METHOD FOR PRODUCING Fc-BINDING PROTEIN USING GENETICALLY ENGINEERED ESCHERICHIA COLI

Abstract

To provide a method for efficiently producing an Fc-binding protein using genetically engineered Escherichia coli capable of expressing the Fc-binding protein.SOLUTION: To solve the problem, an Fc-binding protein is produced through a method comprising: a step of culturing a recombinant Escherichia coli containing a polynucleotide encoding an Fc-binding protein to express the protein; and a step of recovering the protein, wherein a medium used for culturing the recombinant Escherichia coli comprises at least peptone and yeast extract, and wherein the peptone is derived from soybean, cotton, broad bean, lupin, corn, potato, pea, or barley.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for efficiently industrially producing a protein using a genetically engineered Escherichia coli capable of expressing an Fc-binding protein obtained by genetic engineering techniques. In particular, the present invention relates to a particularly efficient production method when the Fc-binding protein is a human neonatal Fc receptor (FcRn).

Background Art

[0002] An Fc-binding protein (Fc receptor) is a receptor protein that binds to the Fc region of an immunoglobulin molecule, binds to an immune complex of an antigen and an immunoglobulin, and transmits a signal intracellularly (Non-Patent Document 1). Each molecule recognizes a single or the same group of immunoglobulin isotypes by a recognition domain belonging to the immunoglobulin superfamily on the recognition domain of the Fc receptor. This determines which accessory cells are activated in the immune response.

[0003] Fc receptors can be further classified into several subtypes, including Fcγ receptors, which are receptors for immunoglobulin G (IgG), Fcα receptors, Fcε receptors, etc. (Non-Patent Documents 1 and 2). Among them, the human neonatal Fc receptor (FcRn) is a major histocompatibility complex (MHC) class I-related molecule different from the human Fcγ receptor belonging to the immunoglobulin superfamily, and is composed of a heavy chain (α chain) and β2-microglobulin (β chain) (Non-Patent Document 3). FcRn is involved in the recycling mechanism of IgG and has the function of suppressing the degradation of IgG. In addition, FcRn binds to IgG in a pH-dependent manner and binds at pH 6.5 or lower (Non-Patent Document 4).

[0004] The antibody adsorption ability of such Fc-binding proteins can also be used as a protein responsible for the capture function of various antibody purification chromatography gels. Therefore, manufacturing methods using gene recombinants capable of expressing Fc-binding proteins have been studied so far. For example, a method of expressing an Fc-binding protein by culturing genetically engineered E. coli has been disclosed, and a general culture medium containing peptone or the like has been used (Non-Patent Document 5). However, when tryptone, which is generally widely used as a medium component, is included in the E. coli culture medium, there has been a problem that industrial production of a predetermined Fc-binding protein cannot be efficiently carried out.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a method for efficiently producing the protein using a genetically engineered E. coli capable of expressing an Fc-binding protein. [Means for Solving the Problems]

[0007] As a result of earnestly studying the culture conditions of a genetically engineered Escherichia coli capable of expressing an Fc-binding protein and the expression conditions of the Fc-binding protein with respect to the above problems, the present inventors have completed the present invention.

[0008] That is, the present invention includes the following aspects: [1] A method for producing an Fc-binding protein, comprising a step of culturing a genetically engineered Escherichia coli containing a polynucleotide encoding an Fc-binding protein to express the protein, and a step of recovering the protein, wherein the medium used for culturing the genetically engineered Escherichia coli is a medium containing at least peptone and yeast extract, and the peptone is derived from any one of soybean, cotton, broad bean, lupin bean, corn, potato, pea and barley.

[0009] [2] The production method according to [1], wherein the medium used for culturing the genetically engineered Escherichia coli further contains saccharides and phosphates.

[0010] [3] The production method according to [1] or [2], wherein the Fc-binding protein is a polypeptide selected from any one of the following (i) to (ix); (i) A polypeptide containing at least the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4 (ii) A polypeptide containing at least the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that one or more substitutions, deletions, insertions and additions of one or several amino acid residues occur at one or several positions in these amino acid residues, and the polypeptide has antibody-binding activity (iii) A polypeptide that contains at least the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that it has an identity of 70% or more with the entire amino acid sequence from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, and has antibody-binding activity. (iv) An Fc-binding protein that contains the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that at least the following amino acid substitutions shown in (1) to (7) occur in said amino acid residues; (1) Cysteine at position 71 in SEQ ID NO: 3 is substituted with arginine (2) Asparagine at position 78 in SEQ ID NO: 3 is substituted with aspartic acid (3) Arginine at position 192 in SEQ ID NO: 3 is substituted with leucine (4) Asparagine at position 196 in SEQ ID NO: 3 is substituted with aspartic acid (5) Glutamine at position 232 in SEQ ID NO: 3 is substituted with leucine (6) Cysteine at position 274 in SEQ ID NO: 3 is substituted with serine (7) Lysine at position 295 in SEQ ID NO: 3 is substituted with glutamic acid (v) An Fc-binding protein that contains the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in said (1) to (7) occur in said amino acid residues, and further, one or more substitutions, deletions, insertions and additions of one or several amino acid residues occur at one or several positions other than the amino acid substitutions shown in said (1) to (7), and has antibody-binding activity. (vi) An amino acid sequence having 70% or more identity with the entire amino acid sequence in which the amino acid substitutions shown in (1) to (7) above have occurred in the amino acid sequence from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid sequence from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence containing the amino acid substitutions of (1) to (7) above and having antibody binding activity, an Fc-binding protein. (vii) An Fc-binding protein containing the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that at least the amino acid substitutions shown in (1) to (10) below have occurred in said amino acid residues; (1) Cysteine at position 71 of SEQ ID NO: 3 is substituted with arginine (2) Asparagine at position 78 of SEQ ID NO: 3 is substituted with aspartic acid (3) Arginine at position 192 of SEQ ID NO: 3 is substituted with leucine (4) Asparagine at position 196 of SEQ ID NO: 3 is substituted with aspartic acid (5) Glutamine at position 232 of SEQ ID NO: 3 is substituted with leucine (6) Cysteine at position 274 of SEQ ID NO: 3 is substituted with serine (7) Lysine at position 295 of SEQ ID NO: 3 is substituted with glutamic acid (8) Valine at position 80 of SEQ ID NO: 3 is substituted with aspartic acid (9) Lysine at position 96 of SEQ ID NO: 3 is substituted with glutamic acid (10) Asparagine at position 172 of SEQ ID NO: 3 is substituted with aspartic acid. (viii) An Fc-binding protein that contains the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (10) occur in the amino acid residues, and further, one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or several positions other than the amino acid substitutions shown in the above (1) to (10), and that has antibody-binding activity. (ix) An amino acid sequence having 70% or more identity to the entire amino acid sequence in which the amino acid substitutions shown in the above (1) to (10) occur in the amino acid sequence from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid sequence from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence containing the amino acid substitutions remaining from the above (1) to (10), and that has antibody-binding activity, and that is an Fc-binding protein. [Advantages of the Invention]

[0011] The present invention uses a genetically engineered Escherichia coli containing a polynucleotide encoding a polypeptide containing at least an Fc-binding protein to industrially produce the polypeptide. The medium used for culturing the genetically engineered Escherichia coli is a medium containing at least peptone and yeast extract, and the peptone is derived from any of soybean, cottonseed, broad bean, lupin bean, corn, potato, pea, and barley. The production method of the present invention is characterized in that the expression level is significantly improved compared to the conventional production method of Fc-binding protein using tryptone, which is a widely used peptone, and thus the protein can be industrially produced efficiently.

[0012] In addition, since the medium does not contain components derived from animals, the risk of infection with bovine spongiform encephalopathy (BSE), which is one of transmissible spongiform encephalopathies (TSE), is minimized, it is highly safe, and it is more suitable for industrial production of proteins.

Brief Description of Drawings

[0013]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0014] Hereinafter, the present invention will be described in detail.

[0015] Preferred embodiments of the Fc-binding protein produced in the present invention include polypeptides shown in any of the following (i) to (ix). (i) A polypeptide comprising at least the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4 (ii) A polypeptide comprising at least the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that one or more substitutions, deletions, insertions and additions (hereinafter also referred to as "modifications") of one or more amino acid residues occur at one or several positions in these amino acid residues, and having antibody-binding activity (iii) A polypeptide comprising at least the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that it has at least 70% identity to the entire amino acid sequence from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, and having antibody-binding activity. (iv) An Fc-binding protein comprising the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, provided that at least the following amino acid substitutions shown in (1) to (7) occur in the amino acid residues (1) Cysteine at position 71 in SEQ ID NO: 3 is substituted with arginine (2) Asparagine at position 78 in SEQ ID NO: 3 is substituted with aspartic acid (3) Arginine at position 192 in SEQ ID NO: 3 is substituted with leucine (4) Asparagine at position 196 in SEQ ID NO: 3 is substituted with aspartic acid (5) The 232nd glutamine of SEQ ID NO: 3 is replaced with leucine (6) The 274th cysteine of SEQ ID NO: 3 is replaced with serine (7) The 295th lysine of SEQ ID NO: 3 is replaced with glutamic acid (v) An Fc-binding protein comprising amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 3 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (7) occur in said amino acid residues, and further one or several amino acid residues are modified (any one or more of substitution, deletion, insertion and addition) at one or several positions other than the amino acid substitutions shown in the above (1) to (7), and having antibody-binding activity. (vi) An amino acid sequence having 70% or more identity to the entire amino acid sequence in which the amino acid substitutions shown in the above (1) to (7) occur in the amino acid sequence from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 4, comprising an amino acid sequence in which the amino acid substitutions shown in the above (1) to (7) remain, and having antibody-binding activity. (vii) An Fc-binding protein comprising amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 3 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 4, provided that at least the amino acid substitutions shown in the following (1) to (10) occur in said amino acid residues; (1) The 71st cysteine of SEQ ID NO: 3 is replaced with arginine (2) The 78th asparagine of SEQ ID NO: 3 is replaced with aspartic acid (3) The 192nd arginine of SEQ ID NO: 3 is replaced with leucine (4) The 196th asparagine of SEQ ID NO: 3 is replaced with aspartic acid (5) The 232nd glutamine of SEQ ID NO: 3 is replaced with leucine (6) The 274th cysteine of SEQ ID NO: 3 is replaced with serine (7) The 295th lysine of SEQ ID NO: 3 is replaced with glutamic acid (8) The 80th valine of SEQ ID NO: 3 is replaced with aspartic acid (9) The 96th lysine of SEQ ID NO: 3 is replaced with glutamic acid (10) The 172nd asparagine of SEQ ID NO: 3 is replaced with aspartic acid. (viii) An Fc-binding protein having antibody-binding activity, comprising the amino acid residues from the 24th alanine to the 297th serine of the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from the 21st isoleucine to the 119th methionine of the amino acid sequence set forth in SEQ ID NO: 4, provided that the amino acid substitutions shown in the above (1) to (10) have occurred in said amino acid residues, and further one or more substitutions, deletions, insertions and additions of one or more amino acid residues have occurred at one or several positions other than the amino acid substitutions shown in the above (1) to (10). (ix) An amino acid sequence having at least 70% identity to the entire amino acid sequence in which the amino acid substitutions shown in the above (1) to (10) have occurred in the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence set forth in SEQ ID NO: 4, comprising the amino acid sequence in which the amino acid substitutions shown in the above (1) to (10) remain, and having antibody-binding activity.

[0016] The amino acid sequence set forth in SEQ ID NO: 3 is the amino acid sequence of the human FcRn α-chain (UniProt No. P55899), and the amino acid sequence set forth in SEQ ID NO: 4 is the amino acid sequence of the human FcRn β-chain (UniProt No. P61769). Among the amino acid sequence set forth in SEQ ID NO: 3, the amino acid residues from alanine (A) at position 24 to serine (S) at position 297 correspond to the extracellular (EC) region of the human FcRn α-chain, and among the amino acid sequence set forth in SEQ ID NO: 4, the amino acid residues from isoleucine (I) at position 21 to methionine (M) at position 119 correspond to the β2-microglobulin (B2M) region of the human FcRn β-chain, respectively.

[0017] The polypeptide shown in any one of (i) to (ix) above only needs to contain at least a region corresponding to the EC region of the aforementioned human FcRn α-chain and the B2M region of the human FcRn β-chain. For example, it may contain all or part of the signal peptide region located on the N-terminal side of the EC region of the human FcRn α-chain or the B2M region of the human FcRn β-chain, or may contain all or part of the transmembrane region and the intracellular region located on the C-terminal side of the EC region of the human FcRn α-chain.

[0018] In the present specification, the Fc-binding protein containing at least amino acid residues corresponding to the EC region of the human FcRn α-chain and the B2M region of the human FcRn β-chain only needs to contain at least an amino acid sequence corresponding to the EC region of the human FcRn α-chain and an amino acid sequence corresponding to the B2M region of the human FcRn β-chain in the amino acid sequence of the protein, and the order of the amino acid residues corresponding to the EC region of the human FcRn α-chain and the amino acid residues corresponding to the B2M region of the human FcRn β-chain does not matter. That is, the amino acid residues corresponding to the B2M region of the human FcRn β-chain may be on the N-terminal side or the C-terminal side of the amino acid residues corresponding to the EC region of the human FcRn α-chain. Also, it may be a mode in which the amino acid residues corresponding to the EC region of the human FcRn α-chain and the amino acid residues corresponding to the B2M region of the human FcRn β-chain are directly linked, or may be a mode in which they are linked via a known linker such as a GS linker (a linker consisting of a repeat of an oligopeptide composed of 4 glycine (G) residues and 1 serine (S) residue).

[0019] In the above (ii), (v), and (viii), "one or several" varies depending on the position of amino acid substitution and the type of amino acid residue in the three-dimensional structure of the Fc-binding protein. As an example, it means any one of 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The modification of "one or several" amino acid residues may occur at positions other than those disclosed in JP 2018-183087 A, JP 2021-073883 A, JP 2021-136967 A, and JP 2022-076998 A as long as it has Fc-binding activity.

[0020] In addition, the "modification of one or several amino acid residues" in the above (ii), (v), and (viii) may include conservative substitutions in which substitutions occur between amino acids with similar physical and / or chemical properties in addition to the amino acid modifications at the specific positions described above. It is generally known to those skilled in the art that conservative substitutions generally maintain the function of the protein between those in which the substitution has occurred and those in which the substitution has not occurred. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). Further, the "modification of one or several amino acid residues" in the above (ii), (v), and (viii) includes naturally occurring mutants or variants.

[0021] As an example of the above (ii), (v), and (viii), the Fc-binding protein disclosed in JP 2018-183087 A, the Fc-binding protein disclosed in JP 2021-073883 A, the Fc-binding protein disclosed in JP 2021-136967 A, the Fc-binding protein disclosed in JP 2022-076998 A, and In addition to the amino acid substitutions (1) to (7) described above, at least one or more of the following amino acid substitutions occur in the Fc-binding protein (8) The valine at position 80 of SEQ ID NO: 3 is substituted with aspartic acid (9) The lysine at position 96 of SEQ ID NO: 3 is substituted with glutamic acid (10) The asparagine at position 172 of SEQ ID NO: 3 is substituted with aspartic acid. Examples thereof include.

[0022] The amino acid sequence identity in (iii), (vi), and (ix) above may be 70% or more, and may have a higher identity (for example, 80% or more, 85% or more, 90% or more, or 95% or more).

[0023] In the present specification, the "identity" of an amino acid sequence means that the two amino acid sequences to be compared are aligned so that as many amino acid residues as possible match, and the percentage obtained by dividing the number of matching amino acid residues by the total number of amino acid residues is represented. When performing the above alignment, a gap is appropriately inserted into one or both of the two sequences to be compared as necessary. The method for aligning such sequences is not particularly limited, but can be performed using well-known sequence comparison programs such as BLAST (Basic Local Alignment Search Tool), FASTA, and CLUSTAL W. When a gap is inserted, the total number of amino acid residues described above is the number of residues counted with one gap as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the sequence identity (%) is calculated by dividing the total number of amino acid residues of the longer sequence by the number of matching amino acid residues.

[0024] The Fc-binding protein produced in the present invention may further have an oligopeptide useful for accelerating analysis and purification from a solution containing contaminants or stabilizing the protein added to the N-terminal side or C-terminal side thereof. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, C-myc tag, and the like.

[0025] Furthermore, a signal peptide for promoting efficient expression in a host may be added to the N-terminal side of the Fc-binding protein produced by the present invention, and examples of signal peptides that secrete proteins into the periplasm such as PelB, DsbA, DsbC, MalE, TorT, etc. can be cited (Japanese Patent Application Laid-Open No. 2011-097898). In particular, (I) a polynucleotide encoding a native OmpA signal peptide (region from the 1st to the 21st of UniProt No. P0A910), or (II) an oligonucleotide encoding a polypeptide in which one or several residues of the signal peptide described in (I) above have been modified (any one or more of substitution, deletion, insertion, or addition occurred), can be used for more efficient production.

[0026] In the present invention, the Fc-binding protein is expressed and produced by culturing a genetically engineered Escherichia coli containing a polynucleotide encoding the protein.

[0027] In the present invention, there is no particular limitation on the Escherichia coli (scientific name: Escherichia coli) strain, and representative examples include Escherichia coli JM109 strain, Escherichia coli W3110 strain, Escherichia coli HB101 strain, Escherichia coli MG1655 strain, Escherichia coli BL21 strain, and Escherichia coli BL21(DE3) strain. In addition, for the above-mentioned Escherichia coli, an Escherichia coli mutant strain obtained by mutagenesis treatment by conventionally known means such as chemical substances such as nitrosoguanidine and ethyl methanesulfonate, ultraviolet rays, and radiation may be used. The genetically engineered Escherichia coli only needs to contain at least a polynucleotide encoding the Fc-binding protein in a state where it can be expressed, and the polynucleotide may be present on an expression vector that replicates autonomously outside the genomic DNA. The expression vector may be any vector capable of expressing a heterologous protein in Escherichia coli, and examples include pUC plasmid vector, pCDF plasmid vector, pTrc plasmid vector, and pET plasmid vector.

[0028] The polynucleotide encoding the Fc-binding protein can be prepared, for example, (α) by converting the amino acid sequence of the Fc-binding protein into a nucleotide sequence and artificially synthesizing a polynucleotide containing the nucleotide sequence, or (β) by directly and artificially preparing a polynucleotide containing the whole or a partial sequence of the Fc-binding protein, or by using a DNA amplification method such as the PCR method from cDNA of the Fc-binding protein or the like, and ligating the prepared polynucleotide by an appropriate method. It can be prepared by the methods described above.

[0029] In the method (α) above, when converting from an amino acid sequence to a nucleotide sequence, it is preferable to perform the conversion in consideration of the codon usage frequency in the Escherichia coli to be transformed. Specifically, for arginine (Arg: R), AGA / AGG / CGG / CGA; for isoleucine (Ile: I), ATA; for leucine (Leu: L), CTA; for glycine (Gly: G), GGA; and for proline (Pro: P), CCC are codons with low usage frequencies (so-called rare codons), and thus the conversion should be performed to avoid these codons.

[0030] The present invention relates to a medium for culturing a recombinant Escherichia coli containing a polynucleotide encoding an Fc-binding protein, which is a medium containing at least peptone and yeast extract, and characterized in that the peptone is derived from any one of soybean, cottonseed, broad bean, lupin bean, corn, potato, pea and barley.

[0031] Media containing at least peptone and yeast extract include, for example, TB (Terrific Broth) medium (peptone: 12 g / L, yeast extract: 24 g / L, glycerol: 10 g / L, dipotassium hydrogen phosphate: 9.4 g / L, potassium dihydrogen phosphate: 2.2 g / L), 2×YT medium (peptone: 16 g / L, yeast extract: 10 g / L, sodium chloride: 5 g / L), LB-Miller medium (peptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L), and SOB medium (peptone: 20 g / L, yeast extract: 5 g / L, sodium chloride: 0.5 g / L, potassium chloride: 0.186 g / L, magnesium sulfate: 2.4 g / L).

[0032] Soybean-derived peptones include, for example, Phytone Supplement, UF (TF: Thermo Fisher Scientific), Phytone Peptone (TF), Soytone (TF), Soy 100 (TF), Proyield Soy SE50MK (FrieslandCampina), Proyield Soy SE70M-UF (FrieslandCampina), and Hypopeptone NS (Shioya MS).

[0033] Cotton-derived peptones include, for example, Cotton 100, UF (TF), Cotton 200, UF (TF), and Cotton Seed Powder (HiMedia).

[0034] Broad bean-derived peptones include, for example, Broadbean peptone (Solabia) and broad bean peptone (Merck Millipore).

[0035] Lupin bean-derived peptones include, for example, Lupin peptone (Solabia).

[0036] Corn-derived peptones include, for example, Solulys 095E (Roquette).

[0037] Potato peptone (Solabia) is an example of a peptone derived from potato.

[0038] Examples of peptones derived from peas include Pea peptone (Solabia), Nutralys F85F (Roquette), Proyield Pea PCE80B (FrieslandCampina), and Vegetable peptone No.1 (Oxoid).

[0039] An example of a peptone derived from barley is Malt Extract (TF).

[0040] Peptone is preferably added to the medium at 80 g / L or less, more preferably 50 g / L or less, still more preferably 5 g / L or more and 35 g / L or less, and even more preferably 8 g / L or more and 25 g / L or less. Yeast extract is preferably added to the medium at 80 g / L or less, more preferably 50 g / L or less, still more preferably 3 g / L or more and 35 g / L or less.

[0041] The above medium may further contain saccharides and phosphates.

[0042] Examples of saccharides include glycerol, glucose, galactose, fructose, maltose, lactose, sucrose, and trehalose.

[0043] Examples of phosphates include dipotassium hydrogen phosphate and potassium dihydrogen phosphate.

[0044] In the present invention, the culturing method of the genetically engineered Escherichia coli is not particularly limited, and it may be cultured by any of batch culture, semi-batch culture (also referred to as fed-batch culture), and perfusion culture, or a combination thereof. However, if nutrient sources such as a carbon source and a nitrogen source are added to the medium all at once at the start of the culture, the growth of Escherichia coli and the expression of the Fc-binding protein by the Escherichia coli are inhibited, and by-products such as organic acids are also produced, which may have an adverse effect on the expression efficiency of the protein and the quality of the obtained protein. Therefore, it is preferable to culture the genetically engineered Escherichia coli by fed-batch culture in which the nutrient source added at the start of the culture is minimized and the nutrient source is appropriately supplied (fed) during the culture.

[0045] In the present invention, the culturing conditions of the genetically engineered Escherichia coli are not particularly limited as long as Escherichia coli can grow and express the Fc-binding protein. However, the culturing temperature is preferably 15°C or higher and 50°C or lower, and particularly preferably 20°C or higher and 33°C or lower. The pH is preferably 6 or higher and 8 or lower. The culturing time can be arbitrarily set, but is usually set to several hours or more and 100 hours or less.

[0046] When the genetically engineered Escherichia coli contains an inducible promoter and expresses an Fc-binding protein under the control of the promoter, it is preferable to induce the expression so that the protein can be expressed well. As an example of the inducible promoter, there are the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter, which are promoters that function in Escherichia coli. For expression induction, for example, an inducer corresponding to the type of promoter can be used. As the inducer, IPTG (Isopropyl-β-D-thiogalactopyranoside) can be exemplified. Specifically, when the turbidity of the culture solution (absorbance at 600 nm) becomes 0.03 or more and 2 or less, an appropriate amount of IPTG is added, and the culture is continued, whereby the expression of the protein of the present invention can be induced. The added concentration of IPTG is, for example, 0.005 mmol / L or more and 1.0 mmol / L or less, preferably 0.01 mmol / L or more and 0.5 mmol / L or less, at the final concentration. When the absorbance at 600 nm of the culture solution at the time of IPTG addition is 0.03 or more and 2 or less, preferably 0.05 or more and 1 or less, it is preferable in terms of enabling good expression of the Fc-binding protein.

[0047] To recover the Fc-binding protein expressed by the method described above, it may be separated / purified from the culture by a method suitable for the expression form of the protein in the genetically engineered Escherichia coli to recover the protein. For example, when it is expressed in the culture supernatant, the cells are separated by a centrifugation operation, and the Fc-binding protein is purified from the obtained culture supernatant. When it is expressed intracellularly (including the periplasm), after collecting the cells by a centrifugation operation, the cells are disrupted by adding an enzyme treatment agent, a surfactant, or the like, and after extracting the Fc-binding protein, it may be purified.

[0048] To purify the recovered Fc-binding protein, methods known in the art can be used. As an example, separation / purification using liquid chromatography can be mentioned. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc. By performing a purification operation by combining these chromatographies, the protein can be prepared with high purity.

[0049] As described above, it is advisable to calculate the production amount of the Fc-binding protein by measuring the absorbance of the purified Fc-binding protein solution at a wavelength of 280 nm.

Example

[0050] Hereinafter, the present invention will be described more specifically using examples and comparative examples, but the present invention is not limited to these examples.

[0051] Example 1 Examination of peptone constituting TB medium The influence of the difference in peptone, which is a constituent component of TB (Terrific Broth) medium, on the production amount of Fc-binding protein was evaluated.

[0052] (1) The Escherichia coli W3110 strain was transformed with an expression vector containing a polynucleotide (SEQ ID NO: 2) encoding the Fc-binding protein FcRn_m7GS consisting of the amino acid sequence set forth in SEQ ID NO: 1 and an inducible promoter to produce a recombinant Escherichia coli capable of expressing the protein (hereinafter also referred to as FcRn_m7GS-expressing Escherichia coli). FcRn_m7GS (SEQ ID NO: 1) consists of, from the N-terminal side, the β2-microglobulin region of the native human neonatal Fc receptor (human FcRn) β chain (amino acid residues 21 to 119 of SEQ ID NO: 4 (UniProt No. P61769)), a GS linker (a polypeptide in which five oligopeptides each consisting of 4 glycine (G) residues and 1 serine (S) residue are linked), and the extracellular region of the native human FcRn α chain (amino acid residues 24 to 297 of SEQ ID NO: 3 (UniProt No. P55899)) in this order. The following amino acid substitutions occur in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, and an OmpA signal peptide (amino acid residues 1 to 21 of UniProt No. P0A910) is added to the N-terminal side of the polypeptide, and a tag consisting of 6 histidine (H) residues and a cysteine tag consisting of the amino acid sequence set forth in SEQ ID NO: 6 are added to the C-terminal side, respectively. (i) Cysteine (C) at position 172 of SEQ ID NO: 5 (corresponding to position 193 in SEQ ID NO: 1 and position 71 in SEQ ID NO: 3, respectively) is substituted with arginine (R) (ii) Asparagine (N) at position 179 of SEQ ID NO: 5 (corresponding to position 200 in SEQ ID NO: 1 and position 78 in SEQ ID NO: 3, respectively) is substituted with aspartic acid (D) (iii) Arginine (R) at position 293 of SEQ ID NO: 5 (corresponding to position 314 in SEQ ID NO: 1 and position 192 in SEQ ID NO: 3, respectively) is substituted with leucine (L) (iv) Asparagine (N) at position 297 of SEQ ID NO: 5 (corresponding to position 318 in SEQ ID NO: 1 and position 196 in SEQ ID NO: 3, respectively) is substituted with aspartic acid (D) (v) The glutamine (Q) at the 333rd position of SEQ ID NO: 5 (corresponding to the 354th position in SEQ ID NO: 1 and the 232nd position in SEQ ID NO: 3, respectively) is replaced with leucine (L). (vi) The cysteine (C) at the 375th position of SEQ ID NO: 5 (corresponding to the 396th position in SEQ ID NO: 1 and the 274th position in SEQ ID NO: 3, respectively) is replaced with serine (S). (vii) The lysine (K) at the 396th position of SEQ ID NO: 5 (corresponding to the 417th position in SEQ ID NO: 1 and the 295th position in SEQ ID NO: 3, respectively) is replaced with glutamic acid (E). (2) The E. coli expressing FcRn_m7GS was inoculated into 2×YT medium (Phytone Peptone (Thermo Fisher Scientific, hereinafter abbreviated as TF): 16 g / L, yeast extract: 10 g / L, sodium chloride: 5 g / L, kanamycin sulfate: 50 mg / L), and pre-cultured at 30 °C and 130 rpm for 16 hours.

[0053] (3) The pre-culture solution of (2) was added to TB medium (any one of the peptones shown in Table 1: 12 g / L, yeast extract: 24 g / L, glycerol: 10 g / L, dipotassium hydrogen phosphate: 9.4 g / L, potassium dihydrogen phosphate: 2.2 g / L) so that the final OD600nm was 0.02.

[0054]

Table 1

[0055] (4) After culturing at 30 °C and 130 rpm for 4 hours, IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 0.05 mmol / L, and the culture was further continued at 25 °C and 130 rpm for 24 hours. After the completion of the culture, the cells in 100 mL of the culture solution were collected by centrifugation to obtain a pellet.

[0056] (5) Add 14 mL of the buffer for cell disruption (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 3 kU / L Benzonase Nuclease, 60 mg / L lysozyme, 6 g / L Triton X-100 (trade name), pH 10) to the cell pellets obtained in (4), and shake at 25 °C and 150 rpm for 2 hours.

[0057] (6) After centrifuging the solution obtained in (5) at 15000 rpm for 20 minutes, the supernatant was sterile filtered through a PVDF (polyvinylidene fluoride) membrane with a pore size of 0.22 μm and recovered as a cell extract.

[0058] (7) The cell extract obtained in (6) was purified by the Ni-NTA (Nickel-nitrilotriacetic acid) affinity chromatography method shown below. (7-1) A chromatographic empty column was filled with Ni-NTA agarose (FUJIFILM Wako Pure Chemical Corporation, product number: 141-09764) to prepare a Ni-NTA column. (7-2) Five times the volume of equilibration buffer A (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, pH 10) of Ni-NTA agarose was passed through the prepared Ni-NTA column for equilibration. (7-3) The cell extract recovered in (6) was passed through the equilibrated Ni-NTA column to adsorb FcRn_m7GS to Ni-NTA agarose. (7-4) Five times the volume of equilibration buffer A of Ni-NTA agarose was passed through the Ni-NTA column after passing the cell extract to wash the unadsorbed proteins. (7-5) After washing, a buffer solution A for elution (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, 300 mmol / L imidazole, pH 10) four times the amount of Ni-NTA agarose was passed through the Ni-NTA column, and the eluate was collected as the Ni-NTA affinity purification eluate.

[0059] (8) The Ni-NTA affinity purification eluate recovered in (7) was purified by the IgG affinity chromatography method shown below. (8-1) 2 mol / L phosphoric acid was added to the Ni-NTA affinity purification eluate recovered in (7) and adjusted to pH 6.5. (8-2) An empty column for chromatography was filled with IgG Sepharose (Cytiva, product number: 17096901) to prepare an IgG column. (8-3) A buffer solution B for equilibration (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, pH 6.5) five times the amount of IgG Sepharose was passed through the prepared IgG column for equilibration. (8-4) The Ni-NTA affinity purification eluate adjusted to pH 6.5 in (8-1) was passed through the equilibrated IgG column to adsorb FcRn_m7GS to IgG Sepharose. (8-5) A buffer solution B for equilibration ten times the amount of IgG Sepharose was passed through the IgG column after passing the eluate to wash the unadsorbed proteins. (8-6) A buffer solution B for elution (50 mmol / L 1,3-bis[tris(hydroxymethyl)methylamino]propane, 150 mmol / L sodium chloride, pH 8.5) six times the amount of IgG Sepharose was passed through the washed IgG column, and the eluate was collected as the IgG affinity purification eluate. (9) The absorbance at a wavelength of 280 nm of the IgG affinity purification eluate recovered in (8) was measured to calculate the production amount of FcRn_m7GS.

[0060] Comparative Example 1 In Example 1(3), FcRn_m7GS-producing Escherichia coli was cultured using the peptone derived from animals shown in Table 2 (the peptone shown as "MC-1" or "MC-2" in Table 2), and the same operations as in Example 1 were performed to calculate the production amount of FcRn_m7GS.

[0061] [Table 2]

[0062] Comparative Example 2 In Example 1(3), FcRn_m7GS-producing Escherichia coli was cultured without adding peptone (shown as "N" in Table 2), and the same operations as in Example 1 were performed to calculate the production amount of FcRn_m7GS.

[0063] The results of Example 1 and Comparative Examples 1 and 2 are collectively shown in FIG. 1. The production amount of FcRn (FcRn_m7GS) is shown as a relative value (ratio) to the production amount of FcRn when using peptone derived from milk casein (tryptone from Nacalai Tesque, shown as "MC-1" in Table 2).

[0064] When using peptones derived from soybean (peptones shown as "S-1" to "S-7" in Table 1), cotton (peptones shown as "CT-1" to "CT-3" in Table 1), broad bean (peptone shown as "B" in Table 1), lupin bean (peptone shown as "L" in Table 1), corn (peptone shown as "CR" in Table 1), potato (peptone shown as "PT" in Table 1), vegetable (peptone shown as "V" in Table 1), pea (peptones shown as "PA-1" to "PA-3" in Table 1) and barley (peptone shown as "MA" in Table 1), in each case, compared with when using peptone derived from milk casein (Comparative Example 1), the production amount of FcRn was improved. In particular, when using peptones derived from soybean, cotton, broad bean, lupin bean, corn and potato, in each case, compared with when using peptone derived from milk casein (Comparative Example 1), the production amount of FcRn was significantly improved (specifically, 2.5 times or more).

[0065] On the other hand, when using peptone derived from wheat (peptone shown as "W-1" to "W-3" in Table 1), there were cases where the FcRn production amount was equal to or lower than that when using peptone derived from milk casein.

[0066] From the above results, when producing an Fc-binding protein by culturing a recombinant Escherichia coli containing a polynucleotide encoding the Fc-binding protein in a medium containing at least peptone and yeast extract and expressing the protein, as the peptone, by using peptone derived from any of soybean, cottonseed, broad bean, lupin bean, corn, potato, pea and barley, it can be seen that compared with the conventional production method using animal-derived peptone, the production amount of the Fc-binding protein is significantly improved and more efficient production can be achieved.

[0067] The FcRn production amount when no peptone was added ( "N" in Table 2, Comparative Example 2) was about 70% of that when using peptone derived from milk casein (Comparative Example 1).

[0068] Example 2 Examination of peptone constituting 2×YT medium, LB medium and SOB medium It was verified whether the same effect as that of TB medium (Example 1) can be obtained even when using a medium containing at least peptone and yeast extract other than TB medium. Specifically, in Example 1(3), except that FcRn_m7GS-expressing Escherichia coli was cultured using any of the media shown in Table 3, the same operations as in Example 1 were performed, and the FcRn_m7GS production amount was calculated.

[0069]

Table 3

[0070] Comparative Example 3 In Example 1(3), except that FcRn_m7GS-expressing Escherichia coli was cultured using any of the media shown in Table 4, the same operations as in Example 1 were performed, and the FcRn_m7GS production amount was calculated.

[0071]

Table 4

[0072] The results of Example 2 and Comparative Example 3 are summarized and shown in FIG. 2. Note that the FcRn (FcRn_m7GS) production amount is shown as a relative value (ratio) to the FcRn production amount (Comparative Example 3) when tryptone is used as the peptone. As the medium containing at least peptone and yeast extract, regardless of whether 2×YT medium, LB-Miller medium, or SOB medium is used, the production amount of the Fc-binding protein is improved when soy-derived peptone is used as the peptone (Example 2) compared to when animal-derived peptone is used (Comparative Example 3).

[0073] Examination of Peptone Constituting TB Medium in Example 3 (Part 2) Using Escherichia coli capable of expressing an Fc-binding protein, which is different from the genetically engineered Escherichia coli used in the previous examples (Examples 1 and 2 and Comparative Examples 1 to 3), the effect of the difference in peptone, which is a component of TB medium, on the production amount of the Fc-binding protein was evaluated.

[0074] (1) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7, FcRn_m10a, which is an Fc-binding protein, was used to transform Escherichia coli BL21(DE3) with an expression vector containing a polynucleotide (SEQ ID NO: 8) encoding the protein and an inducible promoter, thereby producing a recombinant Escherichia coli capable of expressing the protein (hereinafter also referred to as FcRn_m10a-expressing Escherichia coli). FcRn_m10a (SEQ ID NO: 7) consists of, from the N-terminal side, the β2-microglobulin region of the natural human neonatal Fc receptor (human FcRn) β-chain (amino acid residues 21 to 119 of SEQ ID NO: 4 (UniProt No. P61769)), a GS linker (a polypeptide in which five oligopeptides each consisting of 4 glycine (G) residues and 1 serine (S) residue are linked), and the extracellular region of the natural human FcRn α-chain (amino acid residues 24 to 297 of SEQ ID NO: 3 (UniProt No. P55899)), in this order. The following amino acid substitutions (i) to (x) occurred in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, and an OmpA signal peptide (amino acid residues 1 to 21 of UniProt No. P0A910) was added to the N-terminal side of the polypeptide, and a cysteine tag consisting of the amino acid sequence set forth in SEQ ID NO: 6 was added to the C-terminal side. (i) The cysteine (C) at position 172 of SEQ ID NO: 5 (corresponding to position 193 in SEQ ID NO: 7 and position 71 in SEQ ID NO: 3, respectively) was substituted with arginine (R). (ii) The asparagine (N) at position 179 of SEQ ID NO: 5 (corresponding to position 200 in SEQ ID NO: 7 and position 78 in SEQ ID NO: 3, respectively) was substituted with aspartic acid (D). (iii) The valine (V) at position 181 of SEQ ID NO: 5 (corresponding to position 202 in SEQ ID NO: 7 and position 80 in SEQ ID NO: 3, respectively) was substituted with aspartic acid (D). (iv) The lysine (K) at position 197 of SEQ ID NO: 5 (corresponding to position 218 in SEQ ID NO: 7 and position 96 in SEQ ID NO: 3, respectively) was substituted with glutamic acid (E). (v) The asparagine (N) at position 273 of SEQ ID NO: 5 (corresponding to position 294 in SEQ ID NO: 7 and position 172 in SEQ ID NO: 3, respectively) was substituted with aspartic acid (D). (vi) The arginine (R) at the 293rd position of SEQ ID NO: 5 (corresponding to the 314th position in SEQ ID NO: 7 and the 192nd position in SEQ ID NO: 3, respectively) is substituted with leucine (L). (vii) The asparagine (N) at the 297th position of SEQ ID NO: 5 (corresponding to the 318th position in SEQ ID NO: 7 and the 196th position in SEQ ID NO: 3, respectively) is substituted with aspartic acid (D). (viii) The glutamine (Q) at the 333rd position of SEQ ID NO: 5 (corresponding to the 354th position in SEQ ID NO: 7 and the 232nd position in SEQ ID NO: 3, respectively) is substituted with leucine (L). (ix) The cysteine (C) at the 375th position of SEQ ID NO: 5 (corresponding to the 396th position in SEQ ID NO: 7 and the 274th position in SEQ ID NO: 3, respectively) is substituted with serine (S). (x) The lysine (K) at the 396th position of SEQ ID NO: 5 (corresponding to the 417th position in SEQ ID NO: 7 and the 295th position in SEQ ID NO: 3, respectively) is substituted with glutamic acid (E). (2) The Escherichia coli expressing FcRn_m10a was inoculated into 2×YT medium (Phytone Peptone (TF): 16 g / L, yeast extract: 10 g / L, sodium chloride: 5 g / L, kanamycin sulfate: 50 mg / L), and pre-cultured at 30 °C and 130 rpm for 16 hours.

[0075] (3) The pre-culture solution of (2) was added to TB medium (any one of the peptones shown in Table 5: 12 g / L, yeast extract: 24 g / L, glycerol: 10 g / L, dipotassium hydrogen phosphate: 9.4 g / L, potassium dihydrogen phosphate: 2.2 g / L, kanamycin sulfate: 50 mg / L) so that the final OD600nm was 0.02.

[0076]

Table 5

[0077] (4) After culturing at 30 °C and 130 rpm for 4 hours, IPTG was added to a final concentration of 0.05 mmol / L, and further cultured at 25 °C and 130 rpm for 24 hours. After the culture was completed, the cells in 100 mL of the culture solution were collected by centrifugation to obtain a pellet.

[0078] (5) From the cell pellets obtained in (4), a cell extract was obtained by the method described in Example 1 (5) and (6).

[0079] (6) To the cell extract obtained in (5), 1 mol / L phosphoric acid was added and adjusted to pH 6.0, and then purification by IgG affinity chromatography was performed by the same method as described in Example 1 (8-2) to (8-6).

[0080] (7) The absorbance at a wavelength of 280 nm of the IgG affinity-purified eluate recovered in (6) was measured, and the production amount of FcRn_m10a was calculated.

[0081] Comparative Example 4 In Example 3 (3), Escherichia coli expressing FcRn_m10a was cultured using the peptone derived from animals shown in Table 6 (the peptone shown as "MC-1" in Table 6), and the same operations as in Example 3 were performed to calculate the production amount of FcRn_m10a.

[0082]

Table 6

[0083] The results of Example 3 and Comparative Example 4 are summarized and shown in Figure 3. The production amount of FcRn (FcRn_m10a) is shown as a relative value (ratio) to the production amount of FcRn when using peptone derived from milk casein (tryptone from Nacalai Tesque, "MC-1" in Table 5). Even when the Fc-binding protein expressed in recombinant Escherichia coli was changed from FcRn_m7GS (SEQ ID NO: 1) to FcRn_m10a (SEQ ID NO: 7), similar to the case of FcRn_m7GS (Example 1 and Comparative Examples 1 and 2), when using peptones derived from soybean (peptones shown as "S-2" and "S-7" in Table 5), cotton (peptones shown as "CT-1" and "CT-2" in Table 5), and broad bean (peptone shown as "B" in Table 5), in each case, compared with the case of using peptone derived from milk casein (Comparative Example 4), the production amount of FcRn was improved.

Claims

1. A method for producing an Fc-binding protein, comprising the steps of: culturing a recombinant Escherichia coli containing a polynucleotide encoding an Fc-binding protein to express the protein; and recovering the protein, The method for producing a recombinant Escherichia coli, wherein the medium used for culturing the recombinant Escherichia coli contains at least peptone and yeast extract, and the peptone is derived from any one of soybean, cotton, broad bean, lupin bean, corn, potato, pea and barley.

2. The method according to claim 1, wherein the medium used for culturing the recombinant E. coli further contains sugars and phosphates.

3. The method according to claim 1 or 2, wherein the Fc binding protein is a polypeptide selected from any one of the following (i) to (vi): (i) a polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 4 (ii) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 4, with the proviso that any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in these amino acid residues occurs, and has antibody binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 4, wherein the amino acid sequence has an identity of 70% or more to the entire amino acid sequence from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 3 and the entire amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 4, and having antibody-binding activity. (iv) an Fc binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, with the proviso that at least one of the amino acid substitutions shown in (1) to (7) below has occurred in said amino acid residues; (1) Substitution of arginine for cysteine ​​at position 71 of SEQ ID NO:3 (2) Substitution of asparagine at position 78 of SEQ ID NO:3 with aspartic acid (3) Substitution of arginine at position 192 of SEQ ID NO:3 with leucine (4) Substitution of asparagine at position 196 of SEQ ID NO:3 with aspartic acid (5) Substitution of glutamine at position 232 of SEQ ID NO:3 with leucine (6) Substitution of cysteine ​​at position 274 of SEQ ID NO:3 with serine (7) Lysine at position 295 of SEQ ID NO:3 is substituted with glutamic acid (v) An Fc binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, with the proviso that the amino acid residues have the amino acid substitutions shown in (1) to (7) above, and further having any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions other than the amino acid substitutions shown in (1) to (7) above, and having antibody binding activity. (vi) An Fc binding protein having antibody binding activity, comprising an amino acid sequence having an identity of 70% or more to the entire amino acid sequence in which the amino acid substitutions shown in (1) to (7) have occurred in the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence set forth in SEQ ID NO: 4, and in which the amino acid substitutions shown in (1) to (7) remain. (vii) an Fc binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, with the proviso that at least one of the amino acid substitutions shown in (1) to (10) below has occurred in said amino acid residues; (1) Substitution of arginine for cysteine ​​at position 71 of SEQ ID NO:3 (2) Substitution of asparagine at position 78 of SEQ ID NO:3 with aspartic acid (3) Substitution of arginine at position 192 of SEQ ID NO:3 with leucine (4) Substitution of asparagine at position 196 of SEQ ID NO:3 with aspartic acid (5) Substitution of glutamine at position 232 of SEQ ID NO:3 with leucine (6) Substitution of cysteine ​​at position 274 of SEQ ID NO:3 with serine (7) Lysine at position 295 of SEQ ID NO:3 is substituted with glutamic acid (8) Valine at position 80 of SEQ ID NO:3 is substituted with aspartic acid (9) Lysine at position 96 of SEQ ID NO:3 is substituted with glutamic acid (10) Asparagine at position 172 of SEQ ID NO: 3 is replaced with aspartic acid. (viii) An Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 3 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 4, with the proviso that the amino acid residues have the amino acid substitutions shown in (1) to (10) above, and further having any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions other than the amino acid substitutions shown in (1) to (10) above, and having antibody-binding activity. (ix) An Fc binding protein having antibody binding activity, comprising an amino acid sequence having an identity of 70% or more to the entire amino acid sequence in which the amino acid substitutions shown in (1) to (10) have occurred in the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence set forth in SEQ ID NO: 4, wherein the amino acid sequence has an amino acid sequence in which the amino acid substitutions shown in (1) to (10) remain.

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