JP2024097398A - Immunoglobulin-binding proteins - Google Patents

Abstract

[Problem] To provide an immunoglobulin adsorbent comprising a protein that is a polymerized protein of the immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia and has a high antibody adsorption capacity, and an insoluble carrier to which said protein is bound. [Solution] The above problem is solved by an immunoglobulin-binding protein in which a polypeptide containing at least the immunoglobulin-binding domain of FpL is linked via an oligopeptide linker consisting of 2 to 8 residues, which contains at least two or more consecutive proline residues but does not contain any lysine residues. [Selected Figure] None

Description

The present invention relates to a protein that specifically binds to immunoglobulin. More specifically, the present invention relates to a protein that is a polymerized version of the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia, and that has improved antibody adsorption capacity compared to conventional polymerized proteins.

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 then formulated after intermediate purification, final purification, and virus removal.

In the purification process, an affinity carrier that specifically recognizes antibody molecules is used. As the ligand protein used in the carrier, Staphylococcus bacteria-derived Protein A, which has the property of binding to antibodies (immunoglobulins), is often used (Patent Document 1). However, since Protein A is a protein that specifically binds to the Fc region of an antibody, it cannot be applied to the purification of antibodies that do not have an Fc region, such as single chain Fv (scFv), Fab, F(ab') ₂ , IgA, and bispecific T cell inducer (BiTE) antibodies. On the other hand, Finegoldia bacteria-derived Protein L is a protein that binds to the κ light chain of immunoglobulins, and by using Protein L as a ligand protein, it is possible to purify antibodies that do not have an Fc region, which cannot be purified with the above-mentioned Protein A (Patent Document 2).

From the viewpoint of improving the productivity of antibody drugs, there is also a demand for improving the amount of antibody adsorption per affinity adsorbent. As ligands with improved antibody adsorption, a polypeptide in which immunoglobulin-binding domains are linked in tandem (Non-Patent Document 1) and a polypeptide in which two or more immunoglobulin-binding domains are linked via a linker (Patent Document 3) have been disclosed. However, in the case of Protein L, the effect of the linker in improving the amount of adsorption cannot be said to be sufficient.

Special Publication No. 2010-504754 WO2017/191748 WO2015/050153

Freiherr von Roman M et al., Journal of chromatography A, 1347, 80-86, 2014

The object of the present invention is to provide a protein obtained by multimerizing the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia, which has an improved antibody adsorption amount compared to conventional multimerized proteins, and an immunoglobulin adsorbent comprising an insoluble carrier and the protein immobilized on the carrier.

It has been known from prior art that the amount of antibody adsorption increases as the linker length between immunoglobulin-binding domains increases (WO2015/050153). However, when proteins are linked with a long linker, the molecular weight increases, which is problematic as it may reduce productivity in an E. coli expression system. As a result of extensive research to solve the above problem, the present inventors have optimized the amino acid sequence of the linker peptide for linking the immunoglobulin-binding domains, thereby completing the present invention.

That is, the present invention includes the following aspects:
[1] An immunoglobulin-binding protein comprising two or more polypeptides each including at least an immunoglobulin-binding domain of Protein L derived from a bacterium belonging to the genus Finegoldia, and a linker for linking the polypeptides,
The above-mentioned protein, wherein the linker is an oligopeptide consisting of 2 to 8 residues, which contains at least two or more consecutive proline residues but does not contain any lysine residues.

[2] The protein according to [1], wherein the immunoglobulin-binding domain of Protein L derived from a bacterium belonging to the genus Finegoldia is any one of the following (A) to (D):
(A) a polypeptide comprising at least the amino acid sequence set forth in SEQ ID NO:1;
(B) a polypeptide comprising at least a partial sequence of the amino acid sequence set forth in SEQ ID NO: 1 and having immunoglobulin activity;
(C) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a partial sequence thereof, provided that said sequence contains substitution, deletion and/or addition of one or several amino acid residues at one or several positions, and has immunoglobulin-binding activity;
(D) A polypeptide comprising at least an amino acid sequence having 70% or more homology to the amino acid sequence set forth in SEQ ID NO: 1 or a partial sequence thereof, and having immunoglobulin activity.

[3] A polynucleotide encoding the protein according to [1] or [2].

[4] An expression vector comprising the polynucleotide described in [3].

[5] A genetically modified host comprising the polynucleotide described in [3].

[6] The genetically modified host according to [5], wherein the host is Escherichia coli.

[7] A method for producing the protein, comprising the steps of culturing the recombinant host according to [5] to express the protein according to [1] or [2], and recovering the expressed protein.

[8] An immunoglobulin adsorbent comprising an insoluble carrier and the protein according to [1] or [2] immobilized on the insoluble carrier.

[9] 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 [8], thereby adsorbing the immunoglobulins to the adsorbent, and eluting the immunoglobulins adsorbed to the adsorbent.

The protein of the present invention is an immunoglobulin-binding protein in which two or more polypeptides containing at least the immunoglobulin-binding domain of Protein L derived from the genus Finegoldia are linked via a linker, and the linker is an oligopeptide consisting of 2 to 8 residues that contains at least two or more consecutive proline residues but does not contain any lysine residues.

The protein of the present invention has an improved immunoglobulin (antibody) adsorption capacity compared to conventional immunoglobulin-binding proteins, so immunoglobulins contained in a sample can be obtained with high efficiency by using an antibody adsorbent containing an insoluble carrier and the protein of the present invention immobilized on the carrier, and immunoglobulins can be produced at lower cost.

The present invention is described in detail below.

In this specification, when a protein (polypeptide) contains an amino acid sequence, it is also referred to as "the protein (polypeptide) contains amino acid residues consisting of an amino acid sequence," and when a protein (polypeptide) or an amino acid sequence has an amino acid substitution, it is also referred to as "amino acid substitutions occur in the protein (polypeptide) or amino acid sequence." Additionally, the amino acids that make up a protein (polypeptide) or an amino acid sequence are also referred to as "amino acid residues."

The immunoglobulin-binding domain of Protein L (hereinafter, referred to as FpL) derived from the genus Finegoldia, which constitutes the protein of the present invention, is
(a) a polypeptide comprising the entire amino acid sequence of the domain;
As long as it has binding activity to immunoglobulins,
(b) It may be a polypeptide comprising a partial amino acid sequence of the domain.
Furthermore, as long as it has binding activity to immunoglobulin,
(c) The polypeptide according to (a) or (b) may be a polypeptide containing one or several substitutions, deletions and/or additions of amino acid residues at one or several positions;
(d) It may be a polypeptide comprising an amino acid sequence having a high homology of 70% or more with the amino acid sequence of the polypeptide according to (a) or (b) above.
Hereinafter, the polypeptides described in (c) and (d) above are collectively referred to as "variants".

Regarding (a) above, an example of the Finegoldia bacterium from which FpL is derived is Finegoldia magna. The immunoglobulin-binding domain of Protein L derived from Finegoldia magna is:
Domain B1 (amino acid residues 104 to 173 of SEQ ID NO: 46 (GenBank No. AAA25612)),
Domain B2 (amino acid residues 176 to 245 of SEQ ID NO: 46);
Domain B3 (amino acid residues 248 to 317 of SEQ ID NO: 46);
Domain B4 (amino acid residues 320 to 389 of SEQ ID NO: 46);
Domain B5 (amino acid residues 393 to 462 of SEQ ID NO: 46);
Domain C1 (amino acid residues 249 to 317 of SEQ ID NO: 47 (GenBank No. AAA67503)),
Domain C2 (amino acid residues 320 to 389 of SEQ ID NO:47),
Examples include domain C3 (amino acid residues 394 to 463 of SEQ ID NO: 47: SEQ ID NO: 1), and domain C4 (amino acid residues 468 to 537 of SEQ ID NO: 47).

The immunoglobulin-binding domain of FpL in this specification may, for example, contain a portion of another immunoglobulin-binding domain in addition to the selected immunoglobulin-binding domain. For example, when the immunoglobulin-binding domain of FpL contains the amino acid sequence of immunoglobulin-binding domain C3 of Protein L derived from Finegoldia magna, the protein of the present invention may further contain a portion of the N-terminal region of the domain (domain C1, domain C2), or a portion of the C-terminal region of the domain (domain C4).

Regarding (b) above, the immunoglobulin-binding domain of FpL ((a) above) is composed of four β-sheets and one α-helix, a loop portion connecting them, and an N-terminal loop region, but amino acid residues in regions unrelated to binding to immunoglobulins, such as the N-terminal loop region, may be deleted. As a specific example, when the immunoglobulin-binding domain of FpL ((a) above) is domain C3 (SEQ ID NO: 1), it is known that even if the amino acid residues from the first glutamic acid to the ninth glutamic acid, which correspond to the N-terminal loop region, are deleted, the immunoglobulin-binding ability is retained (Housden N.G. et al., Biochemical Society Transaction, 31, 716-718, 2003). In other words, it can be said that the partial amino acid sequence in (b) above is sufficient as long as it contains at least the amino acid sequence of the binding site to immunoglobulins. In other words, the partial amino acid sequence in (b) above is a partial sequence containing the amino acid sequence of the binding site to immunoglobulins.

The term "one or several" in (c) above means, depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, specifically, for example, 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 or 2, or 1.

The "homology" in (d) may mean similarity or identity, and may particularly mean identity. "Homology to amino acid sequence" means homology to the entire amino acid sequence. The "high homology" may mean homology of 70% or more, 80% or more, 90% or more, or 95% or more. 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). Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

When the immunoglobulin-binding domain of FpL is a polypeptide containing the entire amino acid sequence of the immunoglobulin-binding domain C3 (SEQ ID NO: 1) of Protein L derived from Finegoldia magna, preferred embodiments of the variant (above (c) and (d)) include the polypeptides shown in any of (α) to (δ) below.

(α) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, provided that in said amino acid sequence, there is at least one amino acid substitution selected from the following (1) to (53), and having immunoglobulin-binding activity: (1) an amino acid residue corresponding to asparagine at position 50 in SEQ ID NO: 1 is replaced with tyrosine; (2) an amino acid residue corresponding to glutamic acid at position 1 in SEQ ID NO: 1 is replaced with glycine or valine; (3) an amino acid residue corresponding to proline at position 3 in SEQ ID NO: 1 is replaced with serine; (4) an amino acid residue corresponding to glutamic acid at position 4 in SEQ ID NO: 1 is replaced with lysine or glycine; (5) an amino acid residue corresponding to glutamic acid at position 5 in SEQ ID NO: 1 is replaced with valine; (6) an amino acid residue corresponding to lysine at position 7 in SEQ ID NO: 1 is replaced with alanine; (7) an amino acid residue corresponding to glutamic acid at position 8 in SEQ ID NO: 1 is replaced with glycine; (8) an amino acid residue corresponding to SEQ ID NO: 1 (9) The amino acid residue corresponding to the 9th glutamic acid in SEQ ID NO:1 is replaced by valine. (10) The amino acid residue corresponding to the 17th isoleucine in SEQ ID NO:1 is replaced by phenylalanine. (11) The amino acid residue corresponding to the 27th glutamic acid in SEQ ID NO:1 is replaced by glycine or arginine. (12) The amino acid residue corresponding to the 29th lysine in SEQ ID NO:1 is replaced by proline. (13) The amino acid residue corresponding to the 31st threonine in SEQ ID NO:1 is replaced by leucine. (14) The amino acid residue corresponding to the 36th threonine in SEQ ID NO:1 is replaced by alanine. (15) The amino acid residue corresponding to the 41st alanine in SEQ ID NO:1 is replaced by threonine. (16) The amino acid residue corresponding to the 44th asparagine in SEQ ID NO:1 is replaced by either serine, aspartic acid, or glycine. (17) (18) The amino acid residue corresponding to the 50th asparagine in SEQ ID NO:1 is replaced by either serine, lysine or aspartic acid (19) The amino acid residue corresponding to the 51st glycine in SEQ ID NO:1 is replaced by valine (20) The amino acid residue corresponding to the 52nd glutamic acid in SEQ ID NO:1 is replaced by either valine, glycine or aspartic acid (21) The amino acid residue corresponding to the 53rd tyrosine in SEQ ID NO:1 is replaced by phenylalanine (22) The amino acid residue corresponding to the 54th threonine in SEQ ID NO:1 is replaced by isoleucine or methionine (23) The amino acid residue corresponding to the 62nd asparagine in SEQ ID NO:1 is replaced by either tyrosine, histidine, arginine, leucine, methionine or tryptophan (24) sequence (25) the amino acid residue corresponding to the 69th alanine in SEQ ID NO:1 is replaced by threonine; (26) the amino acid residue corresponding to the 2nd threonine in SEQ ID NO:1 is replaced by serine or proline; (27) the amino acid residue corresponding to the 3rd proline in SEQ ID NO:1 is replaced by arginine; (28) the amino acid residue corresponding to the 5th glutamic acid in SEQ ID NO:1 is replaced by lysine or arginine; (29) the amino acid residue corresponding to the 27th glutamic acid in SEQ ID NO:1 is replaced by either valine, lysine or isoleucine; (30) the amino acid residue corresponding to the 32nd phenylalanine in SEQ ID NO:1 is replaced by leucine; (31) the amino acid residue corresponding to the 38th lysine in SEQ ID NO:1 is replaced by alanine; (32) the amino acid residue corresponding to the 44th asparagine in SEQ ID NO:1 is replaced by isoleucine;
(33) The amino acid residue corresponding to the 47th alanine in SEQ ID NO:1 is replaced by threonine or arginine. (34) The amino acid residue corresponding to the 52nd glutamic acid in SEQ ID NO:1 is replaced by asparagine. (35) The amino acid residue corresponding to the 2nd threonine in SEQ ID NO:1 is replaced by alanine. (36) The amino acid residue corresponding to the 5th glutamic acid in SEQ ID NO:1 is replaced by aspartic acid. (37) The amino acid residue corresponding to the 6th proline in SEQ ID NO:1 is replaced by leucine or threonine. (38) The amino acid residue corresponding to the 7th lysine in SEQ ID NO:1 is replaced by (39) The amino acid residue corresponding to valine at position 14 of SEQ ID NO:1 is replaced by alanine. (40) The amino acid residue corresponding to isoleucine at position 23 of SEQ ID NO:1 is replaced by either arginine, threonine, or valine. (41) The amino acid residue corresponding to lysine at position 29 of SEQ ID NO:1 is replaced by phenylalanine. (42) The amino acid residue corresponding to threonine at position 31 of SEQ ID NO:1 is replaced by methionine. (43) The amino acid residue corresponding to glutamic acid at position 33 of SEQ ID NO:1 is replaced by asparagine. (44) The amino acid residue corresponding to the 35th alanine in SEQ ID NO:1 is replaced by threonine. (45) The amino acid residue corresponding to the 36th threonine in SEQ ID NO:1 is replaced by serine. (46) The amino acid residue corresponding to the 37th alanine in SEQ ID NO:1 is replaced by threonine. (47) The amino acid residue corresponding to the 41st alanine in SEQ ID NO:1 is replaced by any of isoleucine, arginine, and tyrosine. (48) The amino acid residue corresponding to the 44th asparagine in SEQ ID NO:1 is replaced by asparagine. (49) the amino acid residue corresponding to glutamic acid at position 52 of SEQ ID NO:1 is replaced with leucine or tyrosine; (50) the amino acid residue corresponding to glycine at position 60 of SEQ ID NO:1 is replaced with arginine; (51) the amino acid residue corresponding to isoleucine at position 64 of SEQ ID NO:1 is replaced with leucine; (52) the amino acid residue corresponding to isoleucine at position 66 of SEQ ID NO:1 is replaced with valine; (53) the amino acid residue corresponding to alanine at position 69 of SEQ ID NO:1 is replaced with leucine or valine;
(β) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, provided that in said amino acid sequence, there is at least one amino acid substitution selected from the following (54) to (64), and having immunoglobulin-binding activity: (54) The amino acid residue corresponding to the 7th lysine in SEQ ID NO: 1 is substituted with glutamine; (55) The amino acid residue corresponding to the 13th lysine in SEQ ID NO: 1 is substituted with valine; (56) The amino acid residue corresponding to the 29th lysine in SEQ ID NO: 1 is substituted with valine or isoleucine; (57) The amino acid residue corresponding to the 6th proline in SEQ ID NO: 1 is substituted with alanine; (58) The amino acid residue corresponding to the 8th glutamic acid in SEQ ID NO: 1 is substituted with alanine; (59) the amino acid residue corresponding to asparagine at position 15 of SEQ ID NO:1 is replaced by valine; (60) the amino acid residue corresponding to isoleucine at position 23 of SEQ ID NO:1 is replaced by phenylalanine or leucine; (61) the amino acid residue corresponding to glutamic acid at position 34 of SEQ ID NO:1 is replaced by any of aspartic acid, phenylalanine, leucine, and valine; (62) the amino acid residue corresponding to lysine at position 38 of SEQ ID NO:1 is replaced by leucine; (63) the amino acid residue corresponding to asparagine at position 50 of SEQ ID NO:1 is replaced by methionine; (64) the amino acid residue corresponding to tyrosine at position 53 of SEQ ID NO:1 is replaced by serine;
(γ) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, provided that in the amino acid sequence, at least one or more amino acid substitutions selected from (1) to (64) above are present, and further comprising substitutions, deletions, insertions and/or additions of one or several amino acid residues other than the amino acid substitutions, and having immunoglobulin-binding activity;
(δ) A polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid sequence has a homology of 70% or more to the entire amino acid sequence having at least one amino acid substitution selected from (1) to (64) above, wherein at least one of the amino acid substitutions remains, and wherein the polypeptide has immunoglobulin-binding activity.

In this specification, the term "amino acid residue corresponding to the Xth amino acid in SEQ ID NO: 1" in a specific amino acid sequence refers to an amino acid residue in the specific amino acid sequence that is arranged at the same position as the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1 in the alignment of the specific amino acid sequence with the amino acid sequence of SEQ ID NO: 1. In addition, the term "amino acid residue corresponding to the Xth amino acid in SEQ ID NO: 1" in the amino acid sequence described in SEQ ID NO: 1 refers to the Xth amino acid itself in the amino acid sequence described in SEQ ID NO: 1. In other words, the positions of the above-exemplified amino acid substitutions (i.e., the amino acid substitutions at the specific positions and optionally other amino acid substitutions) do not necessarily indicate absolute positions in the FpL immunoglobulin binding domain, but indicate relative positions based on the amino acid sequence described in SEQ ID NO: 1. In other words, for example, when the domain contains an insertion, deletion, or addition of an amino acid residue on the N-terminal side of the positions of the above-exemplified amino acid substitutions, the absolute positions of the amino acid substitutions may vary accordingly. The positions of the above-exemplified amino acid substitutions in the domain can be identified, for example, by alignment of the amino acid sequence of the domain with the amino acid sequence described in SEQ ID NO: 1. The alignment can be performed, for example, using the alignment program described above. The same applies to the positions of the amino acid substitutions exemplified above in any amino acid sequence, such as a variant sequence of the amino acid sequence described in SEQ ID NO: 1.

In (α), there is no limit to the number of amino acid substitutions, and it may have 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from the amino acid substitutions described in (1) to (53), or it may have 10 or more amino acid substitutions. There is also no limit to the number of amino acid substitutions in (β), and it may have 1 or 2 amino acid substitutions selected from the amino acid substitutions described in (54) to (64), or it may have all three amino acid substitutions.

An example of "further substitution of one or several amino acid residues other than the amino acid substitution" in (γ) above is a conservative substitution between amino acids with similar physical and/or chemical properties. In the case of conservative substitution, it is generally known to those skilled in the art that the function of a protein is maintained between a substitution and an unsubstituted one. An example of conservative substitution is a substitution between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). In addition, "further substitution, deletion, insertion, and/or addition of one or several amino acid residues other than the amino acid substitution" in (γ) above also includes those caused by naturally occurring mutations (mutants or variants), such as those caused by individual differences or species differences in the microorganisms from which the protein or the gene encoding it is derived.

The "homology" in (δ) above is synonymous with the "homology" in (d) above.

The protein of the present invention is a protein comprising two or more polypeptides comprising at least the immunoglobulin-binding domain of FpL described above and a linker for linking the polypeptides, and the linker is an oligopeptide comprising at least two or more consecutive proline residues, but no lysine residues, and comprising 2 to 8 residues, more preferably 2 to 7 residues. The immunoglobulin-binding domain of FpL may comprise two or more, for example, 2 or more, 3 or more, 4 or more, or 5 or more. When the immunoglobulin-binding domain of FpL is a variant (embodiments (c) and (d) above), the amino acid sequence of the domain may be the same or different.

The protein of the present invention may contain, for example, at its N-terminus or C-terminus, an oligopeptide that is useful for specifically detecting or separating a target substance. Examples of such oligopeptides include polyhistidine and polyarginine. The protein of the present invention may also contain, for example, at its N-terminus or C-terminus, an oligopeptide that is useful for immobilizing the protein of the present invention on a solid phase such as a support for chromatography. Examples of such oligopeptides include oligopeptides that contain lysine residues or cysteine residues.

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

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

The polynucleotide of the present invention can be obtained, for example, by a DNA amplification method such as chemical synthesis or 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 protein of the present invention. Examples of the polynucleotide used as a template include the genomic DNA of an organism that expresses the protein of the present invention, the cDNA of the protein 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 protein of the present invention. When converting from an amino acid sequence to a nucleotide sequence, a standard codon table can be used, but it is preferable to convert taking into consideration 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, AGA/AGG/CGG/CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) are used infrequently (so-called rare codons), so conversion may be performed to avoid these codons. Analysis of the frequency of codon usage can also be performed using public databases (for example, the Codon Usage Database on the website of the Kazusa DNA Research Institute).

The polynucleotide of the present invention may be obtained by obtaining the entire length of the polynucleotide in one go, or by obtaining a polynucleotide consisting of a partial sequence of the polynucleotide and then linking them. The above explanation of the method for obtaining the polynucleotide of the present invention is not limited to the case where the entire sequence is obtained in one go, but can also be applied mutatis mutandis to the case where a partial sequence is obtained.

The polynucleotide of the present invention may be designed to be linked to a polynucleotide encoding the amino acid sequence of any polypeptide to encode a fusion amino acid sequence.

The protein of the present invention can be produced, for example, by culturing a recombinant host containing the polynucleotide of the present invention (hereinafter, also simply referred to as the "recombinant host of the present invention") to express the protein, and then recovering the expressed protein. The recombinant host of the present invention can be obtained, for example, by transforming a host with the polynucleotide of the present invention. The host is not particularly limited as long as it is capable of expressing the protein of the present invention by being transformed with the polynucleotide of the present invention. Examples of hosts include animal cells, insect cells, and microorganisms. Among these, 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, and examples of microorganisms include yeast and bacteria. Further, 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, and 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.

In the genetically modified host of the present invention, the polynucleotide of the present invention may be held in an expressible manner. Specifically, the polynucleotide of the present invention may be held so as to be expressed under the control of a promoter that functions in the host. When the host is Escherichia coli, 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.

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 recombinant host of the present invention may be prepared by transforming a host with an expression vector containing the polynucleotide of the present invention (hereinafter, also simply 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 replicate in the host to be transformed. When the host is Escherichia coli, examples of the expression vector include a pET plasmid vector, a pUC plasmid vector, and a pTrc plasmid vector. The expression vector of the present invention may be provided with a selection marker such as an antibiotic resistance gene. The appropriate position means a position that does not destroy the replication function, selection marker, and regions related to transmissibility of the expression vector. When the polynucleotide of the present invention is inserted into the expression vector, it is preferable to insert it in a state 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 into genomic DNA. The polynucleotide of the present invention can be introduced into genomic DNA, for example, by a method of gene introduction 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. Natl. 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 a competent cell method, a heat shock method, an electroporation method, or the like. After transformation, a host containing the polynucleotide of the present invention can be screened by an appropriate method to obtain a genetically modified host of the present invention.

Information on genetic engineering techniques such as expression vectors and promoters that can be used in various microorganisms can be obtained from publicly known literature, such as "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan, 1987."

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 protein of the present invention can be expressed by culturing the recombinant host of the present invention. The protein of the present invention can be produced by culturing the recombinant host of the present invention to express the protein of the present invention and recovering the expressed protein. That is, this specification discloses a method for producing the protein of the present invention, which includes a step of expressing the protein of the present invention by culturing the recombinant host of the present invention and a step of recovering the expressed protein. The medium composition and culture conditions may be appropriately set according to various conditions such as the type of host and the characteristics of the protein of the present invention. The medium composition and culture conditions may be set, for example, under conditions in which the host can grow and the protein of the present invention can be expressed. The medium may be, for example, a medium containing a carbon source, a nitrogen source, inorganic salts, and various other organic and inorganic components as appropriate. Specifically, 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 depending on whether or not the expression vector of the present invention has been introduced, it is preferable to culture the host by adding an antibiotic corresponding to the antibiotic resistance gene contained in the expression vector to the medium. For example, if the expression vector contains a kanamycin resistance gene, kanamycin can 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 protein secretion from the transformant into the culture medium. For example, when the host is E. coli, it is preferable to add glycine to the medium at 2% (w/v) or less. For example, when the host is E. coli, the culture temperature is generally 10°C to 40°C, preferably 20°C to 37°C, and more preferably around 25°C. For example, when the host is E. coli, the pH of the medium is 6.8 to 7.4, preferably around 7.0.

When the protein of the present invention is expressed under the control of an inducible promoter, it is preferable to induce the protein of the present invention so that it can be well expressed. For example, an inducer according to the type of promoter can be used for expression induction. An example of an inducer is IPTG (isopropyl-β-D-thiogalactopyranoside). When the host is Escherichia coli, for example, the expression of the protein of the present invention can be induced by adding an appropriate amount of IPTG when the turbidity (absorbance at 600 nm) of the culture solution becomes about 0.5 to 1.0, and continuing to culture. The concentration of IPTG added is, for example, 0.005 mM or more and 1.0 mM or less, preferably 0.01 mM or more and 0.5 mM or less. Expression induction such as IPTG induction can be performed, for example, under conditions well known in the art.

The protein of the present invention may 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 protein of the present invention. Examples of the part include the cells of the cultured transformant of the present invention and the medium after culturing (i.e., the culture supernatant). For example, when the protein of the present invention accumulates in the culture supernatant, the cells may be separated by centrifugation, and the protein of the present invention may be recovered from the culture supernatant obtained. When the protein of the present invention accumulates within the cells (including the periplasm), the cells may be recovered by centrifugation, and then the cells may be disrupted by adding an enzyme treatment agent or a surfactant, and the protein of the present invention may be recovered from the disrupted cells. The protein of the present invention may be recovered from the culture supernatant or cell disrupted product described above by a known method used for separating and purifying proteins. Examples of the recovery method include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation.

The protein of the present invention can be used, for example, for the separation or analysis of immunoglobulins (antibodies). When used for the above purpose, it can be used, for example, in the form of an immunoglobulin adsorbent (hereinafter also simply referred to as the "immunoglobulin adsorbent of the present invention") that contains an insoluble carrier and the protein of the present invention immobilized on the insoluble carrier. Note that, in this specification, "separation of immunoglobulins" is not limited to separation of immunoglobulins from a solution in the presence of contaminants, but also includes separation between immunoglobulins based on structure, properties, activity, etc.

The insoluble carrier is not particularly limited as long as it is insoluble in an aqueous solution. 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 Cytiva), 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 into a column, for example. The insoluble carrier may be, for example, granular or non-granular. The insoluble carrier may be, for example, porous or non-porous.

The immunoglobulin (antibody) to be separated or analyzed in the present invention is not particularly limited as long as it has binding ability to the protein of the present invention. Specifically, the immunoglobulin may have a κ light chain. In addition to the κ light chain, the immunoglobulin may or may not include other regions such as an Fc region. The immunoglobulin may not have an Fc region, such as single chain Fv (scFv), Fab, F(ab') ₂ , IgA, or bispecific T cell-inducing (BiTE) antibody. The immunoglobulin may be a monoclonal antibody or a polyclonal antibody. The immunoglobulin may be, for example, any of IgG, IgM, IgA, IgD, and IgE. The IgG may be, for example, any of IgG1, IgG2, IgG3, and IgG4. The origin of the immunoglobulin is not particularly limited. The immunoglobulin may be derived from a single organism or may be derived from a combination of two or more organisms. Immunoglobulins include chimeric antibodies, humanized antibodies, human antibodies, and variants thereof (e.g., amino acid substitutions). Immunoglobulins also include artificially structurally modified antibodies, such as bispecific antibodies, fusion antibodies of κ light chains with other proteins, and κ light chain-drug complexes (ADCs). The term "immunoglobulin" also includes antibody drugs.

The protein of the present invention may be immobilized on an insoluble carrier, for example, by covalent bonding. As a specific example, the protein of the present invention can be immobilized on an insoluble carrier by covalently bonding the protein to the insoluble carrier via an active group possessed by the insoluble carrier. That is, the insoluble carrier may have an active group on its surface or the like. 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 Cytiva), 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 hydroxy groups, epoxy groups, carboxy groups, 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, 3-maleimidopropionic acid, Examples include pionic 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 groups or amino groups present on the surface of the carrier 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-hydroxybenztriazole.

The protein of the present invention can be immobilized on an insoluble carrier in, for example, a buffer solution. Examples of buffer solutions include acetate buffer, phosphate buffer, MES (2-morpholinoethanesulfonic acid) buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris 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 protein of the present invention. The reaction temperature during immobilization may be, for example, 5°C or higher and 50°C or lower, and is preferably 10°C or higher and 35°C or lower.

The immunoglobulin adsorbent of the present invention can be used for separating immunoglobulins (antibodies) by, for example, forming a column packed with the adsorbent. 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 eluting the immunoglobulins adsorbed to the adsorbent. That is, this specification discloses a method for separating immunoglobulins contained in the solution, which includes 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 immunoglobulin adsorbent of the present invention has an improved immunoglobulin adsorption amount compared to conventional adsorbents, and therefore can separate immunoglobulins contained in the solution more efficiently.

The solution containing immunoglobulin can be added to the column using a liquid delivery means such as a pump. The solution containing immunoglobulin may be solvent-substituted with an appropriate buffer solution before being added to the column. The column may be equilibrated with an appropriate buffer solution before being added to the column. By equilibrating the column, it is expected that, for example, immunoglobulins can be separated with a higher purity. Examples of buffer solutions used for solvent substitution and equilibration include phosphate buffer, acetate buffer, and MES buffer. For example, inorganic salts such as sodium chloride of 1 mM to 1000 mM may be added to such buffer solutions. The buffer solutions used for solvent substitution and equilibration may be the same or different. In addition, if components other than immunoglobulins, such as impurities, remain in the column after the solution containing immunoglobulins is passed through the column, such components may be removed from the column before eluting the immunoglobulins adsorbed to the immunoglobulin adsorbent. Components other than immunoglobulins can be removed from the column using, for example, an appropriate buffer solution. The buffer used to remove components other than immunoglobulins can be eluted, for example, by weakening the interaction between the immunoglobulin and the ligand (the protein of the present invention). Examples of means for weakening the interaction between the immunoglobulin and the ligand (the protein 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, for example, by using an appropriate elution solution. Examples of the elution solution include a buffer that is more acidic than the buffer used for solvent replacement or equilibration. Examples of the acidic buffer include a citrate buffer, a glycine hydrochloride buffer, and an acetate buffer. The pH of the elution solution can be set, for example, within a range that does not impair the function of the immunoglobulin (such as binding to an antigen).

By separating immunoglobulins (antibodies) by the above-mentioned method, separated immunoglobulins can be obtained. That is, in one embodiment, the method for separating immunoglobulins may be a method for producing immunoglobulins, specifically, 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. The fractions can be separated, for example, by a conventional method. Methods for separating fractions include a method of replacing the collection container at regular intervals or at regular volumes, a method of replacing the 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 recovered from fractions containing immunoglobulins. The recovery of immunoglobulins from fractions containing immunoglobulins can be performed, 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.

Comparative Example 1
(1) Preparation of immunoglobulin-binding protein expression vector (1-1) A polypeptide (C3KX7d) 2 -IT-6H3K (SEQ ID NO: 5) was designed in which two polypeptides FpL_C3KX7d consisting of the amino acid sequence set forth in SEQ ID NO: 2, which is a variant of the immunoglobulin-binding domain C3 (SEQ ID NO: 1) of Protein L derived from Finegoldia magna (hereinafter also referred to as FpL), are directly linked together, and an oligopeptide (oligopeptide consisting of the amino acid sequence from amino acid 297 to amino acid 319 of SEQ ID NO: 36 in WO2017/069158, IT, SEQ ID NO: 4) for improving the immobilization rate to an insoluble carrier is added to the C-terminus thereof, a histidine tag ( _6H , SEQ ID NO: 6) as a purification tag, and three lysine residues (3K, hereinafter also referred to as "lysine tag") for improving the immobilization rate to an insoluble carrier are added. The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2 is a polypeptide having the following amino acid substitutions in the amino acid sequence of the immunoglobulin-binding domain C3 (SEQ ID NO:1) of Protein L derived from Finegoldia magna: Glu4Gly (this notation indicates that the amino acid residue corresponding to the fourth glutamic acid in SEQ ID NO:1 is substituted with glycine; the same applies below), Pro6Ser, Lys7Ala, Lys13Arg, Lys22Arg, Lys29Ile, Lys38Glu, Lys48Arg, Glu49Asp, Asn50Tyr, Glu52Asp, Tyr53Phe, Asn62Tyr, and Lys67Arg.

(1-2) A polynucleotide (SEQ ID NO: 7) was designed that encodes (C3KX7d) ₂ -IT-6H3K (SEQ ID NO: 5) designed in (1-1). Of the polynucleotides consisting of the sequence described in SEQ ID NO: 7, from 1 to 210 and from 211 to 420 are polynucleotides (SEQ ID NO: 3) encoding FpL_C3KX7d (SEQ ID NO: 2), from 421 to 489 are polynucleotides (SEQ ID NO: 8) encoding an oligopeptide (IT) consisting of the amino acid sequence described in SEQ ID NO: 4, from 490 to 507 are oligonucleotides encoding a histidine tag (6H, SEQ ID NO: 6), and from 508 to 516 are oligonucleotides encoding a lysine tag (3K). In addition, in SEQ ID NO: 7, the sequence of the polynucleotide encoding FpL_C3KX7d (SEQ ID NO: 2) is a sequence converted from the amino acid sequence of FpL_C3KX7d using Escherichia coli type codons, except that from the 211th to 232nd positions of SEQ ID NO: 7, codons different from the above-mentioned codons have been used to facilitate cloning.

(1-3) A primer (oligonucleotide) for synthesizing the polynucleotide consisting of the sequence shown in SEQ ID NO: 7 designed in (1-2) was designed and synthesized. Specifically,
In order to prepare a polynucleotide fragment consisting of the NcoI restriction enzyme site (CCATGG) of the pET26b vector and the sequence from base 1 to base 231 of SEQ ID NO:7, primers consisting of the sequences shown in SEQ ID NO:9 and SEQ ID NO:10 were used.
In order to prepare a polynucleotide fragment consisting of the sequence from base 211 to base 471 of SEQ ID NO:7, primers consisting of the sequences shown in SEQ ID NO:11 and SEQ ID NO:12 were used.
In order to prepare a polynucleotide fragment consisting of the sequence from base 451 to base 492 of SEQ ID NO:7, primers consisting of the sequences shown in SEQ ID NO:13 and SEQ ID NO:14 were used.
In order to prepare a pET26b vector containing bases 472 to 516 of SEQ ID NO:7 and the downstream of the HindIII restriction enzyme site (AAGGTT) of the pET26b vector and the NcoI restriction enzyme site and its upstream of the pET26b vector by inverse PCR, primers consisting of the sequences shown in SEQ ID NO:15 and SEQ ID NO:16 were used:
were designed and synthesized.

(1-4) After preparing a reaction solution having the composition shown in Table 1, PCR was performed by repeating 30 cycles of reaction with the reaction solution, with the first step being at 98°C for 10 seconds, the second step being at 50°C for 5 seconds, and the third step being at 68°C for 40 seconds. Note that the combination of template DNA/forward primer/reverse primer in Table 1 is any of the combinations shown in <1> to <3> in Table 2.

(1-5) Using the combinations of <1> to <3>, a total of three types of PCR products (polynucleotides) were purified, and TE buffer (10 mM Tris-HCl buffer containing 1 mM EDTA) (pH 8.0) containing 0.05 pmol of each polynucleotide was prepared. Furthermore, 0.05 pmol each of polynucleotides having the sequences described in SEQ ID NO: 13 and SEQ ID NO: 14 was added, and these polynucleotides were linked by a Gibson Assembly reaction using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs).

(1-6) The ligation product from the reaction in (1-5) was used to transform E. coli BL21 (DE3) strain, and the strain was cultured (37°C, 16 hours) on LB (Luria-Bertani) plate medium containing 50 μg/mL kanamycin.

(1-7) After preparing a reaction solution having the composition shown in Table 3, the transformant obtained in (1-6) was added and colony PCR was performed under the same conditions as in (1-4). As a result of colony PCR, transformants (genetically modified E. coli) in which amplification of a PCR product of about 600 to 800 base pairs was confirmed were selected and cultured in LB medium containing 50 μg/mL kanamycin, and then purified with a QIAprep Spin Miniprep kit (manufactured by QIAGEN) to obtain a vector (designated pET-(C3KX7d) ₂ -IT-6H3K) capable of expressing the immunoglobulin-binding protein (C3KX7d) ₂ -IT-6H3K (SEQ ID NO: 5).

(1-8) Of the expression vector pET-(C3KX7d) ₂ -IT-6H3K obtained in (1-7), the polynucleotide encoding the immunoglobulin-binding protein and its surrounding region were subjected to cycle sequencing reaction using a Big Dye Terminator Cycle Sequencing ready Reaction kit (manufactured by Life Sciences Co., Ltd.) based on the chain terminator method, and the nucleotide sequence was analyzed using a fully automated DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Life Sciences Co., Ltd.). In this analysis, an oligonucleotide consisting of the sequence described in SEQ ID NO: 17 or SEQ ID NO: 18 was used as a sequencing primer. As a result of the sequence analysis, it was confirmed that a polynucleotide consisting of the sequence described in SEQ ID NO: 7 was inserted into the expression vector.

(2) Preparation of Immunoglobulin-Binding Protein (2-1) The recombinant E. coli containing pET-(C3KX7d)2-IT-6H3K prepared in (1) 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 pre-cultured (30°C, 16 hours).
(2-2) 10 mL of the culture solution after preculture was inoculated into 1 L of 2xYT liquid medium containing 50 μg/mL kanamycin and cultured at 37°C for 2 to 3 hours, after which 200 μL of 0.5 M IPTG (isopropyl-β-D-thiogalactopyranoside) was added and further cultured overnight at 20°C with shaking.

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

(2-4) The supernatant clarified in (2-3) was added to a column packed with Ni Sepharose 6 Fast Flow (manufactured by Cytiva) previously equilibrated with 0.02 M Tris hydrochloride buffer (pH 7.5) containing 0.5 M sodium chloride and 0.02 M imidazole (hereinafter also referred to simply as "equilibration solution"), and the column was washed with 10 times the amount of equilibration solution as the carrier packed in the column. Then, 0.02 M Tris hydrochloride buffer (pH 7.5) containing 0.5 M sodium chloride and 0.5 M imidazole was passed through the column, and a fraction corresponding to immunoglobulin-binding proteins was collected.

(2-5) The absorbance of the collected fractions was measured using a spectrophotometer to quantify the amount of protein contained in the fractions. SDS-PAGE (SDS-polyacrylamide electrophoresis) was also performed to confirm that the immunoglobulin-binding protein was highly purified.

(3) Preparation of Immunoglobulin Adsorbent The fraction containing the purified immunoglobulin-binding protein (C3KX7d) ₂ -IT-6H3K (SEQ ID NO: 5) prepared in (3-1)(2) was concentrated to 1/10 of the original volume using Amicon Ultra (Merck Millipore), and then a 10-fold amount of sodium phosphate buffer was added to the concentrated solution, followed by concentrating the solution to 1/10 of the original volume using Amicon Ultra (Merck Millipore), thereby concentrating the protein solution and replacing the buffer.

(3-2) TOYOPEARL AF-Formyl-650 (Tosoh Corporation), an insoluble carrier having a formyl group as an active group, was replaced with pure water to prepare a 50% (v/v) slurry. 100 μL of the prepared 50% (v/v) slurry was added to a mini Biospin column (BIORAD Corporation).

(3-3) The concentrated solution of (C3KX7d) ₂ -IT-6H3K prepared in (3-1) was added to a mini Biospin column (manufactured by BIORAD) containing an insoluble carrier so that the final concentration was 10 g/L, and then the column was shaken at 25° C. to bind the insoluble carrier and the protein contained in the column by forming a Schiff base.

(3-4) An appropriate amount of 1M sodium borohydride aqueous solution was added and the mixture was shaken at room temperature for 2 hours to reduce the remaining Schiff base and prepare an immunoglobulin adsorbent. The reaction solution in the spin column was collected and the absorbance was measured with a spectrophotometer to quantify the amount of unadsorbed immunoglobulin-binding protein and calculate the amount of immunoglobulin-binding protein immobilized on the insoluble carrier. The prepared adsorbent was washed with water, then replaced with 20% (v/v) ethanol and stored.

(4) Evaluation of static adsorption amount of immunoglobulin adsorbent (4-1) A 0.1 M glycine hydrochloride solution (pH 2.0) (hereinafter also referred to as acidic solution) was passed through a column packed with the immunoglobulin adsorbent prepared in (3), and then the column was washed by passing a 0.02 M Tris hydrochloride buffer solution (pH 7.5) containing 0.15 M sodium chloride (hereinafter also referred to as washing buffer).

(4-2) An immunoglobulin solution (manufactured by Japan Blood Products Organization) diluted to 43 mg/mL with washing buffer was added to the washed column, and the column was then shaken at 25°C and 1000 rpm for 2 hours using a thermostatic shaking incubator (manufactured by TAITEC) to adsorb the immunoglobulin to the insoluble carrier.

(4-3) After washing off unadsorbed immunoglobulin by passing a washing buffer, an acidic solution was passed through to elute the immunoglobulin adsorbed to the adsorbent. The absorbance of the eluate (wavelength 280 nm) was measured with a spectrophotometer, and the static adsorption amount of the adsorbent was calculated from the amount of immunoglobulin contained in the eluate.

Example 1
(1) An immunoglobulin-binding protein C3KX7d-PRRNRPP-C3KX7d-IT-6H3K (SEQ ID NO: 20) was designed, which comprises a polypeptide in which two FpL_C3KX7d (SEQ ID NO: 2) are linked via a linker consisting of the amino acid sequence described in SEQ ID NO: 19. In SEQ ID NO: 20, the 1st to 70th and 78th to 147th amino acid sequences of FpL_C3KX7d (SEQ ID NO: 2), the 71st to 77th linker (SEQ ID NO: 19) sequence, the 148th to 170th oligopeptide (IT, SEQ ID NO: 4) sequence for improving the immobilization rate to an insoluble carrier, the 171st to 176th histidine tag (6H, SEQ ID NO: 6) sequence for purification, and the 177th to 179th lysine tag (3K) sequence for improving the immobilization rate.

(2) Using a method similar to that of Comparative Example 1 (1-2), a polynucleotide was designed (SEQ ID NO: 21) that encodes a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 20 designed in (1).

(3) Using a method similar to that of Comparative Example 1 (1-3), primers (oligonucleotides) were designed to synthesize a polynucleotide consisting of the sequence described in SEQ ID NO:21 (SEQ ID NO:9, SEQ ID NO:12 to SEQ ID NO:16, SEQ ID NO:22 and SEQ ID NO:23).

(4) PCR was performed in the same manner as in Comparative Example 1 (1-4), except that one of the combinations shown in <3> to <5> in Table 4 was used as the template DNA/forward primer/reverse primer combination.

(5) Using the combinations of <3> to <5>, three types of PCR products (polynucleotides) were purified, and TE buffer (pH 8.0) containing 0.05 pmol of each polynucleotide was prepared. Furthermore, 0.05 pmol of each of the polynucleotides having the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 was added, and these polynucleotides were linked by Gibson assembly reaction using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs).

(6) Using the ligation product after the reaction in (5), Escherichia coli was transformed and cultured in the same manner as in Comparative Example 1 (1-6). After colony PCR in the same manner as in Comparative Example 1 (1-7), transformants were selected in the same manner as in Comparative Example 1 (1-8), and the selected transformants were cultured and plasmids were extracted to obtain a vector (designated pET-C3KX7d-PRRNRPP-C3KX7d-IT-6H3K) capable of expressing the immunoglobulin-binding protein C3KX7d-PRRNRPP-C3KX7d-IT-6H3K (SEQ ID NO: 20).

(7) The nucleotide sequence of the expression vector pET-C3KX7d-PRRNRPP-C3KX7d-IT-6H3K obtained in (6) was analyzed in the same manner as in Comparative Example 1 (1-9). As a result of the sequence analysis, it was confirmed that a polynucleotide consisting of the sequence set forth in SEQ ID NO:21 was inserted into the expression vector.

(8) An immunoglobulin-binding protein was prepared from recombinant E. coli containing pET-C3KX7d-PRRNRPP-C3KX7d-IT-6H3K prepared in (7) in a manner similar to that of Comparative Example 1 (2), and the protein was immobilized on an insoluble carrier in a manner similar to that of Comparative Example 1 (3) to prepare an immunoglobulin adsorbent. The amount of adsorption of the adsorbent was then evaluated in a manner similar to that of Comparative Example 1 (4).

Example 2
(1) An immunoglobulin-binding protein C3KX7d-INRPP-C3KX7d-IT-6H3K (SEQ ID NO: 25) was designed, which comprises a polypeptide in which two FpL_C3KX7d (SEQ ID NO: 2) are linked via a linker consisting of the amino acid sequence described in SEQ ID NO: 24. In SEQ ID NO: 25, the 1st to 70th and 76th to 145th amino acid sequences of FpL_C3KX7d (SEQ ID NO: 2), the 71st to 75th linker (SEQ ID NO: 24) sequence, the 146th to 168th oligopeptide (IT, SEQ ID NO: 4) sequence for improving the immobilization rate to an insoluble carrier, the 169th to 174th histidine tag (6H, SEQ ID NO: 6) sequence for purification, and the 175th to 177th lysine tag (3K) sequence for improving the immobilization rate.

(2) Using a method similar to that of Comparative Example 1 (1-2), a polynucleotide was designed (SEQ ID NO: 26) that encodes a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 25 designed in (1).

(3) Using a method similar to that of Comparative Example 1 (1-3), primers (oligonucleotides) were designed to synthesize a polynucleotide consisting of the sequence set forth in SEQ ID NO:26 (SEQ ID NO:9, SEQ ID NO:12 to SEQ ID NO:16, SEQ ID NO:27 and SEQ ID NO:28).

(4) PCR was performed in the same manner as in Comparative Example 1 (1-4), except that the template DNA/forward primer/reverse primer combination shown in <3>, <6>, or <7> in Table 5 was used.

(5) Using the combinations of <3>, <6>, and <7>, three types of PCR products (polynucleotides) were purified, and TE buffer (pH 8.0) containing 0.05 pmol of each polynucleotide was prepared. Furthermore, 0.05 pmol of each of the polynucleotides having the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 was added, and these polynucleotides were linked by Gibson assembly reaction using NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs).

(6) Using the ligation product after the reaction in (5), Escherichia coli was transformed and cultured in the same manner as in Comparative Example 1 (1-6). After colony PCR in the same manner as in Comparative Example 1 (1-7), transformants were selected in the same manner as in Comparative Example 1 (1-8), and the selected transformants were cultured and plasmids were extracted to obtain a vector (designated pET-C3KX7d-INRPP-C3KX7d-IT-6H3K) capable of expressing the immunoglobulin-binding protein C3KX7d-INRPP-C3KX7d-IT-6H3K (SEQ ID NO: 25).

(7) The nucleotide sequence of the expression vector pET-C3KX7d-INRPP-C3KX7d-IT-6H3K obtained in (6) was analyzed in the same manner as in Comparative Example 1 (1-9). As a result of the sequence analysis, it was confirmed that a polynucleotide consisting of the sequence set forth in SEQ ID NO:26 was inserted into the expression vector.

(8) An immunoglobulin-binding protein was prepared from recombinant E. coli containing pET-C3KX7d-INRPP-C3KX7d-IT-6H3K prepared in (7) in a manner similar to that of Comparative Example 1 (2), and the protein was immobilized on an insoluble carrier in a manner similar to that of Comparative Example 1 (3) to prepare an immunoglobulin adsorbent. The amount of adsorption of the adsorbent was then evaluated in a manner similar to that of Comparative Example 1 (4).

Example 3
(1) An immunoglobulin-binding protein C3KX7d-RPP-C3KX7d-IT-6H3K (SEQ ID NO: 29) was designed, which contains a polypeptide in which two FpL_C3KX7d (SEQ ID NO: 2) are linked together via a linker consisting of an arginine residue and two consecutive proline residues (Arg-Pro-Pro). In SEQ ID NO: 29, the first to seventieth and seventy-fourth to 143rd amino acid sequences of FpL_C3KX7d (SEQ ID NO: 2), the seventy-first to seventy-third sequences are the linker sequence, the 144th to 166th sequences are oligopeptide (IT, SEQ ID NO: 4) sequences for improving the immobilization rate on an insoluble carrier, the 167th to 172nd sequences are histidine tag (6H, SEQ ID NO: 6) sequences for purification, and the 173rd to 175th sequences are lysine tag (3K) sequences for improving the immobilization rate.

(2) Using a method similar to that of Comparative Example 1 (1-2), a polynucleotide was designed (SEQ ID NO: 30) that encodes a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 29 designed in (1).

(3) Using a method similar to that of Comparative Example 1 (1-3), primers (oligonucleotides) were designed to synthesize a polynucleotide consisting of the sequence set forth in SEQ ID NO:30 (SEQ ID NO:9, SEQ ID NO:12 to SEQ ID NO:16, SEQ ID NO:31 and SEQ ID NO:32).

(4) PCR was performed in the same manner as in Comparative Example 1 (1-4), except that the template DNA/forward primer/reverse primer combination shown in <3>, <8>, or <9> in Table 6 was used.

(5) Using the combinations of <3>, <8>, and <9>, three types of PCR products (polynucleotides) were purified, and TE buffer (pH 8.0) containing 0.05 pmol of each polynucleotide was prepared. Furthermore, 0.05 pmol of each of the polynucleotides having the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 was added, and these polynucleotides were linked by Gibson assembly reaction using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs).

(6) Using the ligation product after the reaction in (5), Escherichia coli was transformed and cultured in the same manner as in Comparative Example 1 (1-6). After colony PCR in the same manner as in Comparative Example 1 (1-7), transformants were selected in the same manner as in Comparative Example 1 (1-8), and the selected transformants were cultured and plasmids were extracted to obtain a vector (designated pET-C3KX7d-RPP-C3KX7d-IT-6H3K) capable of expressing the immunoglobulin-binding protein C3KX7d-RPP-C3KX7d-IT-6H3K (SEQ ID NO: 29).

(7) The nucleotide sequence of the expression vector pET-C3KX7d-RPP-C3KX7d-IT-6H3K obtained in (6) was analyzed in the same manner as in Comparative Example 1 (1-9). As a result of the sequence analysis, it was confirmed that a polynucleotide consisting of the sequence set forth in SEQ ID NO: 30 was inserted into the expression vector.

(8) An immunoglobulin-binding protein was prepared from recombinant E. coli containing pET-C3KX7d-RPP-C3KX7d-IT-6H3K prepared in (7) in a manner similar to that of Comparative Example 1 (2), and the protein was immobilized on an insoluble carrier in a manner similar to that of Comparative Example 1 (3) to prepare an immunoglobulin adsorbent. The amount of adsorption of the adsorbent was then evaluated in a manner similar to that of Comparative Example 1 (4).

Example 4
(1) An immunoglobulin-binding protein C3KX7d-PP-C3KX7d-IT-6H3K (SEQ ID NO: 33) was designed, which contains a polypeptide in which two FpL_C3KX7d (SEQ ID NO: 2) are linked together via a linker consisting of two consecutive proline residues (Pro-Pro). In SEQ ID NO: 33, the first to seventieth and seventy-third to eleventh 142nd amino acid sequences of FpL_C3KX7d (SEQ ID NO: 2), the seventy-first and seventy-second sequences are the linker sequences, the 143rd to 165th sequences are oligopeptide (IT, SEQ ID NO: 4) sequences for improving the immobilization rate on an insoluble carrier, the 166th to 171st sequences are histidine tag (6H, SEQ ID NO: 6) sequences for purification, and the 172nd to 174th sequences are lysine tag (3K) sequences for improving the immobilization rate.

(2) Using a method similar to that of Comparative Example 1 (1-2), a polynucleotide encoding a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 33 designed in (1) was designed (SEQ ID NO: 34).

(3) Using a method similar to that of Comparative Example 1 (1-3), primers (oligonucleotides) were designed to synthesize a polynucleotide consisting of the sequence set forth in SEQ ID NO:34 (SEQ ID NO:9, SEQ ID NO:12 to SEQ ID NO:16, SEQ ID NO:35 and SEQ ID NO:36).

(4) PCR was performed in the same manner as in Comparative Example 1 (1-4), except that the template DNA/forward primer/reverse primer combination shown in <3>, <10>, or <11> in Table 7 was used.

(5) Using the combinations of <3>, <10>, and <11>, three types of PCR products (polynucleotides) obtained were purified, and TE buffer (pH 8.0) containing 0.05 pmol of each polynucleotide was prepared. Furthermore, 0.05 pmol of each of the polynucleotides having the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 was added, and these polynucleotides were linked by Gibson assembly reaction using NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs).

(6) Using the ligation product after the reaction in (5), Escherichia coli was transformed and cultured in the same manner as in Comparative Example 1 (1-6). After colony PCR in the same manner as in Comparative Example 1 (1-7), transformants were selected in the same manner as in Comparative Example 1 (1-8), and the selected transformants were cultured and plasmids were extracted to obtain a vector (designated pET-C3KX7d-PP-C3KX7d-IT-6H3K) capable of expressing the immunoglobulin-binding protein C3KX7d-PP-C3KX7d-IT-6H3K (SEQ ID NO: 33).

(7) The nucleotide sequence of the expression vector pET-C3KX7d-PP-C3KX7d-IT-6H3K obtained in (6) was analyzed in the same manner as in Comparative Example 1 (1-9). As a result of the sequence analysis, it was confirmed that a polynucleotide consisting of the sequence set forth in SEQ ID NO: 34 was inserted into the expression vector.

(8) An immunoglobulin-binding protein was prepared from recombinant E. coli containing pET-C3KX7d-PP-C3KX7d-IT-6H3K prepared in (7) in a manner similar to that of Comparative Example 1 (2), and the protein was immobilized on an insoluble carrier in a manner similar to that of Comparative Example 1 (3) to prepare an immunoglobulin adsorbent. The amount of adsorption of the adsorbent was then evaluated in a manner similar to that of Comparative Example 1 (4).

Comparative Example 2
(1) An immunoglobulin-binding protein C3KX7d-INKPP-C3KX7d-IT-6H3K (SEQ ID NO: 38) was designed, which comprises a polypeptide in which two FpL_C3KX7d (SEQ ID NO: 2) are linked via a linker consisting of the amino acid sequence described in SEQ ID NO: 37. In SEQ ID NO: 38, the 1st to 70th and 76th to 145th amino acid sequences of FpL_C3KX7d (SEQ ID NO: 2) are the amino acid sequence of FpL_C3KX7d, the 71st to 75th are the linker (SEQ ID NO: 37) sequence, the 146th to 168th are the oligopeptide (IT, SEQ ID NO: 4) sequence for improving the immobilization rate to an insoluble carrier, the 169th to 174th are the histidine tag (6H, SEQ ID NO: 6) sequence for purification, and the 175th to 177th are the lysine tag (3K) sequence for improving the immobilization rate.

(2) Using a method similar to that of Comparative Example 1 (1-2), a polynucleotide encoding a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 38 designed in (1) was designed (SEQ ID NO: 39).

(3) Using a method similar to that of Comparative Example 1 (1-3), primers (oligonucleotides) were designed to synthesize a polynucleotide consisting of the sequence set forth in SEQ ID NO:39 (SEQ ID NO:9, SEQ ID NO:12 to SEQ ID NO:16, SEQ ID NO:40 and SEQ ID NO:41).

(4) PCR was performed in the same manner as in Comparative Example 1 (1-4), except that the template DNA/forward primer/reverse primer combination shown in <3>, <12>, or <13> in Table 8 was used.

(5) Using the combinations of <3>, <12>, and <13>, three types of PCR products (polynucleotides) were purified, and TE buffer (pH 8.0) containing 0.05 pmol of each polynucleotide was prepared. Furthermore, 0.05 pmol of each of the polynucleotides having the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 was added, and these polynucleotides were linked by Gibson assembly reaction using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs).

(6) Using the ligation product after the reaction in (5), Escherichia coli was transformed and cultured in the same manner as in Comparative Example 1 (1-6). After colony PCR in the same manner as in Comparative Example 1 (1-7), transformants were selected in the same manner as in Comparative Example 1 (1-8), and the selected transformants were cultured and plasmids were extracted to obtain a vector (designated pET-C3KX7d-INKPP-C3KX7d-IT-6H3K) capable of expressing the immunoglobulin-binding protein C3KX7d-INKPP-C3KX7d-IT-6H3K (SEQ ID NO: 38).

(7) The nucleotide sequence of the expression vector pET-C3KX7d-INKPP-C3KX7d-IT-6H3K obtained in (6) was analyzed in the same manner as in Comparative Example 1 (1-9). As a result of the sequence analysis, it was confirmed that a polynucleotide consisting of the sequence set forth in SEQ ID NO: 39 was inserted into the expression vector.

(8) An immunoglobulin-binding protein was prepared from recombinant E. coli containing pET-C3KX7d-INKPP-C3KX7d-IT-6H3K prepared in (7) in a manner similar to that of Comparative Example 1 (2), and the protein was immobilized on an insoluble carrier in a manner similar to that of Comparative Example 1 (3) to prepare an immunoglobulin adsorbent. The amount of adsorption of the adsorbent was then evaluated in a manner similar to that of Comparative Example 1 (4).

Comparative Example 3
(1) An immunoglobulin-binding protein C3KX7d-P-C3KX7d-IT-6H3K (SEQ ID NO: 42) was designed, which contains a polypeptide in which two FpL_C3KX7d (SEQ ID NO: 2) are linked via a linker consisting of one proline residue (Pro). In SEQ ID NO: 42, the first to the seventieth and the seventy-second to the fourteenth are the amino acid sequence of FpL_C3KX7d (SEQ ID NO: 2), the seventy-first is the proline residue (linker), the fourteenth to the sixteenth is an oligopeptide (IT, SEQ ID NO: 4) sequence for improving the immobilization rate to an insoluble carrier, the sixteenth to the seventeenth is a histidine tag (6H, SEQ ID NO: 6) sequence for purification, and the seventeenth to the seventeenth is a lysine tag (3K) sequence for improving the immobilization rate.

(2) Using a method similar to that of Comparative Example 1 (1-2), a polynucleotide was designed (SEQ ID NO: 43) that encodes a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 42 designed in (1).

(3) Using a method similar to that of Comparative Example 1 (1-3), primers (oligonucleotides) were designed to synthesize a polynucleotide consisting of the sequence set forth in SEQ ID NO:43 (SEQ ID NO:9, SEQ ID NO:12 to SEQ ID NO:16, SEQ ID NO:44 and SEQ ID NO:45).

(4) PCR was performed in the same manner as in Comparative Example 1 (1-4), except that the template DNA/forward primer/reverse primer combination shown in <3>, <14>, or <15> in Table 9 was used.

(5) Using the combinations of <3>, <14>, and <15>, three types of PCR products (polynucleotides) were purified, and TE buffer (pH 8.0) containing 0.05 pmol of each polynucleotide was prepared. Furthermore, 0.05 pmol of each of the polynucleotides having the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 was added, and these polynucleotides were linked by Gibson assembly reaction using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs).

(6) Using the ligation product after the reaction in (5), Escherichia coli was transformed and cultured in the same manner as in Comparative Example 1 (1-6). After colony PCR in the same manner as in Comparative Example 1 (1-7), transformants were selected in the same manner as in Comparative Example 1 (1-8), and the selected transformants were cultured and plasmids were extracted to obtain a vector (designated pET-C3KX7d-P-C3KX7d-IT-6H3K) capable of expressing the immunoglobulin-binding protein C3KX7d-P-C3KX7d-IT-6H3K (SEQ ID NO: 42).

(7) The nucleotide sequence of the expression vector pET-C3KX7d-P-C3KX7d-IT-6H3K obtained in (6) was analyzed in the same manner as in Comparative Example 1 (1-9). As a result of the sequence analysis, it was confirmed that a polynucleotide consisting of the sequence set forth in SEQ ID NO: 43 was inserted into the expression vector.

(8) An immunoglobulin-binding protein was prepared from recombinant E. coli containing pET-C3KX7d-P-C3KX7d-IT-6H3K prepared in (7) in a manner similar to that of Comparative Example 1 (2), and the protein was immobilized on an insoluble carrier in a manner similar to that of Comparative Example 1 (3) to prepare an immunoglobulin adsorbent. The amount of adsorption of the adsorbent was then evaluated in a manner similar to that of Comparative Example 1 (4).

The static adsorption amounts of the immunoglobulin adsorbents in Examples 1 to 4 and Comparative Examples 1 to 3 are summarized in Table 10. Note that in Table 10, the linker length refers to the length of the peptide inserted at the linking position between the immunoglobulin-binding domains FpL_C3KX7d (SEQ ID NO: 2) of FpL. Therefore, deletions or additions to the terminal sequences of each domain are not included in the linker length.

When FpL immunoglobulin-binding domains were linked together, the static adsorption amount was improved by linking them via an oligopeptide consisting of the amino acid sequence described in any one of SEQ ID NO: 19 (Example 1), SEQ ID NO: 24 (Example 2), Arg-Pro-Pro (Example 3), and Pro-Pro (Example 4) compared to when the FpL immunoglobulin-binding domains were directly linked together (Comparative Example 1) (Comparative Example 1: 44.8 g/L, Example 1: 47.2 g/L, Example 2: 47.8 g/L, Example 3: 51.9 g/L, Example 4: 48.8 g/L). On the other hand, when the FpL immunoglobulin-binding domains were linked together via an oligopeptide consisting of the sequence described in SEQ ID NO: 37 (Comparative Example 2) or one proline residue (Comparative Example 3), the static adsorption amount was reduced compared to when the FpL immunoglobulin-binding domains were directly linked together (Comparative Example 1) (Comparative Example 1: 44.8 g/L, Comparative Example 2: 39.9 g/L, Comparative Example 3: 43.3 g/L). The amount of immunoglobulin-binding protein immobilized on the insoluble carrier in Examples 1 to 4 and Comparative Examples 1 to 3 was almost the same.

The above results show that in an immunoglobulin-binding protein that contains two or more polypeptides that contain at least the immunoglobulin-binding domain of FpL and a linker for linking the polypeptides, the static adsorption amount of an immunoglobulin adsorbent that contains an insoluble carrier and the protein immobilized on the insoluble carrier is improved by using an oligopeptide consisting of 2 to 8 residues that contains at least two or more prolines but does not contain any lysine residues as the linker.

Claims (9)

An immunoglobulin-binding protein comprising two or more polypeptides each including at least an immunoglobulin-binding domain of Protein L derived from a bacterium belonging to the genus Finegoldia, and a linker for linking the polypeptides,
The above-mentioned protein, wherein the linker is an oligopeptide consisting of 2 to 8 residues, which contains at least two or more consecutive proline residues but does not contain any lysine residues. The protein according to claim 1, wherein the immunoglobulin-binding domain of Protein L derived from a bacterium belonging to the genus Finegoldia is any one of the following (1) to (4):
(1) A polypeptide comprising at least the amino acid sequence set forth in SEQ ID NO:1;
(2) A polypeptide comprising at least a partial sequence of the amino acid sequence set forth in SEQ ID NO: 1 and having immunoglobulin activity;
(3) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a partial sequence thereof, provided that said sequence contains substitution, deletion and/or addition of one or several amino acid residues at one or several positions, and has immunoglobulin-binding activity;
(4) A polypeptide comprising at least an amino acid sequence having 70% or more homology to the amino acid sequence set forth in SEQ ID NO: 1 or a partial sequence thereof, and having immunoglobulin activity. A polynucleotide encoding the protein according to claim 1 or 2. An expression vector comprising the polynucleotide of claim 3. A genetically modified host comprising the polynucleotide of claim 3. The genetically modified host according to claim 5, wherein the host is Escherichia coli. A method for producing the protein, comprising the steps of culturing the recombinant host according to claim 5 to express the protein according to claim 1 or 2, and recovering the expressed protein. An immunoglobulin adsorbent comprising an insoluble carrier and the protein according to claim 1 or 2 immobilized on the insoluble carrier. 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 8 to adsorb the immunoglobulins to the adsorbent, and eluting the immunoglobulins adsorbed to the adsorbent.