protein of the present invention has a high immunoglobulin binding capacity.

[0060] In one embodiment, the variant immunoglobulin-binding protein of the present invention may include an immunoglobulin-binding domain of SpA other than the B, Z, and C domains (e.g., the A, D, or E domain) or its variants. In another embodiment, the variant immunoglobulin-binding protein of the present invention may include the B, Z, or C domains without an amino acid residue inserted between the 3rd Asn and 4th Lys positions, or their variants, but preferably does not include these domains or variants. In a preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises entirely modified immunoglobulin-binding domains derived from the B, Z, or C domains or their variants, with an amino acid residue inserted between the Asn and Lys positions.

[0061] In a preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence in which one, two, three, or four amino acid residues selected from Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr are inserted between the 3rd Asn and 4th Lys positions in the amino acid sequence of the B domain represented by Serial Number 1. In another preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence in which one to four amino acid residues selected from Phe, Leu, Ile, and Pro are inserted between the 3rd Asn and 4th Lys positions in the amino acid sequence of the B domain represented by Serial Number 1. In another preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide having an amino acid sequence that functions as an immunoglobulin-binding domain, wherein the amino acid sequence is obtained by inserting one, two, three, or four amino acid residues selected from Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between Asn and Lys at positions 3 to 4 corresponding to Serial No. 1 in an amino acid sequence having at least 70% homology with the amino acid sequence of the B domain represented by Serial No. 1. Furthermore, in another preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide having an amino acid sequence that functions as an immunoglobulin-binding domain, wherein the amino acid sequence is obtained by inserting one to four amino acid residues selected from Phe, Leu, Ile, and Pro between Asn and Lys at positions 3 to 4 corresponding to Serial No. 1 in an amino acid sequence having at least 70% homology with the amino acid sequence of the B domain represented by Serial No. 1. In a further preferred embodiment, the amino acid sequence having at least 70% homology with the amino acid sequence of the B domain represented by Serial No. 1 is an amino acid sequence having a Val1 / Ala29 variation relative to the amino acid sequence represented by Serial No. 1.

[0062] In a preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence in which one, two, three, or four amino acid residues selected from Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr are inserted between the 3rd Asn and 4th Lys positions in the amino acid sequence of the Z domain represented by Serial Number 2. In another preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence in which one to four amino acid residues selected from Phe, Leu, Ile, and Pro are inserted between the 3rd Asn and 4th Lys positions in the amino acid sequence of the Z domain represented by Serial Number 2. Furthermore, in another preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence that functions as an immunoglobulin-binding domain. This amino acid sequence is obtained by inserting one, two, three, or four amino acid residues selected from Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between Asn and Lys positions corresponding to positions 3-4 of Serial No. 2, in an amino acid sequence that has at least 70% homology with the amino acid sequence of the Z domain represented by Serial No. 2. Furthermore, in another preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence that functions as an immunoglobulin-binding domain. This amino acid sequence is obtained by inserting one to four amino acid residues selected from Phe, Leu, Ile, and Pro between Asn and Lys positions corresponding to positions 3-4 of Serial No. 2, in an amino acid sequence that has at least 70% homology with the amino acid sequence of the Z domain represented by Serial No. 2. In a further preferred embodiment, the amino acid sequence having at least 70% homology with the amino acid sequence of the Z domain represented by sequence number 2 is an amino acid sequence having a variation of Val1 / Ala29 with respect to the amino acid sequence represented by sequence number 2.

[0063] In a preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence in which one, two, three, or four amino acid residues selected from Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr are inserted between the 3rd Asn and 4th Lys positions in the C domain represented by Serial Number 3. In another preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence in which one to four amino acid residues selected from Phe, Leu, Ile, and Pro are inserted between the 3rd Asn and 4th Lys positions in the C domain represented by Serial Number 3. Furthermore, in another preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence that functions as an immunoglobulin-binding domain, wherein the amino acid sequence is obtained by inserting one, two, three, or four amino acid residues selected from Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between Asn and Lys positions corresponding to positions 3 and 4 of Serial No. 3. Furthermore, in another preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises a polypeptide consisting of an amino acid sequence that functions as an immunoglobulin-binding domain, wherein the amino acid sequence is obtained by inserting one to four amino acid residues selected from Phe, Leu, Ile, and Pro between Asn and Lys positions corresponding to positions 3 and 4 of Serial No. 3. In a further preferred embodiment, the amino acid sequence having at least 70% homology with the amino acid sequence of the C domain represented by sequence number 3 is an amino acid sequence having a variation of Val1 / Ala29 relative to the amino acid sequence represented by sequence number 3.

[0064] The polypeptide contained in the variant immunoglobulin-binding protein of the present invention preferably has an amino acid sequence in which an Asn-Lys residue is preserved at position 3 to 4 corresponding to any of the amino acid sequences represented by sequence numbers 1 to 3, and an amino acid is inserted between these residues. More preferably, it has an Asp-Asn-Lys or Gln-Asn-Lys residue at position 2 to 4, and an amino acid is inserted between the Asn and Lys residues. Even more preferably, it has an Ala-Asp-Asn-Lys, Gln-Gln-Asn-Lys, or Val-Asp-Asn-Lys residue at position 1 to 4, and an amino acid is inserted between the Asn and Lys residues. When the Asn residue at position 3 is deleted or mutated, although the alkali resistance of the polypeptide is improved, the immunoglobulin binding capacity is sometimes reduced.

[0065] Preferably, the polypeptide contained in the variant immunoglobulin binding protein of the present invention has a Val residue at position 1 of the amino acid sequence corresponding to any one of the sequences 1 to 3, and / or has an Ala residue at position 29 of the amino acid sequence corresponding to any one of the sequences 1 to 3.

[0066] In a preferred embodiment, the variant immunoglobulin-binding protein of the present invention comprises 2 to 12 of the aforementioned polypeptides, more preferably 3 to 8. These polypeptides may be the same or different. Preferably, the N-terminus of each polypeptide is linked to the C-terminus of an adjacent polypeptide. Each polypeptide may be directly linked to an adjacent polypeptide, or linked via a peptide having 1 to 10 amino acid residues. An example of such a peptide is the peptide represented by EF.

[0067] As a preferred example of the variant immunoglobulin-binding protein of the present invention, a polypeptide composed of the amino acid sequences represented by serial numbers 4 to 20 can be cited. The amino acid sequences represented by serial numbers 4 to 19 are amino acid sequences obtained by linking variants of four C domains. The variant of the C domain is formed by replacing Ala at position 1 with Val and Gly at position 29 with Ala in the amino acid sequence represented by serial number 3, and inserting one or two amino acid residues selected from Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between Asn at position 3 and Lys at position 4. The amino acid sequence represented by sequence number 20 is an amino acid sequence obtained by linking four variants of the Z domain. The variant of the Z domain is formed by replacing Ala at position 1 with Val and Gly at position 29 with Ala in the amino acid sequence represented by sequence number 2, and inserting Ile between Asn at position 3 and Lys at position 4.

[0068] As another preferred example of the variant immunoglobulin-binding protein of the present invention, a polypeptide having immunoglobulin-binding ability and consisting of an amino acid sequence having at least 70% homology with any of the amino acid sequences represented by serial numbers 4 to 18, and having Asn-X-Lys (where X is Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, or Tyr) at positions 4 to 6, 63 to 65, 122 to 124, and 181 to 183 corresponding to serial numbers 4 to 18, respectively. Preferably, these polypeptides have Val at position 1 corresponding to serial number 3 and Ala at position 29.

[0069] As another preferred example of the variant immunoglobulin-binding protein of the present invention, a polypeptide having immunoglobulin-binding ability and consisting of an amino acid sequence having at least 70% homology with the amino acid sequence represented by Serial No. 19, and having Asn-Ile-Thr-Lys arranged at positions 4-7, 64-67, 124-127, and 184-187 corresponding to Serial No. 19, respectively. Preferably, these polypeptides have Val at position 1 corresponding to Serial No. 3 and Ala at position 29.

[0070] As another preferred example of the variant immunoglobulin-binding protein of the present invention, a polypeptide having immunoglobulin-binding ability and consisting of an amino acid sequence having at least 70% homology with the amino acid sequence represented by Serial No. 20, and having Asn-Ile-Lys arranged at positions 4-6, 63-65, 122-124, and 181-183 corresponding to Serial No. 20, respectively. Preferably, these polypeptides have Val at position 1 corresponding to Serial No. 2 and Ala at position 29.

[0071] 1.2. Polynucleotides, vectors

[0072] The present invention further provides a polynucleotide (DNA, etc.) encoding the aforementioned variant immunoglobulin-binding protein of the present invention. One embodiment of the present invention relates to a polynucleotide encoding the aforementioned variant immunoglobulin-binding protein or its equivalent functional variant. In this specification, an "equifunctional variant" of an immunoglobulin-binding protein refers to an immunoglobulin-binding protein modified by the addition, deletion, substitution, or chemical modification of some amino acid residues, maintaining at least 70% homology with the amino acid sequence of the unmodified immunoglobulin-binding protein, maintaining the structure with at least one amino acid residue inserted between Asn and Lys, and exhibiting equivalent immunoglobulin-binding activity to the unmodified immunoglobulin-binding protein.

[0073] Furthermore, as described above, the variant immunoglobulin-binding protein of the present invention includes a protein having one or more, preferably two to twelve, and more preferably three to eight immunoglobulin-binding domains. The polynucleotide encoding such a protein and the expression plasmid comprising a vector containing the polynucleotide can be prepared using known methods.

[0074] In one embodiment, the polynucleotide encoding the variant immunoglobulin-binding protein of the present invention encodes a polypeptide represented by any one of SEQ ID NO. 1 to 20. In another embodiment, the polynucleotide encoding the variant immunoglobulin-binding protein of the present invention encodes an isofunctional variant of an immunoglobulin-binding protein represented by any one of SEQ ID NO. 1 to 20. In yet another embodiment, the polynucleotide encoding the variant immunoglobulin-binding protein of the present invention is a polynucleotide composed of a nucleotide sequence represented by any one of SEQ ID NO. 23 to 39. In yet another embodiment, the polynucleotide encoding the variant immunoglobulin-binding protein of the present invention is a polynucleotide composed of a nucleotide sequence having at least 70% homology to a nucleotide sequence represented by any one of SEQ ID NO. 23 to 39, and encodes an isofunctional variant of an immunoglobulin-binding protein represented by any one of SEQ ID NO. 4 to 20.

[0075] 1.3. Production of variant immunoglobulin-binding proteins

[0076] As a standard technique for producing the variant immunoglobulin-binding protein of the present invention from the aforementioned polynucleotides or vectors, known gene recombination techniques, such as those described in *Current Protocols in Molecular Biology* by Frederick M. Ausbel et al. and *Molecular Cloning* edited by Sambrook et al. (Cold Spring Harbor Laboratory Press, 3rd edition, 2001), can be used. Specifically, an expression vector containing a polynucleotide (DNA, etc.) encoding the variant immunoglobulin-binding protein of the present invention is transformed into a host such as *E. coli*, and the resulting recombinant is cultured in a suitable liquid culture medium, thereby enabling the large-scale and economical acquisition of the target modified protein from the cultured cells. As preferred expression vectors, any known vector capable of replicating within host cells can be used, such as the plasmids described in U.S. Patent No. 5,151,350 and *Molecular Cloning* edited by Sambrook et al. (Cold Spring Harbor Laboratory Press, 3rd edition, 2001). Furthermore, there are no particular limitations on the host used for transformation; any known host used for expressing recombinant proteins, such as bacteria like *E. coli*, fungi, insect cells, and mammalian cells, can be used. To transform the host by introducing nucleic acids, any method known in the art can be used depending on the host. For example, *Molecular Cloning* (Cold Spring Harbor Laboratory Press, 3rd edition), edited by Sambrook et al., can be used. rd The methods described in publications such as (ed., 2001) are well-known to those skilled in the art. Methods for culturing transformed recombinants (bacteria, etc.) and recovering expressed proteins are also exemplified in the embodiments of this invention.

[0077] Alternatively, the variant immunoglobulin-binding protein of the present invention can also be expressed using a cell-free protein synthesis system.

[0078] 1.4. Carrier

[0079] This invention further provides an affinity carrier obtained by immobilizing the mutant immunoglobulin-binding protein of the present invention onto a water-insoluble substrate (carrier). The carrier can be in the form of particles, which can be porous or non-porous. The particle-shaped carrier can be used as a packed bed or in a suspension. Suspension states include those known as expanded beds and pure suspensions, in which the particles are free to move. When using a monolithic column, a packed bed, or a expanded bed, the separation sequence generally follows conventional chromatography utilizing concentration gradients. When using a pure suspension, an intermittent chromatography method is used. Preferably, the carrier is a packing material. Alternatively, the carrier can also be in the form of a chip, capillary, or filter. Magnetic particles can also be used as the carrier. As for magnetic particles, there are no particular limitations as long as they are easily magnetized by magnetic induction. Examples include ferric oxide (Fe3O4), ferric oxide (γ-Fe2O3), various ferrites, metals such as iron, manganese, nickel, cobalt, and chromium, magnetic microparticles composed of alloys of cobalt, nickel, and manganese, or hydrophobic polymers and hydrophilic polymers containing these magnetic particles. As a suitable example, Japanese Patent Application Publication No. 2008-32411 describes a method in which a hydrophobic first polymer layer is formed on the surface of a parent particle containing superparamagnetic particles, and a second polymer layer having at least a glycidyl group on its surface is formed on the first polymer layer. Magnetic particles containing at least one polar group selected from oxygen, nitrogen, and sulfur atoms are introduced by chemically modifying the glycidyl group. In a preferred embodiment, the affinity carrier of the present invention is an affinity chromatography carrier.

[0080] In one embodiment of the present invention, the affinity carrier preferably has a particle size of 10–500 μm, more preferably 20–200 μm. When the carrier is a synthetic polymer, a particle size of 20–100 μm is even more preferred, and a particle size of 30–80 μm is still more preferred. When the carrier is a polysaccharide, a particle size of 50–200 μm is even more preferred, and a particle size of 60–150 μm is still more preferred. When the particle size is less than 10 μm, the column pressure becomes high at high flow rates, making it impractical. If the particle size is greater than 500 μm, there is a time lag in the amount of immunoglobulin binding to the affinity carrier (binding capacity). It should be noted that "particle size" in this specification refers to the volume average particle size obtained by a laser diffraction scattering particle size distribution measuring device.

[0081] One embodiment of the present invention involves an affinity carrier that is preferably porous, having a diameter of 50–150 μm. 2 / g, more preferably 80-130m 2 Specific surface area per g. Here, if the specific surface area is less than 50 m² / g. 2 / g, then considering the capacity, there is a time lag; on the other hand, it is greater than 150m2 At / g, the carrier strength is poor, and sometimes the carrier is destroyed at high flow rates, causing the column pressure to rise. It should be noted that "specific surface area" in this specification refers to the value obtained by dividing the surface area with fine pores of 10–5000 nm obtained by mercury porosimetry by the dry weight of the particles.

[0082] In one embodiment of the present invention, the affinity carrier preferably has a volume average pore size of 100–1400 nm. When the carrier is a synthetic polymer, a volume average pore size of 100–400 nm is more preferred, and a volume average pore size of 200–300 nm is even more preferred. When the carrier is a polysaccharide, a volume average pore size of 500–1400 nm is more preferred, and a volume average pore size of 800–1200 nm is even more preferred. Here, when the volume average pore size is less than 100 nm, the binding capacity at high flow rates sometimes decreases significantly. On the other hand, if it exceeds 1400 nm, the binding capacity decreases regardless of the flow rate. It should be noted that "volume average pore size" in this specification refers to the volume average pore size of pores with a pore size of 10–5000 nm obtained by mercury porosimetry.

[0083] When the carrier meets the above-mentioned range of particle size, specific surface area, and pore size distribution, the balance between the interparticle gaps and the relatively large pore size within the particles that form the flow path of the purified solution and the binding surface area of ​​the purified molecules is optimized, and the binding capacity is maintained at a high level at high flow rates.

[0084] The carrier material can be, for example, a polymer with a hydrophilic surface, such as a polymer having hydroxyl (-OH), carboxyl (-COOH), amino carbonyl (-CONH2, or N-substituted), amino (-NH2, or substituted), oligomeric, or polyvinyloxy groups on its outer surface (and, if present, on its inner surface). In one embodiment, the polymer can be a synthetic polymer such as polymethacrylate, polyacrylamide, polystyrene, or polyvinyl alcohol, preferably a synthetic polymer such as a copolymer obtained by crosslinking polyfunctional monomers such as polyfunctional (meth)acrylate or divinylbenzene. The above-mentioned synthetic polymers can be readily manufactured using known methods (for example, see the method described in J. MATER. CHEM 1991, 1(3), 371-374). Alternatively, commercially available products such as TOYOPEARL (TOSOH Corporation) can also be used. In other embodiments, the polymers are polysaccharides such as dextran, starch, cellulose, pullulan, or agarose. The aforementioned polysaccharides can be readily manufactured using known methods (e.g., referring to the method described in Patent No. 4081143). Alternatively, commercially available products such as Sepharose (GEHEALTHCAREBIO-SCIENCES) can be used. In other embodiments, inorganic carriers such as silica or zirconium oxide may also be used.

[0085] In one embodiment of the present invention, a specific example of a porous particle used as a support in the affinity carrier includes, for example, a particle containing 10-50% by mass of a crosslinked vinyl monomer and 3-90% by mass of an epoxy-containing vinyl monomer, with a particle size of 20-80 μm and a specific surface area of ​​50-150 m². 2 / g, porous organic polymer particles with a volume average pore size of 100-400nm.

[0086] It should be noted that, when measuring the immersion volume (pore volume) of the affinity carrier with a pore size of 10 to 5000 nm according to an embodiment of the present invention using a mercury porosimeter, it is preferably 1.3 to 7.0 mL / g. When the carrier is a synthetic polymer, it is more preferably 1.3 to 2.5 mL / g. When the carrier is a polysaccharide, it is more preferably 3.0 to 6.0 mL / g.

[0087] 1.5. Immobilization of ligands onto a carrier

[0088] As a method for binding the variant immunoglobulin-binding protein (ligand) of the present invention to the aforementioned carrier, a general method for immobilizing the protein on the carrier can be used. Examples of immobilization methods include, for instance, physical adsorption of the ligand to the carrier, and chemical bonding between the carrier and the ligand. Examples of methods for chemical bonding between the carrier and the ligand include, for instance, using a carrier having a carboxyl group and activating the carboxyl group with N-hydroxysuccinimide to react with the amino group of the ligand; using a carrier having an amino or carboxyl group and reacting with the carboxyl or amino group of the ligand in the presence of a dehydrating condensing agent such as a water-soluble carbodiimide to form an amide bond; using a carrier having a hydroxyl group and activating it with a cyanide halide such as cyanogen bromide to react with the amino group of the ligand; or sulfonating the hydroxyl group of the carrier with toluenesulfonate or trifluoroethane sulfonate to react with the amino group of the ligand; introducing an epoxy group into the carrier using a diepoxide, epichlorohydrin, etc., to react with the amino, hydroxyl, or thiol group of the ligand; and using a carrier having an epoxy group to react with the amino, hydroxyl, or thiol group of the ligand. From the viewpoint of stability in the aqueous solution in which the reaction is carried out, the binding method of introducing ligands via epoxy groups is preferred.

[0089] The ring-opening epoxy groups, i.e., alcoholic hydroxyl groups, generated by ring-opening of epoxy groups serve the following functions: hydrophilizing the support surface to prevent non-specific adsorption of proteins, etc., and improving the toughness of the support in water to prevent damage to the support at high flow rates. Therefore, when residual epoxy groups that have not bound to the ligands exist in the support after ligand immobilization, it is preferable to ring-open these residual epoxy groups. For example, a method for ring-opening epoxy groups in the support can be described by stirring the support in an aqueous solvent using an acid or alkali under heating or at room temperature. Alternatively, thioglycerol or other thiol-containing blocking agents, or monoethanolamine or other amino-containing blocking agents, can be used to ring-open the epoxy groups. The most preferred ring-opening epoxy group is the ring-opening epoxy group obtained by ring-opening the epoxy groups contained in the support using thioglycerol. Thioglycerol has the following advantages: it has lower toxicity than thioglycerol and the like; the ring-opening epoxy group formed by adding thioglycerol has lower non-specific adsorption compared to the ring-opening group using an amino-containing blocking agent; and the dynamic binding amount of the support is higher.

[0090] Furthermore, molecules of arbitrary length (spacers) can be introduced between the support and the ligand as needed. Examples of spacers include polymethylene chains, polyethylene glycol chains, and sugars. Spacers can also be, for example, difunctional compounds chemically bonded to the support surface and also bonded to the ligand.

[0091] 1.6. Effects

[0092] One embodiment of the present invention includes an immobilized affinity carrier of the variant immunoglobulin-binding protein of the present invention, which has a high initial immunoglobulin dynamic binding capacity (DBC) and does not significantly reduce its performance under alkaline conditions (e.g., washing with an alkaline solution such as 0.01–0.8 M sodium hydroxide).

[0093] 2. Methods for isolating immunoglobulins

[0094] A method for separating immunoglobulins according to one embodiment of the present invention will be described. The method for separating immunoglobulins according to this embodiment includes: contacting a sample containing immunoglobulins with an affinity carrier immobilized with the aforementioned variant immunoglobulin-binding protein of the present invention, thereby adsorbing the immunoglobulins onto the carrier (first step); and eluting the immunoglobulins from the carrier (second step), preferably further comprising, after the second step, washing the carrier with an alkaline solution (third step). The affinity carrier of the present invention used in the immunoglobulin separation method of the present invention can be in suspension form, packed in a chromatographic column, or in the form of a chip, capillary, filter, or magnetic particles.

[0095] In a preferred embodiment, in the first step, a sample containing immunoglobulins is brought into contact with the affinity carrier of the present invention under conditions where immunoglobulins are adsorbed onto the ligands. In this first step, substances in the sample other than immunoglobulins are hardly adsorbed onto the ligands and remain on the carrier. Subsequently, if necessary, the carrier can be washed with a neutral buffer solution containing salts such as NaCl to remove any substances that are weakly retained on the ligands.

[0096] In the second step, an appropriate buffer solution with a pH of 2–5 is passed through the sample to elute the immunoglobulins adsorbed onto the ligands. By recovering this eluent, the immunoglobulins can be separated from the sample.

[0097] In the method for separating immunoglobulins according to this embodiment, it is preferable to perform the third step immediately after the second step described above. In the third step, the carrier is washed with an alkaline solution (CIP washing). Examples of alkaline solutions used in the third step include aqueous solutions of sodium hydroxide, aqueous solutions of potassium hydroxide, triethylamine, and tetrabutylammonium hydroxide.

[0098] Because the affinity carrier of the present invention has improved alkali resistance of the protein ligand, it can stably maintain its immunoglobulin binding ability after the washing in the third step described above, and therefore can be reused in the immunoglobulin separation method of the present invention.

[0099] In one embodiment of the immunoglobulin separation method of the present invention, the immunoglobulin to be separated may be an antibody or a pharmaceutical product containing the antibody. Therefore, in one embodiment, the present invention provides a method for manufacturing an antibody pharmaceutical product using the affinity carrier of the present invention. The sequence of this method is substantially the same as that of the immunoglobulin separation method described above, except that it uses a sample containing the target antibody pharmaceutical product.

[0100] Example

[0101] The present invention will be further described in detail below with examples. Furthermore, the following description generally represents the manner in which the invention is carried out, and unless otherwise specified, the invention is not limited to this description.

[0102] Refer to Example 1 for the synthesis of porous particles.

[0103] An organic monomer solution was prepared by dissolving 8.2 g of glycidyl methacrylate (manufactured by MITSUBISHI RAYON), 65.9 g of trimethylolpropane trimethacrylate (manufactured by Sartomer), and 90.6 g of glyceryl monomethacrylate (manufactured by Nippon Oil Co., Ltd.) in 245.8 g of 2-octanone (manufactured by Toyo Synthetic Industries Co., Ltd.) and 62 g of acetophenone (manufactured by Wako Pure Chemical Industries Co., Ltd.), and then adding 2 g of 2,2'-azobisisobutyronitrile (manufactured by Wako Pure Chemical Industries Co., Ltd.).

[0104] Next, add 8.5g of polyvinyl alcohol (PVA-217 manufactured by Kuraray), 0.43g of sodium dodecyl sulfate (Emal 10G manufactured by Kao Corporation), and 21.3g of sodium sulfate (manufactured by Wako Pure Chemical Industries Co., Ltd.) to 4240g of pure water, stir overnight, and prepare an aqueous solution.

[0105] Next, the obtained aqueous solution was placed into a 7L separable flask, equipped with a thermometer, stirrer, and cooling tube, and placed in a warm water bath under nitrogen conditions, stirred at 600 rpm. Then, the separable flask was heated in the warm water bath until the aqueous solution reached 85°C. The aforementioned organic monomer solution was then added to the aqueous solution using a dropping funnel, and stirring was continued for 5 hours.

[0106] Next, after cooling the reaction solution, it was transferred to a 5L polypropylene bottle and allowed to stand until the particles were suspended. Excess water was removed from the bottom and discarded. Acetone was then added to the reaction solution to allow the particles to settle. The reaction solution was then allowed to stand for 3 minutes, and the acetone was removed by decantation. This process was repeated twice, followed by the addition of water to allow the particles to settle. The particles were then allowed to stand for 3 minutes and decanted. This process was repeated twice to wash the particles. The particle dispersion was then replaced with acetone again, air-dried overnight, and then dried using a vacuum dryer to obtain 90g of porous particles (hereinafter referred to as PB). The average particle size of PB was 53μm, and the specific surface area was 95m². 2 / g.

[0107] Example 1: Preparation of C-domain recombinant immunoglobulin-binding protein 1 (IgGBPC1)

[0108] A plasmid encoding the amino acid sequence shown in SEQ ID NO: 4 was prepared, and this plasmid was used to transform *E. coli* competent cells BL21(DE3) (manufactured by STRATAGENE) to obtain recombinant cells. The recombinant cells were incubated at 37°C until the absorbance (OD600) reached approximately 10. Subsequently, IPTG (manufactured by Sigma-Aldrich) was added to a final concentration of 1 mM, and the cells were incubated at 37°C for 4 hours to express recombinant immunoglobulin-binding protein. After protein expression, the cells were recovered and lysed in Tris buffer at pH 9.5. The immunoglobulin-binding protein was purified from the cell lysate using anion exchange chromatography (Q-Sepharose FF, manufactured by GE HEALTHCARE BIO-SCIENCES) and cation exchange chromatography (SP-Sepharose FF, manufactured by GE HEALTHCARE BIO-SCIENCES). The purified immunoglobulin-binding protein was dialyzed against 10 mM citrate buffer at pH 6.6 for 16 hours. The purity of the immunoglobulin-binding protein, confirmed by SDS-PAGE, was above 95%. The purified immunoglobulin-binding protein was designated as C-domain recombinant immunoglobulin-binding protein 1 (IgGBPC1). This protein contains a variant with four C-domains (Sequence No. 3), in which Phe is inserted between Asn and Lys positions corresponding to positions 3-4 of Sequence No. 3 in each variant domain. Furthermore, each domain in IgGBPC1 exhibits an alkali-enhancing variant, A1V / G29A (Protein Science, 2013, 22, 1230-1238).

[0109] Examples 2-16: Preparation of recombinant immunoglobulin-binding proteins 2-16 (IgGBPC2-IgGBPC16) with C domain

[0110] Plasmids encoding the amino acid sequences shown in sequence numbers 5–19 were prepared, and recombinant immunoglobulin-binding proteins 2–16 (IgGBPC2–IgGBPC16) were manufactured using substantially the same procedures as in Example 1. These proteins contain variants of four C domains (sequence number 3), with amino acids as shown in Table 1 inserted between Asn and Lys at positions 3–4 of sequence number 3 in each variant domain. Additionally, each domain in IgGBPC2–16 exhibits the alkali-strengthening variant A1V / G29A.

[0111] Example 17: Preparation of Z-domain recombinant immunoglobulin-binding protein 1 (IgGBPZ1)

[0112] A plasmid encoding the amino acid sequence shown in SEQ ID NO: 20 was prepared, and the Z-domain recombinant immunoglobulin-binding protein 1 (IgGBPZ1) was manufactured using substantially the same procedure as in Example 1. This protein contains a variant with four Z-domains (SEQ ID NO: 2), with an Ile inserted between Asn and Lys at positions 3-4 of SEQ ID NO: 2 in each variant domain. Additionally, each domain in IgGBPZ1 exhibits an alkali-enhancing variant, A1V / G29A.

[0113] Comparative Example 1: Preparation of C-domain recombinant immunoglobulin-binding protein O (IgGBPC0)

[0114] A plasmid encoding the amino acid sequence shown in SEQ ID NO: 21 was prepared, and the recombinant C-domain immunoglobulin-binding protein 0 (IgGBPC0) was manufactured using substantially the same procedure as in Example 1. This protein contains a variant of four C-domains (SEQ ID NO: 3), with no inserted amino acid residues between Asn and Lys at positions 3-4 of SEQ ID NO: 3 in each variant domain. It should be noted that IgGBPC0 is a highly alkali-resistant immunoglobulin-binding protein with an alkali-enhanced variant domain A1V / G29A (Protein Science, 2013, 22, 1230-1238).

[0115] Comparative Example 2: Preparation of Z-domain recombinant immunoglobulin-binding protein 0 (IgGBPZ0)

[0116] A plasmid encoding the amino acid sequence shown in SEQ ID NO. 22 was prepared, and the Z-domain recombinant immunoglobulin-binding protein 0 (IgGBPZ0) was manufactured using substantially the same procedure as in Example 1. This protein contains a variant of four Z-domains (SEQ ID NO. 2), with no inserted amino acid residues between Asn and Lys at positions 3-4 of SEQ ID NO. 2 in each variant domain. It should be noted that IgGBPZ0 is a highly alkali-resistant immunoglobulin-binding protein with an alkali-enhanced variant domain A1V / G29A (Protein Science, 2013, 22, 1230-1238).

[0117] The immunoglobulin-binding proteins of Examples 1-17 and Comparative Examples 1-2 are shown in Table 1.

[0118] [Table 1]

[0119]

[0120] Experimental Example 1: Immunoglobulin binding capacity of the carrier used in affinity chromatography

[0121] 1) Immobilization of carriers by immunoglobulin-binding proteins

[0122] The PB prepared in Example 1 was suspended in 150 μL of pure water and transferred to a filter tube (Millipore) for centrifugation to remove the pure water. 450 μL of 0.1 M carbonate buffer (pH 9.8) containing 450 μL of 0.85 M sodium sulfate was added to the solution containing 1 mg of immunoglobulin-binding protein 1 (IgGBPC1) prepared in Example 1. The mixture was shaken at 25°C for 5 hours to allow the immunoglobulin-binding protein to bind to the PB. After filtering the resulting particles, the mixture was mixed with 450 μL of 1 M thioglycerol and reacted at 25°C for 16 hours to block residual epoxy groups. The mixture was washed with 0.5 M NaOH, followed by washing with 0.1 M sodium citrate buffer (pH 3.2) and 0.1 M sodium phosphate buffer (pH 7.6) to obtain 450 μL of bound porous particles (IgGBPC1 / PB). Porous particles (IgGBPC1-16 / PB and IgGBPZ0-1 / PB) containing any one of IgGBPC0, IgGBPC2-16 and IgGBPZ0-1 were obtained in the same order.

[0123] 2) Determination of the amount of immunoglobulin-binding protein introduced into the carrier.

[0124] For each 150 μL suspension containing 1 mg of any one of IgG BPC1-16 / PB and IgG BPZ0-1 / PB, the amount of immunoglobulin-binding protein introduced into the vector was determined using the BCA Assay kit (PIERCE).

[0125] 3) Determination of IgG dynamic binding capacity

[0126] IgG BPC0-16 / BP and IgG BPZ0-1 / PB were packed into chromatographic columns with an inner diameter of 0.5 cm until the bed height was 20 cm. After equilibration with 20 mM phosphate buffer (pH 7.5), the columns were flowed at a linear flow rate of 300 cm / h through 20 mM phosphate buffer (pH 7.5) containing human polyclonal IgG (5 mg / mL). The dynamic binding capacity (DBC) was determined by monitoring the amount of human polyclonal IgG adsorbed and the carrier volume when the concentration of human polyclonal IgG in the eluent exceeded 10% using absorbance.

[0127] 4) Alkali resistance test

[0128] The carrier-packed column used in 3) was installed in AKTAprime plus, and 20 mL of 0.5 M sodium hydroxide was flowed through the column. After removing the column from the apparatus and sealing it, it was left at room temperature for a certain period of time (15, 30, 45 hours). The DBC of human polyclonal IgG was then determined in the same order as in 3) at a linear flow rate of 300 cm / h. The binding capacity retention rate (%DBC) was calculated when the DBC before treatment with 0.5 M sodium hydroxide was set as 100%.

[0129] The results of immunoglobulin-binding protein delivery, DBC, and alkali resistance tests for each affinity chromatography carrier are shown in Tables 2 and 3.

[0130] [Table 2]

[0131]

[0132] [Table 3]

[0133]

[0134] The immunoglobulin-binding proteins of Examples 1-17 and Comparative Examples 1-2 showed almost equal binding amounts to the carriers. On the other hand, the carriers immobilized with the immunoglobulin-binding proteins of Examples 1-17 (IgGBPC1-16 / PB and IgGBPZ1 / PB) exhibited improved DBC compared to the carriers immobilized with the proteins of Comparative Examples 1 or 2 (IgGBPC0 / PB and IgGBPZ0 / PB). Furthermore, IgGBPC1-16 / PB and IgGBPZ1 / PB maintained high alkali resistance, comparable to IgGBPC0 / PB or IgGBPZ0 / PB, even under extremely harsh alkaline conditions such as exposure to 0.5M sodium hydroxide for up to 45 hours, thus maintaining a relatively high DBC. Therefore, by using immunoglobulin-binding proteins with a modified domain formed by inserting amino acid residues between the 3rd Asn and 4th Lys positions of the amino acid sequence containing the SpA immunoglobulin-binding domain as ligands, affinity chromatography carriers with high DBC and alkali resistance can be obtained. sequence list <110> JSR Co., Ltd. JSR Life Sciences Co., Ltd. <120> Immunoglobulin-binding proteins and their affinity carriers <130> JSR0071 <150> JP2015-063519 <151> 2015-03-26 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 58 <212> PRT <213> Staphylococcus aureus <220> <223> Protein A and B domains <400> 1 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 2 <211> 58 <212> PRT <213> Staphylococcus aureus <220> <223> Protein A and Z domains <400> 2 Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 3 <211> 58 <212> PRT <213> Staphylococcus aureus <220> <223> Protein A and C domains <400> 3 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 4 <211> 237 <212> PRT <213> Staphylococcus aureus <220> <223> IgGBPC1 <400> 4 Met Val Asp Asn Phe Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Phe 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Phe Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Phe Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 5 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC2 <400> 5 Met Val Asp Asn Leu Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Leu 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Leu Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Leu Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 6 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC3 <400> 6 Met Val Asp Asn Ile Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Ile 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Ile Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Ile Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 7 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC4 <400> 7 Met Val Asp Asn Pro Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Pro 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Pro Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Pro Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 8 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC5 <400> 8 Met Val Asp Asn Gln Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Gln 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Gln Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Gln Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 9 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC6 <400> 9 Met Val Asp Asn His Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn His 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn His Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn His Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 10 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC7 <400> 10 Met Val Asp Asn Arg Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Arg 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Arg Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Arg Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 11 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC8 <400> 11 Met Val Asp Asn Thr Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Thr 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Thr Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Thr Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 12 <211> 237 <212> PRT <213> Staphylococcus aureus <220> <223> IgGBPC9 <400> 12 Met Val Asp Asn Tyr Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Tyr 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Tyr Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Tyr Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 13 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC10 <400> 13 Met Val Asp Asn Ala Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Ala 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Ala Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Ala Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 14 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC11 <400> 14 Met Val Asp Asn Met Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Met 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Met Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Met Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 15 <211> 237 <212> PRT <213> Staphylococcus aureus <220> <223> IgGBPC12 <400> 15 Met Val Asp Asn Asp Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Asp 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Asp Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Asp Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 16 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC13 <400> 16 Met Val Asp Asn Trp Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Trp 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Trp Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Trp Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 17 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC14 <400> 17 Met Val Asp Asn Glu Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Glu 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Glu Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Glu Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 18 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC15 <400> 18 Met Val Asp Asn Val Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Val 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Val Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val 145 150 155 160 Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Val Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 19 <211> 241 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC16 <400> 19 Met Val Asp Asn Ile Thr Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 1 5 10 15 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala 20 25 30 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu 35 40 45 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn 50 55 60 Ile Thr Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu 65 70 75 80 His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser 85 90 95 Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys 100 105 110 Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Ile Thr Lys Phe 115 120 125 Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn 130 135 140 Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp 145 150 155 160 Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp 165 170 175 Ala Gln Ala Pro Lys Val Asp Asn Ile Thr Lys Phe Asn Lys Glu Gln 180 185 190 Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu 195 200 205 Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser 210 215 220 Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro 225 230 235 240 Lys <210> 20 <211> 237 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPZ1 <400> 20 Met Val Asp Asn Ile Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala 35 40 45 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Ile 50 55 60 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70 75 80 Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys 85 90 95 Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Ile Lys Phe Asn Lys Glu 115 120 125 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu 130 135 140 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln 145 150 155 160 Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 165 170 175 Pro Lys Val Asp Asn Ile Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 180 185 190 Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 195 200 205 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu 210 215 220 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 235 <210> 21 <211> 233 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPC0 <400> 21 Met Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu 1 5 10 15 Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile 20 25 30 Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu 35 40 45 Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Lys Phe 50 55 60 Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn 65 70 75 80 Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp 85 90 95 Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp 100 105 110 Ala Gln Ala Pro Lys Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn 115 120 125 Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg 130 135 140 Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu 145 150 155 160 Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val 165 170 175 Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu 180 185 190 His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser 195 200 205 Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys 210 215 220 Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 <210> 22 <211> 233 <212> PRT <213> Yellow grape fungus <220> <223> IgGBPZ0 <400> 22 Met Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu 1 5 10 15 Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile 20 25 30 Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu 35 40 45 Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Lys Phe 50 55 60 Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn 65 70 75 80 Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp 85 90 95 Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp 100 105 110 Ala Gln Ala Pro Lys Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn 115 120 125 Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg 130 135 140 Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn 145 150 155 160 Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val 165 170 175 Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu 180 185 190 His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser 195 200 205 Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys 210 215 220 Lys Leu Asn Asp Ala Gln Ala Pro Lys 225 230 <210> 23 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC1 <400> 23 atggtggata actttaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataact ttaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataacttta aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aactttaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 24 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC2 <400> 24 atggtggata acctgaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataacc tgaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataacctga aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacctgaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 25 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC3 <400> 25 atggtggata acattaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataaca ttaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataacatta aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacattaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 26 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC4 <400> 26 atggtggata acccgaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataacc cgaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 gtggataacc cgaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataacccga aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 gataacccga aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacccgaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 aacccgaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 27 <210> 27 <211> 711 <211> 711 <212> DNA <212> DNA <213> 金黄色葡萄球菌 <213> Staphylococcus aureus <220> <220> <223> IgGBPC5 <400> 27 atggtggata accagaaatt taacaaaga cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaataa 180 gtggataacc agaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 360 gataaccaga aattaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aaccagaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 28 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC6 <400> 28 atggtggata accataaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataacc ataaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataaccata aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aaccataaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 29 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC7 <400> 29 atggtggata accgcaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataacc gcaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataaccgca aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aaccgcaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 30 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC8 <400> 30 atggtggata acaccaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataaca ccaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataacacca aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacaccaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 31 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC9 <400> 31 atggtggata actataaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataact ataaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataactata aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aactataaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 32 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC10 <400> 32 atggtggata acgcgaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataacg cgaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataacgcga aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacgcgaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 33 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC11 <400> 33 atggtggata acatgaaatt taacaaaaga cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaataa 180 gtggataaca tgaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 360 gataacatga aattaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacatgaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 34 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC12 <400> 34 atggtggata acgataaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataacg ataaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataacgata aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacgataaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 35 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC13 <400> 35 atggtggata actggaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataact ggaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataactgga aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aactggaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 36 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC14 <400> 36 atggtggata acgaaaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataacg aaaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataacgaaa aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacgaaaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 37 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC15 <400> 37 atggtggata acgtgaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 60 ctgccgaacc tgaccgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 120 agcgtgagca aagaaattct ggaagcgaaa aaactgaacg atgcgcaggc gccgaaataa 180 gtggataacg tgaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga ccgaagaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 gtgagcaaag aaattctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaataagtg 360 gataacgtga aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaccg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagcgtg 480 agcaaagaaa ttctggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacgtgaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaccgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagcgtgagc 660 aaagaaattc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711 <210> 38 <211> 723 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPC16 <400> 38 atggtggata acattaccaa atttaacaaa gaacagcaga acgcgtttta tgaaattctg 60 catctgccga acctgaccga agaacagcgc aacgcgttta ttcagagcct gaaagatgat 120 ccgagcgtga gcaaagaaat tgcggaagcg aaaaaactga acgatgcgca ggcgccgaaa 180 taagtggata acattaccaa atttaacaaa gaacagcaga acgcgtttta tgaaattctg 240 catctgccga acctgaccga agaacagcgc aacgcgttta ttcagagcct gaaagatgat 300 ccgagcgtga gcaaagaaat tgcggaagcg aaaaaactga acgatgcgca ggcgccgaaa 360 taagtggata acattaccaa atttaacaaa gaacagcaga acgcgtttta tgaaattctg 420 catctgccga acctgaccga agaacagcgc aacgcgttta ttcagagcct gaaagatgat 480 ccgagcgtga gcaaagaaat tgcggaagcg aaaaaactga acgatgcgca ggcgccgaaa 540 taagtggata acattaccaa atttaacaaa gaacagcaga acgcgtttta tgaaattctg 600 catctgccga acctgaccga agaacagcgc aacgcgttta ttcagagcct gaaagatgat 660 ccgagcgtga gcaaagaaat tgcggaagcg aaaaaactga acgatgcgca ggcgccgaaa 720 taa 723 <210> 39 <211> 711 <212> DNA <213> Staphylococcus aureus <220> <223> IgGBPZ1 <400> 39 atggtggata acattaaatt taacaaaga cagcagaacg cgttttatga aattctgcat 60 120 agccagagcg cgaacctgct ggaagcgaaa aaactgaacg atgcgcaggc gccgaataa 180 gtggataaca ttaaatttaa caaagaacag cagaacgcgt tttatgaaat tctgcatctg 240 ccgaacctga acgaaaca gcgcaacgcg tttattcaga gcctgaaaga tgatccgagc 300 cagagcgcga acctgctgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaataagtg 360 gataacatta aatttaacaa agaacagcag aacgcgtttt atgaaattct gcatctgccg 420 aacctgaacg aagaacagcg caacgcgttt attcagagcc tgaaagatga tccgagccag 480 agcgcgaacc tgctgggaagc gaaaaaactg aacgatgcgc aggcgccgaa ataagtggat 540 aacattaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 600 ctgaacgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagccagagc 660 gcgaacctgc tggaagcgaa aaaactgaac gatgcgcagg cgccgaaata a 711

Claims

1. An immunoglobulin-binding protein, consisting of an amino acid sequence represented by one of sequence numbers 4 to 20.

2. A polynucleotide encoding the immunoglobulin-binding protein of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A recombinant comprising the vector of claim 3.

5. A method of producing an immunoglobulin-binding protein, comprising: The polynucleotide of claim 2 may be expressed using a cell-free protein synthesis system, or the polynucleotide of claim 2 may be expressed in the recombinant of claim 4.

6. A method for manufacturing an immunoglobulin-binding protein, comprising: (1) For the amino acid sequence represented by sequence number 3, Val is present at position 1 corresponding to the amino acid sequence represented by sequence number 3, Ala is present at position 29 corresponding to the amino acid sequence represented by sequence number 3, and one or two amino acid residues are inserted between positions 3 and 4 corresponding to the amino acid sequence represented by sequence number 3. The one or two amino acid residues are Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, Tyr, or Ile-Thr; or (2) For the amino acid sequence represented by sequence number 2, insert Ile between the positions corresponding to the 3rd and 4th positions of the amino acid sequence represented by sequence number 2; The immunoglobulin-binding protein obtained by the manufacturing method is the immunoglobulin-binding protein according to claim 1.

7. A method for enhancing the immunoglobulin-binding ability of an immunoglobulin-binding protein, comprising: (1) For the amino acid sequence represented by sequence number 3, Val is present at position 1 corresponding to the amino acid sequence represented by sequence number 3, Ala is present at position 29 corresponding to the amino acid sequence represented by sequence number 3, and one or two amino acid residues are inserted between positions 3 and 4 corresponding to the amino acid sequence represented by sequence number 3. The one or two amino acid residues are Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, Tyr, or Ile-Thr; or (2) For the amino acid sequence represented by sequence number 2, insert Ile between the positions corresponding to the 3rd and 4th positions of the amino acid sequence represented by sequence number 2; The immunoglobulin-binding protein obtained by the improvement method is the immunoglobulin-binding protein according to claim 1.

8. An affinity carrier is formed by immobilizing the immunoglobulin-binding protein of claim 1 onto a water-insoluble substrate.

9. A method for isolating immunoglobulins, using the affinity vector as described in claim 8.

10. A method for manufacturing an antibody pharmaceutical product, using the affinity vector as described in claim 8.

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