JP7587200B2 - Immunoglobulin-binding polypeptides - Google Patents

Description

The present invention relates to a polypeptide that specifically binds to immunoglobulin. More specifically, the present invention relates to said polypeptide that has an excellent immunoglobulin adsorption capacity.

Antibody drugs are medicines that utilize antibodies (immunoglobulins), molecules that are responsible for the immune function in the body. Antibody drugs bind to target molecules with high specificity and affinity due to the diversity of the variable regions of antibodies. As a result, antibody drugs have few side effects, and in recent years, the range of diseases for which they can be used has been expanding, leading to a rapid expansion of the market.

The manufacture of antibody drugs involves a culture process and a purification process. In the culture process, efforts are made to improve productivity by modifying the antibody-producing cells and optimizing the culture conditions. In addition, affinity chromatography is used for crude purification in the purification process, and the drug is formulated after going through intermediate purification, final purification, and virus removal.

In the purification process, an affinity adsorbent is also used, which contains an insoluble carrier and a protein (ligand) that specifically recognizes the antibody molecules immobilized on the insoluble carrier. Protein A (hereinafter abbreviated as SpA) derived from bacteria of the genus Staphylococcus and Protein G derived from bacteria of the genus Streptococcus are often used as the ligand.

When manufacturing antibody drugs, these affinity carriers are used multiple times to keep production costs low, and after use, a process is carried out to remove impurities remaining on the carrier. The process of removing the impurities usually involves stationary washing with sodium hydroxide to regenerate the affinity carrier. Therefore, the ligand protein must be chemically stable enough to maintain its binding to the antibody even after the regeneration process.

Examples of chemically stable ligand proteins used in affinity carriers include an alkali-stable chromatography ligand that uses the amino acid sequence of domain C of SpA (Patent Document 1) and an affinity chromatography ligand that is composed of an amino acid sequence in which a portion of the amino acid sequence of any of domains B, C, and Z of SpA is deleted (Patent Document 2). It is also known that chemical stability is improved by substituting lysine with another amino acid in domain C of SpA (Patent Document 3). It is also known that the structure is stabilized by substituting the 29th glycine in domain Z with alanine (Non-Patent Document 1).

On the other hand, from the viewpoint of improving the productivity of antibody drugs, there is also a demand for an increase in the amount of antibody adsorbed per affinity adsorbent. As ligands with improved antibody adsorption, a polypeptide in which immunoglobulin-binding domains are linked in tandem (Non-Patent Document 2) and a polypeptide in which two or more immunoglobulin-binding domains are linked via a linker (Patent Document 4) have been disclosed. Thus, there is a demand for immunoglobulin-binding polypeptides with improved antibody adsorption.

Special Publication No. 2010-504754 JP 2012-254981 A WO2012/133342 WO2015/034000

Bjorn Nilsson et al., Protein Engineering, 1(2), 107-113, 1987 Freiherr von Roman M et al., Journal of chromatography A, (1347), 80-86, 2014

The object of the present invention is to provide an immunoglobulin-binding polypeptide that has immunoglobulin-binding activity and has an improved amount of immunoglobulin adsorption, and an immunoglobulin adsorbent that includes an insoluble carrier and the polypeptide immobilized on the insoluble carrier.

As a result of intensive research to solve the above problems, the present inventors discovered that the amount of immunoglobulin adsorption can be improved by replacing at least the amino acid residue corresponding to the 50th lysine in domain C (domain consisting of the amino acid sequence set forth in SEQ ID NO: 1) of Staphylococcus aureus-derived SpA in the immunoglobulin-binding domain of Staphylococcus genus Protein A (SpA) with tryptophan, and thus completed the present invention.

That is, the present invention includes the following aspects [1] to [13].

[1] An immunoglobulin-binding polypeptide comprising a modified immunoglobulin-binding domain of Protein A derived from a bacterium of the genus Staphylococcus, in which the modification of the immunoglobulin-binding domain comprises substituting the amino acid residue corresponding to the lysine at position 50 of SEQ ID NO:1 with tryptophan.

[2] The immunoglobulin-binding polypeptide according to [1], wherein the modified immunoglobulin-binding domain has an amino acid sequence selected from the group consisting of (a) to (c) below:
(a) an amino acid sequence in which the 50th lysine in the amino acid sequence of SEQ ID NO: 1 has been replaced with tryptophan; (b) an amino acid sequence in which the 50th lysine in the amino acid sequence of SEQ ID NO: 1 has been replaced with tryptophan, and further in which one or more amino acid residues have been substituted, deleted, inserted, and/or added at one or more positions other than the 50th; (c) an amino acid sequence having 70% or more homology to the amino acid sequence of SEQ ID NO: 1 or a partial sequence thereof, in which the amino acid residue corresponding to the 50th lysine in SEQ ID NO: 1 has been replaced with tryptophan.

[3] The modified immunoglobulin binding domain further comprises:
The immunoglobulin-binding polypeptide according to [1] or [2], which comprises a substitution of the amino acid residue corresponding to the 35th lysine of SEQ ID NO: 1 with arginine, and a substitution of the amino acid residue corresponding to the 49th lysine of SEQ ID NO: 1 with methionine.

[4] The modified immunoglobulin binding domain further comprises:
Substitution of the amino acid residue corresponding to lysine at position 35 of SEQ ID NO:1 with arginine;
The immunoglobulin-binding polypeptide according to [1] or [2], which comprises a substitution of the amino acid residue corresponding to the 49th lysine of SEQ ID NO: 1 with methionine, and a substitution of the amino acid residue corresponding to the 11th asparagine of SEQ ID NO: 1 with arginine.

[5] The modified immunoglobulin binding domain further comprises:
Substitution of the amino acid residue corresponding to the third asparagine in SEQ ID NO:1 with histidine;
Substitution of the amino acid residue corresponding to the fourth lysine in SEQ ID NO:1 with arginine;
Substitution of the amino acid residue corresponding to the 7th lysine of SEQ ID NO:1 with valine;
Substitution of the amino acid residue corresponding to glutamic acid at position 15 of SEQ ID NO:1 with alanine;
Substitution of the amino acid residue corresponding to asparagine 21 of SEQ ID NO:1 with tyrosine;
Substitution of the amino acid residue corresponding to glycine at position 29 of SEQ ID NO:1 with alanine;
Substitution of the amino acid residue corresponding to serine at position 39 of SEQ ID NO:1 with glycine;
Substitution of the amino acid residue corresponding to valine at position 40 of SEQ ID NO:1 with alanine;
The immunoglobulin-binding polypeptide according to any one of [1] to [4], which comprises a substitution of the amino acid residue corresponding to lysine 42 of SEQ ID NO:1 with leucine, and a substitution of the amino acid residue corresponding to lysine 58 of SEQ ID NO:1 with aspartic acid.

[6] The peptide according to [1], wherein the modified immunoglobulin-binding domain comprises the amino acid sequence set forth in SEQ ID NO:3.

[7] A polynucleotide encoding an immunoglobulin-binding polypeptide according to any one of [1] to [6].

[8] An expression vector comprising the polynucleotide described in [7].

[9] A genetically modified host comprising the polynucleotide described in [7] or the expression vector described in [8].

[10] The genetically modified host according to [9], wherein the host is Escherichia coli.

[11] A method for producing an immunoglobulin-binding polypeptide, comprising the steps of culturing a recombinant host according to [9] or [10], expressing an immunoglobulin-binding polypeptide according to any one of [1] to [6], and recovering the expressed polypeptide.

[12] An immunoglobulin adsorbent comprising an insoluble carrier and an immunoglobulin-binding polypeptide according to any one of [1] to [6] immobilized on the insoluble carrier.

[13] A method for separating immunoglobulins contained in a solution, comprising the steps of adding a solution containing immunoglobulins to a column packed with the adsorbent described in [12] to adsorb the immunoglobulins to the adsorbent, and eluting the immunoglobulins adsorbed to the adsorbent.

In the immunoglobulin-binding polypeptide of the present invention, the amount of immunoglobulin adsorption is improved by introducing at least a tryptophan substitution for the amino acid residue corresponding to the lysine at position 50 of SEQ ID NO:1. Therefore, it is useful as a ligand polypeptide constituting an adsorbent for separating antibodies (immunoglobulins).

The present invention is described in detail below.

The present invention relates to an immunoglobulin-binding polypeptide that contains a modified immunoglobulin-binding domain (hereinafter also referred to as a "modified immunoglobulin-binding domain") of Protein A derived from a bacterium of the genus Staphylococcus, and the modification of the immunoglobulin-binding domain includes substituting the amino acid residue corresponding to the 50th lysine of SEQ ID NO:1 with tryptophan.

In the present invention, "immunoglobulin binding" means binding to immunoglobulins, and is also referred to as "immunoglobulin binding activity" or "antibody binding activity." It may also mean binding to the Fc region of immunoglobulins. "Immunoglobulin-binding polypeptide" means a polypeptide that has immunoglobulin binding. Note that whether or not a given polypeptide has immunoglobulin binding can be determined by a person skilled in the art by measuring using a known method, for example, the ELISA (Enzyme-Linked Immunosorbent Assay) method.

In the present invention, the "immunoglobulin-binding domain" that is modified by introducing the amino acid substitutions (Lys50Trp, etc.) according to the present invention described below means the immunoglobulin-binding domain of Protein A (hereinafter abbreviated as SpA) derived from bacteria of the genus Staphylococcus.

Staphylococcus aureus is an example of a bacterium of the genus Staphylococcus from which SpA is derived. Examples of immunoglobulin-binding domains include domain C, domain E, domain D, domain A, and domain B/Z. Examples of immunoglobulin-binding domains include domain C in particular. Examples of domain C of Protein A derived from Staphylococcus aureus include amino acid residues 270 to 327 of GenBank No. AAA26676. The amino acid sequence of said domain C is shown in SEQ ID NO: 1. Examples of domain E of Protein A derived from Staphylococcus aureus include a polypeptide consisting of amino acid residues 37 to 92 of GenBank No. AAA26676. The amino acid sequence of said domain E is shown in SEQ ID NO: 4. Examples of domain D of Protein A derived from Staphylococcus aureus include amino acid residues 270 to 327 of GenBank No. AAA26676. An example of a domain A of Protein A derived from Staphylococcus aureus is a polypeptide consisting of amino acid residues from positions 93 to 153 of AAA26676. The amino acid sequence of said domain D is shown in SEQ ID NO: 5. An example of domain A of Protein A derived from Staphylococcus aureus is a polypeptide consisting of amino acid residues from positions 154 to 211 of GenBank No. AAA26676. The amino acid sequence of said domain A is shown in SEQ ID NO: 6. An example of domain B of Protein A derived from Staphylococcus aureus is a polypeptide consisting of amino acid residues from positions 212 to 269 of GenBank No. AAA26676. The amino acid sequence of said domain B is shown in SEQ ID NO: 7. An example of domain Z of Protein A derived from Staphylococcus aureus is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8.

The immunoglobulin-binding domain of SpA according to the present invention, which is modified by introducing Lys50Trp or the like as described below, may be a sequence found in nature, such as the specific amino acid sequence described above, or may be a variant sequence thereof.

Variant sequences include amino acid sequences that include substitutions, deletions, insertions, and/or additions of one or several amino acid residues at one or several positions in the immunoglobulin-binding domain of SpA (e.g., the amino acid sequence set forth in SEQ ID NO:1).

The term "one or several" means, for example, 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 amino acid residue, depending on the position of the amino acid residue in the three-dimensional structure of the polypeptide and the type of amino acid residue. An example of the substitution of the amino acid residue is a conservative substitution in which a substitution occurs between amino acids with similar physical and/or chemical properties. In the case of a conservative substitution, it is generally known to those skilled in the art that the function of a polypeptide is maintained between a substituted and unsubstituted polypeptide. 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). The substitutions, deletions, insertions, and/or additions of amino acid residues also include those that arise due to naturally occurring mutations (mutants or variants), for example, due to individual differences or differences in species of the microorganism from which the polypeptide or the gene encoding it is derived.

In addition, the variant sequence of the immunoglobulin-binding domain of SpA may be an amino acid sequence having high homology to the amino acid sequence of the domain (e.g., the amino acid sequence set forth in SEQ ID NO: 1) or a partial sequence thereof. The "homology" may mean similarity or identity, and may particularly mean identity. The "identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). The "similarity" between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). The amino acid residues with similar side chain properties are as described above. Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA. The term "high homology" may mean a homology of 70% or more, 80% or more, 90% or more, or 95% or more.

The immunoglobulin-binding polypeptide of the present invention is a polypeptide that has been modified by introducing a substitution of at least the amino acid residue corresponding to the 50th lysine of SEQ ID NO:1 with tryptophan in the immunoglobulin-binding domain of the above-mentioned SpA.

In the present invention, the term "the α-th amino acid of SEQ ID NO: 1" refers to an amino acid that is present at the α-position counting from the N-terminus of the amino acid sequence described in SEQ ID NO: 1. In an amino acid sequence, the term "an amino acid residue corresponding to the α-th amino acid of SEQ ID NO: 1" refers to an amino acid residue in an amino acid sequence that is arranged at the same position as the α-th amino acid in the amino acid sequence shown in SEQ ID NO: 1 in an alignment between the amino acid sequence and the amino acid sequence of SEQ ID NO: 1. The alignment can be performed, for example, using an alignment program such as BLAST or FASTA. In addition, in the present invention, "Xaa ₁ αXaa ₂ " refers to the substitution of the amino acid residue corresponding to the α-th Xaa 1 of SEQ ID NO: ₁ with Xaa _2. That is, "substitution of the amino acid residue corresponding to the 50th lysine of SEQ ID NO: 1 with tryptophan" is also expressed as Lys50Trp.

The immunoglobulin-binding polypeptide of the present invention may further have other amino acid substitutions other than Lys50Trp in the modified immunoglobulin-binding domain (Lys50Trp and other amino acid substitutions are also collectively referred to as "amino acid substitutions according to the present invention" or "Lys50Trp, etc."). Examples of other amino acid substitutions include any one or more of Asn11Arg, Lys35Arg, and Lys49Met. Furthermore, any one or more amino acid substitutions selected from the group consisting of Asn3His, Lys4Arg, Lys7Val, Glu15Ala, Asn21Tyr, Ser39Gly, Val40Ala, Lys42Leu, and Lys58Asp, and/or the amino acid substitution Gly29Ala, which is known to increase structural stability (Bjorn Nilsson et al., Protein Engineering, 1(2), 107-113, 1987), may be introduced into the modified immunoglobulin-binding domain as the other amino acid substitutions. Other amino acid substitutions include one or more of Asp2Glu, Asp3Ile, Asp3Thr, Asp4Thr, Asn11Tyr, Ser39Cys, Glu47Val, Lys58Glu, Lys58Val, and Lys58Asn.

When the immunoglobulin-binding polypeptide of the present invention has two or more amino acid substitutions other than Lys50Trp (such as Asn11Arg), the combination of the other amino acid substitutions is not particularly limited. Specific examples of the combination of the other amino acid substitutions include:
Amino acid substitutions of Lys35Arg and Lys49Met,
the amino acid substitutions Asn11Arg, Lys35Arg and Lys49Met;
the amino acid substitutions Asn3His, Lys4Arg, Lys7Val, Asn11Arg, Glu15Ala, Asn21Tyr, Gly29Ala, Lys35Arg, Ser39Gly, Val40Ala, Lys42Leu, Lys49Met and Lys58Asp;
Examples include:

More specifically, the immunoglobulin-binding polypeptide of the present invention includes a modified immunoglobulin-binding domain (a modified immunoglobulin-binding domain including the amino acid sequence set forth in SEQ ID NO:3) that contains the amino acid sequence set forth in SEQ ID NO:1 and that has the following amino acid substitutions introduced into the amino acid sequence: Asn3His, Lys4Arg, Lys7Val, Asn11Arg, Glu15Ala, Asn21Tyr, Gly29Ala, Lys35Arg, Ser39Gly, Val40Ala, Lys42Leu, Lys49Met, Lys50Trp, and Lys58Asp.

The amino acid sequence described in SEQ ID NO:3 is a sequence in which the amino acid substitutions Asn3His, Lys4Arg, Lys7Val, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, Ser39Gly, Val40Ala, Lys42Leu, and Lys58Asp have been introduced into the amino acid sequence described in SEQ ID NO:1 (the amino acid sequence described in SEQ ID NO:2), and further amino acid substitutions Asn11Arg, Lys35Arg, Lys49Met, and Lys50Trp have been introduced.

The immunoglobulin-binding polypeptide of the present invention may contain only one of the modified immunoglobulin-binding domains described above, or may contain multiple modified immunoglobulin-binding domains. The immunoglobulin-binding polypeptide of the present invention may contain, for example, 2 or more, 3 or more, 4 or more, or 5 or more modified immunoglobulin-binding domains, or 10 or less, 7 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or any compatible combination thereof. When the immunoglobulin-binding polypeptide of the present invention contains multiple modified immunoglobulin-binding domains, the amino acid sequences of the multiple modified immunoglobulin-binding domains may or may not be identical. The multiple modified immunoglobulin-binding domains may be linked to each other, for example, via a suitable linker. There are no particular limitations on the sequence and length of such linkers, but examples include linkers formed by linking multiple repeating units such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Gly, and Gly-Gly-Gly-Gly-Ser. The most common linker is a linker formed by repeating Gly-Gly-Gly-Gly-Ser three times.

The immunoglobulin-binding polypeptide of the present invention may contain, in addition to a modified immunoglobulin-binding domain derived from a selected immunoglobulin-binding domain, a portion of another immunoglobulin-binding domain and/or a portion of a region other than the immunoglobulin-binding domain of SpA. For example, when the immunoglobulin-binding polypeptide of the present invention contains a modified immunoglobulin-binding domain derived from domain C, it may further contain a portion of the N-terminal region of domain C of SpA (domain E, domain D, domain A, domain B/Z), or may contain a portion of the C-terminal region of domain C of SpA.

The immunoglobulin-binding polypeptide of the present invention may consist of only the modified immunoglobulin-binding domain, or may further contain other amino acid sequences (e.g., oligopeptides). For example, in the immunoglobulin-binding polypeptide of the present invention, other amino acid sequences may be added to at least one of the N-terminus and C-terminus of the modified immunoglobulin-binding domain.

The other amino acid sequences are not particularly limited, for example, in terms of type, length, etc., as long as they do not impair the immunoglobulin-binding ability or stability of the immunoglobulin-binding polypeptide of the present invention. More specifically, for the purpose of specifically detecting or separating a target substance, a polypeptide such as polyhistidine or polyarginine may be added directly or indirectly to the end of the modified immunoglobulin-binding domain. In addition, for the purpose of immobilizing the immunoglobulin-binding polypeptide of the present invention on a solid phase such as a support for chromatography, an oligopeptide containing lysine or cysteine may be added directly or indirectly to the end of the modified immunoglobulin-binding domain.

When the immunoglobulin-binding polypeptide of the present invention contains the other amino acid sequence as described above, the immunoglobulin-binding polypeptide of the present invention may be produced, for example, in a form that contains the other amino acid sequence in advance, or the other amino acid sequence produced separately may be added. When the immunoglobulin-binding polypeptide of the present invention contains the other amino acid sequence as described above, typically, the immunoglobulin-binding polypeptide of the present invention can be produced by expressing it from a polynucleotide that encodes the full-length amino acid sequence of the immunoglobulin-binding polypeptide of the present invention that contains the other amino acid sequence as described above. That is, for example, a polynucleotide that encodes the other amino acid sequence and a polynucleotide that encodes the immunoglobulin-binding polypeptide of the present invention (e.g., one that does not contain the other amino acid sequence) may be linked so that the other amino acid sequence is added to the N-terminus or C-terminus of the immunoglobulin-binding polypeptide of the present invention, and the immunoglobulin-binding polypeptide of the present invention may be expressed. Also, for example, a chemically synthesized other amino acid sequence may be chemically bound to the N-terminus or C-terminus of the immunoglobulin-binding polypeptide of the present invention (e.g., one that does not contain the other amino acid sequence).

The immunoglobulin-binding polypeptide of the present invention can be produced, for example, by expressing a polynucleotide encoding the immunoglobulin-binding polypeptide of the present invention. In this specification, a polynucleotide encoding an immunoglobulin-binding polypeptide of the present invention is also referred to as a "polynucleotide of the present invention." Specifically, the polynucleotide of the present invention may be a polynucleotide that includes a nucleotide sequence encoding the immunoglobulin-binding polypeptide of the present invention.

The polynucleotide of the present invention can be obtained, for example, by chemical synthesis or a DNA amplification method such as PCR. The DNA amplification method can be carried out using, as a template, a polynucleotide containing a nucleotide sequence to be amplified, such as a nucleotide sequence encoding the immunoglobulin-binding polypeptide of the present invention. Examples of polynucleotides used as templates include genomic DNA of an organism expressing the immunoglobulin-binding polypeptide of the present invention, cDNA of the immunoglobulin-binding polypeptide of the present invention, and a vector containing the polynucleotide of the present invention. The nucleotide sequence of the polynucleotide of the present invention can be designed, for example, by conversion from the amino acid sequence of the immunoglobulin-binding polypeptide of the present invention. When converting an amino acid sequence to a nucleotide sequence, a standard codon table can be used, but it is preferable to convert taking into account the frequency of codon usage in the host to be transformed with the polynucleotide of the present invention. As an example, when the host is Escherichia coli, the following codons may be converted to avoid the use of AGA/AGG/CGG/CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) (so-called rare codons). Analysis of codon usage can also be done using public databases (for example, the Codon Usage Database on the website of the Kazusa DNA Research Institute).

The polynucleotide of the present invention can also be obtained, for example, by introducing a mutation into a polynucleotide encoding the above-mentioned immunoglobulin-binding domain (for example, an immunoglobulin-binding domain containing the amino acid sequence set forth in SEQ ID NO: 1 or a variant sequence thereof) so that the domain has an amino acid substitution such as Lys50Trp. A polynucleotide encoding an immunoglobulin-binding domain can be obtained, for example, by chemical synthesis or a DNA amplification method such as PCR. When the immunoglobulin-binding polypeptide of the present invention has two or more amino acid substitutions in its modified immunoglobulin-binding domain, these amino acid substitutions may be introduced, for example, simultaneously or sequentially. For example, a mutation may be introduced into a polynucleotide encoding an immunoglobulin-binding domain so that the domain has two or more amino acid substitutions according to the present invention. An amino acid residue at a certain position may also be modified two or more times. For example, an immunoglobulin-binding polypeptide that already has an amino acid substitution of Asn11Lys may further have an amino acid substitution of Asn11Arg.

In addition, the polynucleotide is not limited to the above-mentioned mutations that result in amino acid substitutions, and any mutation, such as a mutation for constructing a variant sequence, may be introduced and used as the polynucleotide of the present invention or a material for obtaining the polynucleotide.

An example of a method for introducing a mutation into a polynucleotide is the error-prone PCR method. The reaction conditions in the error-prone PCR method are not particularly limited as long as they are conditions that allow the desired mutation to be introduced into the polynucleotide. For example, the concentrations of the four types of substrate deoxynucleotides (dATP/dTTP/dCTP/dGTP) are made non-uniform, and _MnCl2 is added to the PCR reaction solution at a concentration of 0.01 to 10 mM (preferably 0.1 to 1 mM) to perform PCR, thereby introducing a mutation into the polynucleotide.

Another method for introducing mutations into polynucleotides is the inverse PCR method. Inverse PCR involves first denaturing a polynucleotide into which a gene of interest has been inserted, then annealing a mutation primer and carrying out an extension reaction using DNA polymerase. The extended polynucleotide is selectively cleaved using the restriction enzyme DpnI, and the newly synthesized gene is phosphorylated, ligated, and cyclized to introduce a mutation into the polynucleotide.

In addition to the error-prone PCR method and inverse PCR method described above, other methods for introducing mutations into a polynucleotide include applying a mutagenic agent to the polynucleotide, irradiating the polynucleotide with ultraviolet light, and introducing mutations into the polynucleotide. Examples of mutagenic agents include mutagenic agents commonly used by those skilled in the art, such as hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, and hydrazine. Such methods for introducing mutations into a polynucleotide are not limited to the introduction of amino acid substitutions exemplified above, and can also be used to construct variant sequences (for example, the introduction of substitutions, deletions, insertions, and/or additions of amino acid residues, or changes in amino acid sequences within the range of homology as described above).

The polynucleotide of the present invention may be obtained, for example, by obtaining its full-length sequence all at once, or by obtaining and linking its partial sequences. The above description of the method for obtaining the polynucleotide of the present invention is not limited to the case where the full-length sequence is obtained all at once, but can also be applied mutatis mutandis to the case where the partial sequence is obtained.

Specifically, the immunoglobulin-binding polypeptide of the present invention can be produced, for example, by expressing the immunoglobulin-binding polypeptide of the present invention in a recombinant host containing a polynucleotide of the present invention. In this specification, a recombinant host containing a polynucleotide of the present invention is also referred to as a "recombinant host of the present invention." The recombinant host of the present invention can express the immunoglobulin-binding polypeptide of the present invention by virtue of containing the polynucleotide of the present invention. In other words, the recombinant host of the present invention is a host capable of expressing the immunoglobulin-binding polypeptide of the present invention.

The genetically modified host of the present invention can be obtained, for example, by transforming a host with the polynucleotide of the present invention. That is, the genetically modified host of the present invention can be, for example, a host that has been genetically modified (transformed) with the polynucleotide of the present invention, a host that contains the polynucleotide of the present invention, or a host that can express the immunoglobulin-binding polypeptide of the present invention. The host used for genetic recombination is not particularly limited as long as it is a host that can express the immunoglobulin-binding polypeptide of the present invention by being transformed with the polynucleotide of the present invention. Examples of hosts include animal cells, insect cells, and microorganisms. Examples of animal cells include COS cells, CHO (Chinese Hamster Ovary) cells, Hela cells, NIH3T3 cells, and HEK293 cells. Examples of insect cells include Sf9 cells and BTI-TN-5B1-4 cells. Examples of microorganisms include yeast and bacteria. Examples of yeast include yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts of the genus Pichia such as Pichia pastoris, and yeasts of the genus Schizosaccharomyces such as Schizosaccharomyces pombe. Examples of bacteria include bacteria of the genus Escherichia such as Escherichia coli. Examples of Escherichia coli include the JM109 strain and the BL21 (DE3) strain. Note that using yeast or Escherichia coli as a host is preferable in terms of productivity, and using Escherichia coli as a host is even more preferable.

The polynucleotide of the present invention may be retained in the genetically recombinant host of the present invention in an expressible manner. Specifically, the polynucleotide of the present invention may be retained so as to be expressed under the control of a promoter that functions in the host. Examples of promoters that function in the host include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter when Escherichia coli is used as the host.

In the recombinant host of the present invention, the polynucleotide of the present invention may be present on a vector that autonomously replicates outside of genomic DNA. That is, the polynucleotide of the present invention may be introduced into a host as an expression vector containing the polynucleotide of the present invention. That is, in one embodiment, the recombinant host of the present invention may be a host containing an expression vector containing the polynucleotide of the present invention. In this specification, the expression vector containing the polynucleotide of the present invention is also referred to as the "expression vector of the present invention". The expression vector of the present invention can be obtained, for example, by inserting the polynucleotide of the present invention into an appropriate position of the expression vector. The expression vector is not particularly limited as long as it is stable and can be replicated in the host to be transformed. Examples of the expression vector include, for example, pET plasmid vector, pUC plasmid vector, and pTrc plasmid vector when Escherichia coli is used as the host. The expression vector may be provided with a selection marker such as an antibiotic resistance gene. The appropriate position means a position that does not destroy the expression vector replication function, selection marker, and region related to transmissibility. When inserting the polynucleotide of the present invention into the expression vector, it is preferable to insert it in a state where it is linked to a functional polynucleotide such as a promoter required for expression.

In the recombinant host of the present invention, the polynucleotide of the present invention may be introduced, for example, into genomic DNA. The polynucleotide of the present invention can be introduced into genomic DNA, for example, by a gene introduction method using homologous recombination. Examples of gene introduction methods using homologous recombination include a method using linear DNA such as the red-driven integration method (Datsenko, K. A, and Wanner, B. L. Proc. NatI. Acad. Sci. USA. 97: 6640-6645 (2000)), a method using a vector containing a temperature-sensitive replication origin, a method using a vector without a replication origin that functions in the host, and a transduction method using a phage.

Transformation of a host using a polynucleotide such as an expression vector of the present invention can be carried out, for example, by a method commonly used by those skilled in the art. For example, when Escherichia coli is selected as the host, transformation can be carried out by the competent cell method, the heat shock method, electroporation method, etc. After transformation, the genetically modified host of the present invention can be obtained by screening using an appropriate method.

Information on genetic engineering techniques such as expression vectors and promoters that can be used in various microorganisms is described in detail in, for example, "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan, 1987," and these can be used.

When the recombinant host of the present invention contains the expression vector of the present invention, the expression vector of the present invention can be prepared from the recombinant host of the present invention. For example, the expression vector of the present invention can be prepared from a culture obtained by culturing the recombinant host of the present invention by an alkaline extraction method or using a commercially available extraction kit such as the QIAprep Spin Miniprep kit (manufactured by QIAGEN).

The immunoglobulin-binding polypeptide of the present invention can be expressed by culturing the recombinant host of the present invention. The immunoglobulin-binding polypeptide of the present invention can be expressed by culturing the recombinant host of the present invention, and the immunoglobulin-binding polypeptide of the present invention can be produced by recovering the expressed polypeptide. That is, the present invention provides a method for producing the immunoglobulin-binding polypeptide of the present invention, which includes, for example, a step of expressing the immunoglobulin-binding polypeptide of the present invention by culturing the recombinant host of the present invention, and a step of recovering the expressed polypeptide. The medium composition and culture conditions can be appropriately set depending on various conditions such as the type of host and the characteristics of the immunoglobulin-binding polypeptide of the present invention. The medium composition and culture conditions can be set, for example, so that the host can grow and the immunoglobulin-binding polypeptide of the present invention can be expressed. As the medium, for example, a medium containing a carbon source, a nitrogen source, inorganic salts, and various other organic and inorganic components as appropriate can be used. For example, when the host is Escherichia coli, an example of a preferred medium is LB (Luria-Bertani) medium (tryptone 1% (w/v), yeast extract 0.5% (w/v), sodium chloride 1% (w/v)) supplemented with necessary nutrient sources. In order to selectively grow the recombinant host of the present invention based on the presence or absence of introduction of the expression vector of the present invention, it is preferable to add an antibiotic corresponding to the antibiotic resistance gene contained in the expression vector to the medium and culture it. For example, when the expression vector contains a kanamycin resistance gene, kanamycin may be added to the medium. The same applies when the polynucleotide of the present invention is introduced onto genomic DNA. The medium may also contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystatin, thioglycolate, and dithiothreitol. The medium may also contain a reagent such as glycine that promotes the secretion of a polypeptide from the recombinant host into the culture medium. For example, when the host is Escherichia coli, it is preferable to add glycine to the medium at 2% (w/v) or less. The culture temperature may be, for example, 10°C to 40°C, preferably 20°C to 37°C, and more preferably around 25°C when the host is Escherichia coli. The pH of the medium may be, for example, pH 6.8 to pH 7.4, preferably around pH 7.0 when the host is Escherichia coli. In addition, when the immunoglobulin-binding polypeptide of the present invention is expressed under the control of an inducible promoter, it is preferable to induce the immunoglobulin-binding polypeptide of the present invention so that it can be well expressed. For expression induction, for example, an inducer according to the type of promoter can be used. An example of the inducer is IPTG (isopropyl-β-D-thiogalactopyranoside). For example, when the host is Escherichia coli, an appropriate amount of IPTG is added when the turbidity (absorbance at 600 nm) of the culture solution becomes about 0.5 to 1.0, and the culture is continued, whereby the expression of the immunoglobulin-binding polypeptide of the present invention can be induced. The concentration of IPTG added may be, for example, 0.005 to 1.0 mM, preferably 0.01 to 0.5 mM. Expression induction, such as IPTG induction, can be performed, for example, under conditions well known in the art.

The immunoglobulin-binding polypeptide of the present invention can be isolated and recovered from the culture by a method suitable for its expression form. In this specification, the term "culture" refers to the whole or a part of the culture medium obtained by culturing. The part is not particularly limited as long as it contains the immunoglobulin-binding polypeptide of the present invention. Examples of the part include the cultured cells of the genetically modified host of the present invention and the medium after culture (i.e., the culture supernatant). For example, when the immunoglobulin-binding polypeptide of the present invention accumulates in the culture supernatant, the cells can be separated by centrifugation, and the immunoglobulin-binding polypeptide of the present invention can be recovered from the culture supernatant obtained. In addition, when the immunoglobulin-binding polypeptide of the present invention accumulates within the cells (including the periplasm), the cells can be recovered by centrifugation, and then the cells can be disrupted by adding an enzyme treatment agent, a surfactant, or the like, and the immunoglobulin-binding polypeptide of the present invention can be recovered from the disrupted product. The immunoglobulin-binding polypeptide of the present invention can be recovered from the culture supernatant or cell disrupted product by a known method used for, for example, separation and purification of polypeptides. Such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation.

The immunoglobulin-binding polypeptide of the present invention can be used, for example, in the separation or analysis of immunoglobulins (antibodies). The immunoglobulin-binding polypeptide of the present invention can be used, for example, by immobilizing it on an insoluble carrier. That is, the separation or analysis of immunoglobulins (antibodies) can be specifically performed, for example, using an immunoglobulin adsorbent containing an insoluble carrier and an immunoglobulin-binding polypeptide of the present invention immobilized on the insoluble carrier. In this specification, an immunoglobulin adsorbent containing an insoluble carrier and an immunoglobulin-binding polypeptide of the present invention immobilized on the insoluble carrier is also referred to as the "immunoglobulin adsorbent of the present invention." Note that "separation of immunoglobulins" is not limited to separation of immunoglobulins from a solution containing coexisting impurities, but also includes separation between immunoglobulins based on structure, properties, activity, etc. The insoluble carrier is not particularly limited. Examples of insoluble carriers include carriers made from polysaccharides such as agarose, alginate (alginic acid salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and carriers made from ceramics such as silica. Among them, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carriers include polymethacrylate gels with hydroxyl groups introduced such as Toyopearl (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by GE Healthcare), and cellulose gels such as Cellufine (manufactured by JNC Corporation). The shape of the insoluble carrier is not particularly limited. The insoluble carrier may be in a shape that can be packed in a column, for example. The insoluble carrier may be, for example, a granular or non-granular material. Additionally, the insoluble carrier may be, for example, porous or non-porous.

The immunoglobulin-binding polypeptide of the present invention can be immobilized on an insoluble carrier, for example, via a covalent bond. Specifically, the immunoglobulin-binding polypeptide of the present invention can be immobilized on an insoluble carrier, for example, by covalently bonding the immunoglobulin-binding polypeptide of the present invention to the insoluble carrier via an active group possessed by the insoluble carrier. That is, the insoluble carrier may have an active group. The insoluble carrier may have an active group on its surface, for example. Examples of the active group include an N-hydroxysuccinimide (NHS) activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, and a haloacetamide. As an insoluble carrier having an active group, for example, a commercially available insoluble carrier having an active group may be used as it is, or an active group may be introduced into the insoluble carrier and used. Examples of commercially available carriers having active groups include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, Epoxy-activated Sepharose 6B (all manufactured by GE Healthcare), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

An example of a method for introducing active groups onto the carrier surface is to react one of the compounds having two or more active sites with hydroxyl groups, epoxy groups, carboxyl groups, amino groups, etc. present on the carrier surface.

Examples of compounds that introduce epoxy groups into hydroxyl or amino groups present on the surface of a carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

Examples of compounds that introduce carboxy groups into epoxy groups present on the carrier surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

In addition, compounds that introduce maleimide groups into hydroxyl, epoxy, carboxyl, and amino groups present on the carrier surface include N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, 4-(4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, and 3-maleimidopropionic acid. Examples include carboxylic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

Examples of compounds that introduce haloacetyl groups into hydroxyl or amino groups present on the carrier surface include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester.

Another example of a method for introducing active groups onto the carrier surface is a method in which an ω-alkenylalkane glycidyl ether is reacted with a hydroxy group or amino group present on the carrier surface, and then the ω-alkenyl moiety is halogenated with a halogenating agent to activate the group. Examples of ω-alkenylalkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

Another example of a method for introducing active groups onto the carrier surface is a method in which active groups are introduced onto carboxy groups present on the carrier surface using a condensation agent and an additive. Examples of condensation agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenzotriazole.

The immunoglobulin-binding polypeptide of the present invention can be immobilized on an insoluble carrier in, for example, a buffer solution. Examples of the buffer solution include acetate buffer, phosphate buffer, MES (2-morpholinoethanesulfonic acid) buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris (Tris(hydroxymethyl)aminomethane) buffer, and borate buffer. The reaction temperature during immobilization can be appropriately set depending on various conditions, such as the reactivity of the active group and the stability of the immunoglobulin-binding polypeptide. The reaction temperature during immobilization may be, for example, 5°C to 50°C, and preferably 10°C to 35°C.

The immunoglobulin adsorbent of the present invention can be used, for example, in a column to separate immunoglobulins (antibodies). Specifically, for example, immunoglobulins can be separated by adding a solution containing immunoglobulins to a column packed with the immunoglobulin adsorbent of the present invention to adsorb the immunoglobulins to the adsorbent, and then eluting the immunoglobulins adsorbed to the adsorbent. That is, the present invention provides a method for separating immunoglobulins, which includes, for example, a step of adding a solution containing immunoglobulins to a column packed with the immunoglobulin adsorbent of the present invention to adsorb the immunoglobulins to the adsorbent, and a step of eluting the immunoglobulins adsorbed to the adsorbent. The solution containing immunoglobulins can be added to the column using a liquid delivery means such as a pump. In this specification, adding a liquid to a column is also referred to as "delivering a liquid to a column." The solution containing immunoglobulins may be solvent-substituted with an appropriate buffer solution before being added to the column. In addition, the column may be equilibrated with an appropriate buffer solution before being added to the column. The equilibration is expected to separate, for example, immunoglobulins with higher purity. Examples of buffers used for solvent exchange and equilibration include phosphate buffer, acetate buffer, and MES buffer. For example, inorganic salts such as 10 mM to 100 mM sodium chloride may be added to such buffers. The buffer used for solvent exchange and the buffer used for equilibration may be the same or different. In addition, if components other than immunoglobulins, such as impurities, remain in the column after passing the immunoglobulin-containing solution through the column, such components may be removed from the column before eluting the immunoglobulin adsorbed to the immunoglobulin adsorbent. Components other than immunoglobulins can be removed from the column, for example, using an appropriate buffer. For the buffer used for removing components other than immunoglobulins, the description of the buffer used for solvent exchange and equilibration can be applied mutatis mutandis. The immunoglobulin adsorbed to the immunoglobulin adsorbent can be eluted, for example, by weakening the interaction between the immunoglobulin and the ligand (the immunoglobulin-binding polypeptide of the present invention). Examples of means for weakening the interaction between immunoglobulin and a ligand (the immunoglobulin-binding polypeptide of the present invention) include lowering the pH with a buffer, adding a counter peptide, increasing the temperature, and changing the salt concentration. Specifically, the immunoglobulin adsorbed to the immunoglobulin adsorbent can be eluted using, for example, an appropriate elution solution. Examples of the elution solution include buffers that are more acidic than the buffers used for solvent replacement and equilibration. Examples of such buffers include citrate buffer, glycine hydrochloride buffer, and acetate buffer. The pH of the elution solution can be set within a range that does not impair the function of the immunoglobulin (such as binding to an antigen).

By carrying out the separation of immunoglobulins (antibodies) in this manner, for example, separated immunoglobulins can be obtained. That is, in one embodiment, the method for separating immunoglobulins may be a method for producing immunoglobulins, and specifically, may be a method for producing separated immunoglobulins. Immunoglobulins are obtained, for example, as eluted fractions containing immunoglobulins. That is, a fraction containing eluted immunoglobulins can be separated. Separation of fractions can be carried out, for example, by a conventional method. Methods for separating fractions include a method of replacing a collection container at regular intervals or at regular volumes, a method of replacing a collection container according to the shape of the chromatogram of the eluate, and a method of separating fractions using an automatic fraction collector such as an autosampler. Furthermore, immunoglobulins can be collected from fractions containing immunoglobulins. Collection of immunoglobulins from fractions containing immunoglobulins can be carried out, for example, by a known method used for separating and purifying proteins.

The present invention will be explained in more detail below using examples and comparative examples, but the present invention is not limited to these examples.

(Preparation of Immunoglobulin-Binding Polypeptides)
Comparative Example 1 A polypeptide SpA10a-K3 (SEQ ID NO: 9) was designed in which a tag sequence (Lys-Lys-Lys) for immobilization on an insoluble carrier was added to the C-terminus of the immunoglobulin-binding polypeptide SpA10a consisting of the amino acid sequence set forth in SEQ ID NO: 2. SpA10a is a polypeptide in which the following amino acid substitutions have occurred in domain C (amino acid residues 270 to 327 of GenBank No. AAA26676) of Protein A (hereinafter, SpA) derived from naturally occurring Staphylococcus bacteria, which consists of the amino acid sequence set forth in SEQ ID NO:1: Asn3His (this notation indicates that the amino acid residue corresponding to the third asparagine in SEQ ID NO:1 has been substituted with histidine, the same applies below), Lys4Arg, Lys7Val, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, Ser39Gly, Val40Ala, Lys42Leu, and Lys58Asp.

(Example 1) A polypeptide SpA10b-K3 (SEQ ID NO: 13) was designed in which a tag sequence (Lys-Lys-Lys) for immobilization to an insoluble carrier was added to the C-terminus of the immunoglobulin-binding polypeptide SpA10b consisting of the amino acid sequence set forth in SEQ ID NO: 3. SpA10b is a polypeptide in which the amino acid substitutions Asn11Arg, Lys35Arg, Lys49Met, and Lys50Trp have occurred in SpA10a consisting of the amino acid sequence set forth in SEQ ID NO: 2.

(1) After synthesizing the polynucleotides encoding SpA10a-K3 and SpA10b designed in (Comparative Example 1) and (Example 1), polynucleotides containing the nucleotide sequences were prepared using PCR. PCR was performed by using each of the synthesized polynucleotides as template DNA, and using oligonucleotides consisting of the nucleotide sequences described in SEQ ID NO: 10 (5'-TAGCCATGGGCGCCGATATTCGCTTTA-3') and SEQ ID NO: 11 (5'-CTACTCGAGATCCGGTGCTTGAGCATC-3') as PCR primers to prepare a reaction solution having the composition shown in Table 1, and then heat-treating the reaction solution at 98°C for 30 seconds, carrying out 30 cycles of reaction consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 60 seconds, and finally heat-treating the reaction solution at 72°C for 2 minutes.

(2) The resulting polynucleotide was purified, digested with the restriction enzymes NcoI and XhoI, and then ligated to the expression vector pET-28a, which had been previously digested with the restriction enzymes NcoI and XhoI, and the ligation product was used to transform E. coli BL21 (DE3) strain (Nippon Gene Co., Ltd.).

(3) The resulting transformants (recombinant E. coli) were cultured in LB medium containing 50 μg/mL kanamycin, and the expression vectors (named pET-SpA10a-K3 and pET-SpA10b-K3, respectively) were extracted using a QIAprep Spin Miniprep kit.

(4) Of the obtained extracts, the polynucleotides encoding the immunoglobulin-binding polypeptides and their surrounding regions were subjected to cycle sequencing reactions using a Big Dye Terminator Cycle Sequencing ready Reaction kit (manufactured by Thermo Fisher Scientific) based on the chain terminator method, and the nucleotide sequences were analyzed using a fully automated DNA sequencer, ABI Prism 3700 DNA analyzer (manufactured by Thermo Fisher Scientific). In this analysis, an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 was used as a sequencing primer.

Sequence analysis confirmed that a polynucleotide encoding SpA10a-K3 and a polynucleotide encoding SpA10b-K3 were inserted into the expression vectors pET-SpA10a-K3 and pET-SpA10b-K3, respectively. The sequence of the polynucleotide encoding SpA10a-K3 is shown in SEQ ID NO: 12. The sequence of the polynucleotide encoding SpA10b-K3 is shown in SEQ ID NO: 14.

(Preparation of Immunoglobulin-Binding Polypeptides)
(1) The recombinant E. coli containing pET-SpA10a-K3 or pET-SpA10b-K3 prepared above was inoculated into 20 mL of 2xYT liquid medium (tryptone 1.6% (w/v), yeast extract 1% (w/v), sodium chloride 0.5% (w/v)) containing 50 μg/mL kanamycin, and precultured (30° C., 16 hours).

(2) 10 mL of the precultured culture was inoculated into 1 L of 2xYT liquid medium containing 50 μg/mL kanamycin, and cultured at 37°C for 2 to 3 hours. Then, 200 μL of 0.5 M IPTG (isopropyl-β-D-thiogalactopyranoside) was added, and the mixture was further cultured overnight at 20°C with shaking.

(3) After the culture, the culture medium was centrifuged to recover 8 g of cultured cells (wet weight). 2 g of the cultured cells were lysed using BugBuster Protein Extraction Reagent (Merck Millipore), and the supernatant obtained by centrifugation was passed through a filter to clarify.

(4) The supernatant clarified in (3) was added to a column packed with IgG Sepharose 6 Fast Flow (GE Healthcare) that had been previously equilibrated with 0.05 M Tris buffer (pH 7.6) containing 0.15 M sodium chloride (hereinafter referred to as washing buffer), and the column was washed with washing buffer in an amount 10 times the amount of the carrier packed in the column. Then, 0.1 M glycine-hydrochloric acid buffer (pH 3.0) (hereinafter referred to as acidic solution) was passed through the column to recover a fraction corresponding to the immunoglobulin-binding polypeptide.

(5) The absorbance of the collected fractions was measured using a spectrophotometer to quantify the amount of polypeptide contained in the fractions. SDS-PAGE was also performed to confirm that the immunoglobulin-binding polypeptides (SpA10a-K3 or SpA10b-K3) were highly purified.

(Preparation of immunoglobulin adsorbent)
(1) The fraction containing the purified immunoglobulin-binding polypeptide (SpA10a-K3 or SpA10b-K3) prepared above was concentrated to 1/10 of the original volume using Amicon Ultra (Merck Millipore). A 10-fold amount of sodium phosphate buffer was added to the concentrated solution, and the solution was again concentrated to 1/10 of the original volume using Amicon Ultra (Merck Millipore). Through these operations, the fraction was concentrated and the buffer was replaced.

(2) TOYOPEARL AF-Formyl-650 (Tosoh Corporation), an insoluble carrier having a formyl group as an active group, was replaced with pure water and then dried by suction filtration. The water content of the suction-dried gel after suction filtration was measured with a water content meter (Mettler Toledo), and 0.5 to 3.0 g of the gel was added to a stoppered Erlenmeyer flask.

(3) A buffer solution (boric acid or phosphate buffer) containing 0.5 M sodium chloride was added to the stoppered Erlenmeyer flask containing the suction-dried gel to swell the suction-dried gel.

(4) The concentrated solution of immunoglobulin-binding polypeptide prepared in (1) was added to a stoppered Erlenmeyer flask so that the final concentration was 5 g/L, and the mixture was shaken at room temperature to bind the gel and polypeptide contained in the flask by forming a Schiff base. After 15 minutes to 4 hours, a portion of the supernatant from the stoppered Erlenmeyer flask was collected, and the amount of the polypeptide immobilized on the gel was calculated from the proportion of unbound immunoglobulin-binding polypeptide.

(5) An appropriate amount of 1M _NaBH4 aqueous solution was added and the mixture was shaken at room temperature for 15 minutes to 2 hours to reduce the remaining Schiff base, thereby preparing an immunoglobulin adsorbent. The prepared adsorbent was washed with water, and then replaced with 20% (v/v) ethanol for storage.

(Measurement of static adsorption amount of immunoglobulin adsorbent)
(1) Each of the immunoglobulin adsorbents prepared above was prepared into a 50% (w/v) slurry, and 100 μL of the slurry was added to a Mini Biospin column (manufactured by BIORAD).

(2) After washing by passing an acidic solution and a washing buffer solution through the column, an immunoglobulin solution (Japan Blood Products Organization) (43 mg/mL) diluted with the washing buffer solution was added, and the column was shaken at 25°C and 1000 rpm for 2 hours using a thermostatic shaking incubator (TAITEC) to adsorb the immunoglobulin to the insoluble carrier.

(3) After washing off unadsorbed immunoglobulin by passing a washing buffer solution, the immunoglobulin adsorbed to the adsorbent was eluted using an acidic solution. The absorbance (wavelength 280 nm) of the eluate was measured using a spectrophotometer, and the static adsorption amount of the adsorbent was calculated from the amount of immunoglobulin contained in the eluate. The results are shown in Table 2.

The static adsorption amount was improved by using SpA10b (SEQ ID NO: 3) as the immunoglobulin-binding polypeptide compared to when SpA10a (SEQ ID NO: 2) was used. This shows that the static adsorption amount of immunoglobulin is improved by introducing the amino acid substitution Lys50Trp into the immunoglobulin-binding polypeptide.

As described above, in the present invention, by introducing the amino acid substitution Lys50Trp into the immunoglobulin-binding domain, an adsorbent with improved immunoglobulin (antibody) adsorption capacity can be obtained. In other words, the present invention makes it possible to obtain immunoglobulins contained in a sample more efficiently.

Therefore, the present invention is useful in the production of immunoglobulins in the fields of antibody drugs and antibody diagnostics, removal of immunoglobulins from biological components, component analysis, etc.

Claims (8)

An immunoglobulin-binding polypeptide comprising a modified immunoglobulin-binding domain of Protein A derived from a bacterium of the genus Staphylococcus, wherein the modified immunoglobulin-binding domain comprises the amino acid sequence set forth in SEQ ID NO:3 . A polynucleotide encoding the immunoglobulin-binding polypeptide of claim 1 . An expression vector comprising the polynucleotide of claim 2 . A genetically modified host comprising the polynucleotide of claim 2 or the expression vector of claim 3 . The genetically recombinant host of claim 4 , wherein the host is Escherichia coli. A method for producing an immunoglobulin-binding polypeptide, comprising the steps of: culturing a genetically recombinant host according to claim 4 or 5 to express the immunoglobulin-binding polypeptide according to claim 1 ; and recovering the expressed polypeptide. An immunoglobulin adsorbent comprising an insoluble carrier and the immunoglobulin-binding polypeptide according to claim 1 immobilized on the insoluble carrier. 8. A method for separating immunoglobulins contained in a solution, comprising the steps of: adding a solution containing immunoglobulins to a column packed with the adsorbent according to claim 7 to adsorb the immunoglobulins to the adsorbent; and eluting the immunoglobulins adsorbed to the adsorbent.