Immunoglobulin-binding protein variants with increased alkaline tolerance and uses thereof

Abstract

To provide immunoglobulin-binding protein variants with increased alkali-tolerance and uses thereof.SOLUTION: Provided are immunoglobulin-binding protein variants with increased alkali tolerance, and uses thereof, and more particularly, immunoglobulin-binding protein variants with increased alkali tolerance relative to the wild type due to mutations of amino acids at specific positions in the B3 domain of the immunoglobulin-binding proteins.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention provides an immunoglobulin-binding protein variant with increased alkaline tolerance, more specifically, an immunoglobulin-binding protein variant with improved alkaline tolerance compared to the wild-type by mutating an amino acid at a specific position in the B3 domain of the immunoglobulin-binding protein, and uses thereof. [Background technology]

[0002] Monoclonal antibodies are secreted into the culture medium during the cultivation of genetically engineered animal cells, and exist at very low concentrations due to the mixture of many proteins secreted by the cells themselves and proteins in the culture medium. Therefore, removing impurities other than the target monoclonal antibody is an important step in antibody production. The monoclonal antibody isolation and purification process mainly uses affinity chromatography, which utilizes antibody affinity ligands that can selectively recover only monoclonal antibodies from the culture medium. Protein A, protein G, protein L, etc. are used as antibody affinity ligands.

[0003] Immunoglobulin-binding bacterial proteins such as protein A, protein G, protein A / G, and protein L are widely used to purify and detect immune proteins (antibodies, antibody fragments, etc.). These immunoglobulin-binding proteins have different antibody-binding patterns, such as the recognition site on the antibody and the type of antibody they bind to.

[0004] Among these, Protein L was first isolated from the bacterium Peptostreptococcus magnus (Finegoldia magna) and was named after its ability to bind to immunoglobulins through light chain interaction (L chain interaction). Unlike Protein A and Protein G, which bind to the Fc region of immunoglobulins (antibodies), Protein L binds to antibodies through light chain (e.g., kappa) interactions. Because Protein L is not affected by the heavy chain portion during antibody binding, it has the advantage of being able to bind to a wider range of antibody types than Protein A and Protein G, including antibody fragments such as scFv (single chain variable fragment) and Fab fragments.

[0005] Generally, cleaning-in-place (CIP) is performed after antibody purification to remove various contaminants such as nucleic acids, lipids, proteins, and microorganisms remaining on the resin. NaOH is the most widely used resin cleaning agent for this purpose. However, since protein-based purification resins are sensitive to alkali, it is necessary to develop an immunoglobulin-binding protein that is stable even under alkaline conditions in order to apply it more effectively to antibody purification. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Republic of Korea Patent No. 10-1857953 Summary of the Invention [Problem to be solved by the invention]

[0007] The present application provides immunoglobulin-binding protein variants with increased alkaline tolerance and uses thereof. The immunoglobulin-binding protein variants may be variants of protein L or functional portions thereof (e.g., the B3 domain), and are characterized by improved (increased) alkaline tolerance (immunoglobulin-binding ability, stability, etc. under alkaline conditions) compared to the wild-type protein or functional portions thereof.

[0008] More specifically, one example of the present application is a B3 domain of protein L (e.g., counting from the second amino acid, proline (Pro, P), in the amino acid sequence of SEQ ID NO: 4),

[0009] The present invention provides a polypeptide in which one or more amino acids selected from the group consisting of glutamic acid (Glu, E) at the 28th amino acid, lysine (Lys, K) at the 43rd amino acid, tyrosine (Tyr, Y) at the 13th amino acid, aspartic acid (Asp, D) at the 51st amino acid, and asparagine (Asn, N) at the 60th amino acid are substituted with amino acids different from those in the original.

[0010] In one example, the polypeptide may be a polypeptide that includes one or more substitutions selected from the following in the B3 domain of protein L (e.g., counting from the second amino acid, proline (Pro, P), in the amino acid sequence of SEQ ID NO: 4):

[0011] substitution of amino acid 28 (E) with glycine (Gly, G), alanine (Ala, A), leucine (Leu, L), proline (Pro, P), or tryptophan (Trp, W); substitution of amino acid 43 (K) with proline (Pro, P) or glutamic acid (Glu, E); The 13th amino acid (Y) is replaced by phenylalanine (Phe, F); The 51st amino acid (D) is substituted with valine (Val, V) or threonine (Thr, T); and The 60th amino acid (N) is replaced with phenylalanine (Phe, F).

[0012] Another example provides a polypeptide multimer containing two or more repeating units (monomers) of the above polypeptide.

[0013] The polypeptide or polypeptide multimer may additionally comprise one or more selected from the group consisting of the A, B1, B2, B4, C, W, and M domains of protein L.

[0014] Another example provides a protein L variant comprising, as the B3 domain, the above polypeptide or a polypeptide multimer comprising two or more repeats of the above polypeptide.

[0015] The above-mentioned polypeptides, polypeptide multimers, and / or protein L mutants may have superior alkaline resistance (e.g., immunoglobulin binding ability, stability, etc. under alkaline conditions) compared to the B3 domain of wild-type protein L (e.g., SEQ ID NO: 4) or a multimer containing the same or wild-type protein L.

[0016] Other examples provide nucleic acid molecules encoding the aforementioned polypeptides, polypeptide multimers, and / or Protein L variants.

[0017] Another example provides a recombinant vector containing the nucleic acid molecule, which can be used as an expression vector to express the polypeptide, polypeptide multimer, and / or protein L variant in a suitable host cell.

[0018] Another example provides a recombinant cell containing the nucleic acid molecule or recombinant vector. The recombinant cell may be a host cell transformed with the nucleic acid molecule or recombinant vector, and may be a cell capable of expressing the polypeptide, polypeptide multimer, and / or Protein L variant.

[0019] Another example is a matrix (resin) comprising the polypeptide, a polypeptide multimer containing two or more repeat units of the polypeptide, or multiple ligands including a protein L mutant containing the polypeptide or polypeptide multimer as a B3 domain coupled to a solid support. The matrix may be used in techniques for separating and / or purifying proteins by adsorption, such as chromatography.

[0020] Other examples include: (1) the polypeptide; (2) A polypeptide multimer containing two or more repeating units consisting of the polypeptide. (3) a protein L mutant containing the polypeptide or polypeptide multimer as a B3 domain; (4) a nucleic acid molecule encoding the polypeptide, polypeptide multimer, or protein L variant; (5) A recombinant vector containing the nucleic acid molecule. (6) a recombinant cell containing the nucleic acid molecule or recombinant vector; and (7) A matrix in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein L variant are coupled to a solid support; The present invention provides a composition for immunoglobulin binding and / or a composition for immunoglobulin separation and / or purification, which comprises one or more members selected from the group consisting of:

[0021] Other examples include: (1) the polypeptide; (2) A polypeptide multimer containing two or more repeating units consisting of the polypeptide. (3) a protein L mutant containing the polypeptide or polypeptide multimer as a B3 domain; (4) a nucleic acid molecule encoding the polypeptide, polypeptide multimer, or protein L variant; (5) A recombinant vector containing the nucleic acid molecule. (6) a recombinant cell containing the nucleic acid molecule or recombinant vector; and (7) A matrix in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein L variant are coupled to a solid support; The present invention provides a use of one or more of the following selected from the group consisting of: for immunoglobulin binding, and / or immunoglobulin separation and / or purification.

[0022] Other examples include: (a) the polypeptide; (b) a polypeptide multimer comprising two or more repeating units consisting of the polypeptide; (c) a protein L mutant comprising the polypeptide or polypeptide multimer as a B3 domain; and (d) a chromatographic matrix in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein L variant are coupled to a solid support; The present invention provides a composition for separating and / or purifying immunoglobulins, comprising one or more members selected from the group consisting of:

[0023] Other examples include: (a) the polypeptide; (b) a polypeptide multimer comprising two or more repeating units consisting of the polypeptide; (c) a protein L mutant comprising the polypeptide or polypeptide multimer as a B3 domain; and (d) a chromatographic matrix in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein L variant are coupled to a solid support; The present invention provides a method for the separation and / or purification of immunoglobulins, comprising the steps of:

[0024] Another example is to add to a sample (e.g., a liquid sample) containing immunoglobulins: (a) the polypeptide; (b) a polypeptide multimer comprising two or more repeating units consisting of the polypeptide; (c) a protein L mutant comprising the polypeptide or polypeptide multimer as a B3 domain; and (d) a chromatographic matrix in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein L variant are coupled to a solid support; and adsorbing the immunoglobulin.

[0025] Another example provides a method for separating or purifying one or more target compounds from a liquid, comprising the step of contacting a sample (e.g., a liquid sample) containing the target compounds with one or more selected from the group consisting of (a) the polypeptide, (b) a polypeptide multimer containing two or more repeating units consisting of the polypeptide, (c) a protein L variant containing the polypeptide or polypeptide multimer as a B3 domain, and (d) a chromatography matrix in which multiple ligands containing the polypeptide, polypeptide multimer, or protein L variant are coupled to a solid support, thereby adsorbing the target compounds. [Means for solving the problem]

[0026] The present application provides a protein L B3 domain mutant with enhanced alkaline tolerance and uses thereof.

[0027] Definition of Terms As used herein, the phrase "a polynucleotide (which may be confused with "gene or nucleic acid molecule") or polypeptide (which may be confused with "protein")" "comprises a specific nucleic acid sequence or amino acid sequence," "consists (essentially) of a specific nucleic acid sequence or amino acid sequence," or "is expressed by a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide essentially comprises the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including "substantially equivalent sequences" in which mutations (deletions, substitutions, modifications, and / or additions) have been made to the specific nucleic acid sequence or amino acid sequence (or as not excluding the mutations) within the scope of maintaining the original function (binding to an immunoglobulin light chain) and / or the desired function (e.g., increased alkaline resistance compared to the wild-type protein L B3 domain) of the polynucleotide or polypeptide.

[0028] In one example, a polynucleotide or polypeptide "comprises" or "consists of or displays a particular nucleic acid or amino acid sequence" means that the polynucleotide or polypeptide is (i) essentially comprising or consisting of the specified nucleic acid or amino acid sequence; or (ii) It may mean that the nucleic acid sequence or amino acid sequence consists of or essentially contains an amino acid sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity to the specific nucleic acid sequence or amino acid sequence, and maintains the original function and / or intended function. In this specification, the original function may be the immunoglobulin (particularly the light chain region) binding ability of the wild-type protein L B3 domain (in the case of an amino acid sequence) or the function of encoding a protein having such immunoglobulin-binding ability (in the case of a nucleic acid sequence), and the target function may be increased alkaline tolerance compared to the wild-type protein L B3 domain (in the case of an amino acid sequence) or the function of encoding a protein having such increased alkaline tolerance (in the case of a nucleic acid sequence).

[0029] The polypeptides provided herein may comprise an amino acid sequence defined as a particular SEQ ID NO: or may be a functional variant thereof. The term "functional variant" includes all similar sequences that contain one or more additional mutations at amino acid positions that do not affect the improved chemical stability and / or affinity (binding ability) of the polypeptide for immunoglobulins in an increased pH environment.

[0030] As used herein, the term "sequence identity" refers to the degree of identity with a given nucleic acid or amino acid sequence, and can be expressed as a percentage (%). Homology for nucleic acid sequences can be determined, for example, using the literature algorithm BLAST (see Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873, 1993) or Pearson's FASTA (see Methods Enzymol., 183, 63, 1990). Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed (see http: / / www.ncbi.nlm.nih.gov).

[0031] As used herein, the term "amino acid residue at a specific position in the amino acid sequence of a polypeptide" may be interpreted as encompassing an amino acid residue at a specific position in the amino acid sequence, or an amino acid residue at a position corresponding to the specific position in an isotype protein derived from the same species and / or a protein derived from a different species that has the same activity as the protein.

[0032] In the amino acid sequence of a polypeptide provided herein, when the first residue at the N-terminus is methionine (M), the methionine may be present in the natural state or may be produced through a recombinant process. When the polypeptide provided herein is produced recombinantly, polypeptides containing an amino acid sequence excluding the first N-terminal residue, methionine (M), and / or a signal peptide located at the N-terminus are also included within the scope of the present specification. Furthermore, when the first N-terminal residue of a protein's amino acid sequence is not methionine, a methionine may be added to the N-terminus of the first residue when the protein is produced recombinantly.

[0033] Unless otherwise specified, amino acid positions used herein are counted from the second amino acid following methionine (M) in the case of a recombinant protein containing methionine (M) as the first amino acid residue at the N-terminus. For example, in the case of a polypeptide of SEQ ID NO: 4 containing methionine (M) as the first amino acid at the N-terminus, the "28th amino acid" refers to the amino acid residue at the 28th position (i.e., the 29th amino acid in SEQ ID NO: 4), counting proline (P), the second amino acid following the first amino acid methionine, as the first amino acid.

[0034] The single letter (three letter) amino acids used herein refer to the following amino acids according to standard abbreviation conventions in the biochemistry field: A (Ala): alanine; C (Cys): cysteine; D (Asp): aspartic acid; E (Glu): glutamic acid; F (Phe): phenylalanine; G (Gly): glycine; H (His): histidine; I (IIe): isoleucine; K (Lys): lysine; L (Leu): leucine; M (Met): methionine; N (Asn): asparagine; O (Ply): pyrrolysine; P (Pro): proline; Q (Gln): glutamine; R (Arg): arginine; S (Ser): serine; T (Thr): threonine; U (Sec): selenocysteine; V (Val): valine; W (Trp): tryptophan; Y (Tyr): tyrosine.

[0035] As used herein, the notation "(single amino acid letter) (amino acid position) (single amino acid letter)" means that the amino acid indicated first at that amino acid position in a native polypeptide is substituted with the amino acid indicated second. For example, E28G means that the glutamic acid (E) corresponding to residue 28 in a native polypeptide is substituted with glycine (G). Furthermore, a slash ( / ) at the amino acid indicated second can mean "or." For example, E28G / A can mean that the glutamic acid (E) corresponding to residue 28 in a native polypeptide is substituted with glycine (G) or alanine (A).

[0036] As used herein, the term "about" is an expression intended to include all numerical values ​​in a range equivalent to or similar to the numerical value given thereafter, and may be interpreted as including a range of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, ±0.05, or ±0.01 based on the numerical value given thereafter, but is not limited thereto.

[0037] The present invention will now be described in more detail.

[0038] Immunoglobulin-binding protein variants: Protein L B3 domain variants, and multimers containing the same, immunoglobulin-binding proteins, and protein L variants The present application provides an immunoglobulin-binding protein variant with enhanced alkaline tolerance and uses thereof. The immunoglobulin-binding protein variant may be a variant of protein L or a functional portion thereof (e.g., the B3 domain), and is characterized by improved (increased) alkaline tolerance (immunoglobulin-binding ability, stability, etc. under alkaline conditions) compared to the wild-type protein or a functional portion thereof.

[0039] The immunoglobulin-binding protein of the present application was first isolated from the bacterium Peptostreptococcus magnus (Finegoldia magna) and was named protein L after it was confirmed to bind to immunoglobulins through light chain interaction. Protein L from Peptostreptococcus magnus (e.g., NCBI Accession No. AAA25612.1) consists of 719 amino acid residues and has a molecular weight of 95 kD (measured by SDS-PAGE in the presence of a reducing agent (2-mercaptoethanol)) or 76 kD (measured by gel chromatography in the presence of 6 M guanidine hydrochloride), and is an acidic protein (pI 4.0). Unlike Protein A and Protein G, which bind to the Fc region of immunoglobulins (antibodies), Protein L binds to antibodies through light chain (e.g., kappa) interactions. Because Protein L is not affected by the heavy chain portion during antibody binding, it can bind to a wider range of antibody types than Protein A and Protein G, including all subtypes of antibodies, including IgG, IgM, IgA, IgE, and IgD, and can also bind to antibody fragments such as scFv (single chain variable fragment) and Fab fragments.

[0040] Due to such immunoglobulin light chain (kappa) binding activity, Protein L can be usefully used for purifying antibodies (full length or fragments) containing kappa type light chains (all or part).

[0041] Thus, the immunoglobulin-binding protein variants of the present application may be those that are improved based on protein L, particularly the B3 domain of protein L. The protein L may be derived from Peptostreptococcus magnus, and may be, for example, represented by the amino acid sequence of NCBI Accession No. AAA25612.1 (SEQ ID NO: 50), or may have an amino acid sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity to the amino acid sequence, and may be a protein that maintains immunoglobulin (particularly, light chain) binding ability, but is not limited thereto.

[0042] The B3 domain of protein L may be the B3 domain of protein L derived from Peptostreptococcus magnus (e.g., SEQ ID NO: 50), and may be represented by the amino acid sequence of SEQ ID NO: 4, or may be a protein having an amino acid sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity to the amino acid sequence, and maintaining immunoglobulin (particularly, light chain) binding ability, but is not limited thereto. The B3 domain of the protein L may be encoded by the nucleic acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0043] By introducing mutations by amino acid substitution at specific positions in the B3 domain, the alkaline resistance of the B3 domain, multimers containing the B3 domain, and / or protein L can be increased while maintaining its binding ability to immunoglobulins (particularly the light chain region).

[0044] One example is a polypeptide (B3 domain mutant of protein L) in which one or more (e.g., 1, 2, 3, 4, or 5) amino acids selected from the group consisting of glutamic acid (Glu, E) corresponding to the 28th amino acid residue, lysine (Lys, K) corresponding to the 43rd amino acid residue, tyrosine (Tyr, Y) corresponding to the 13th amino acid residue, aspartic acid (Asp, D) corresponding to the 51st amino acid residue, and asparagine (Asn, N) corresponding to the 60th amino acid residue in the B3 domain of protein L (based on the B3 domain of protein L) are substituted with amino acids different from the original.

[0045] The other amino acid may be selected from the group consisting of alanine (A, Ala), asparagine (N, Asn), threonine (T, Thr), glutamic acid (E, Glu), serine (S, Ser), valine (V, Val), isoleucine (I, Ile), leucine (L, Leu), aspartic acid (D, Asp), cysteine ​​(C, Cys), glutamine (Q, Gln), methionine (M, Met), phenylalanine (F, Phe), proline (P, Pro), tryptophan (W, Trp), tyrosine (Y, Tyr), arginine (R, Arg), histidine (H, His), lysine (K, Lys), and glycine (G, Gly), and may be an amino acid different from the amino acid residue at that position in the B3 domain of wild-type protein L.

[0046] In one embodiment, the polypeptide can include one or more (e.g., 1, 2, 3, 4, or 5) substitutions in the B3 domain of protein L selected from the following: substitution of amino acid 28 (E) with glycine (Gly, G), alanine (Ala, A), leucine (Leu, L), proline (Pro, P), or tryptophan (Trp, W); substitution of amino acid 43 (K) with proline (Pro, P) or glutamic acid (Glu, E); The 13th amino acid (Y) is replaced by phenylalanine (Phe, F); The 51st amino acid (D) is substituted with valine (Val, V) or threonine (Thr, T); and The 60th amino acid (N) is replaced by phenylalanine (Phe, F).

[0047] In another specific example, the polypeptide may be one in which the 28th amino acid (E) in the B3 domain of protein L is substituted with glycine (Gly, G), alanine (Ala, A), leucine (Leu, L), proline (Pro, P), or tryptophan (Trp, W), e.g., glycine (Gly, G).

[0048] In another embodiment, the polypeptide comprises, in the B3 domain of protein L: The 28th amino acid (E) is substituted with glycine (Gly, G), alanine (Ala, A), leucine (Leu, L), proline (Pro, P), or tryptophan (Trp, W), for example, glycine (Gly, G); It may additionally contain one or more substitutions selected from the following:

[0049] Counting from the second amino acid, proline (Pro, P), in the amino acid sequence of SEQ ID NO: 4, substitution of amino acid 43 (K) with proline (Pro, P) or glutamic acid (Glu, E); The 13th amino acid (Y) is replaced by phenylalanine (Phe, F); The 51st amino acid (D) is substituted with valine (Val, V) or threonine (Thr, T); and The 60th amino acid (N) is replaced by phenylalanine (Phe, F).

[0050] For example, the polypeptide may comprise the following amino acid mutations in the B3 domain of the protein L:

[0051] E28G (substitution of the 28th amino acid, glutamic acid (E), with glycine (G); single amino acid substitutions herein are denoted and interpreted in the same manner), E28A, E28L, E28P, E28W, E28G+Y13F, E28 G+K43P (substitution of the 28th amino acid, glutamic acid (E), with glycine (G) and substitution of the 43rd amino acid, lysine (K), with proline (P); multiple amino acid substitutions herein are denoted and interpreted in the same manner), E28G+D51V, E28G+N60E, E28L, E28P, E28W, E28G+K43E, E28G+D51T, E28G+N60F, or Y13F+E28G+K43P.

[0052] The B3 domain of protein L (wild type) may comprise the amino acid sequence of SEQ ID NO:4.

[0053] The amino acid positions of the B3 domain of protein L described herein are counted from the second amino acid, proline (Pro, P), in the amino acid sequence of SEQ ID NO: 4. That is, the 28th amino acid (E) (E28) described herein corresponds to the 29th amino acid in SEQ ID NO: 4, the 43rd amino acid (K) (K43) corresponds to the 44th amino acid in SEQ ID NO: 4, the 13th amino acid (Y) (Y13) corresponds to the 14th amino acid in SEQ ID NO: 4, the 51st amino acid (D) (D51) corresponds to the 52nd amino acid in SEQ ID NO: 4, and the 60th amino acid (N) (N60) corresponds to the 61st amino acid in SEQ ID NO: 4.

[0054] In one embodiment, the polypeptide (protein L B3 domain mutant) has the following structure based on the amino acid sequence of SEQ ID NO: 4: Amino acid sequence modified by introducing E28A mutation (SEQ ID NO: 7); an amino acid sequence modified by introducing the E28G mutation (SEQ ID NO: 8); Amino acid sequence modified by introducing E28G+Y13F mutations (SEQ ID NO: 9); Amino acid sequence modified by introducing E28G+K43P mutations (SEQ ID NO: 10); Amino acid sequence modified by introducing E28G+D51V mutations (SEQ ID NO: 11); Amino acid sequence modified by introducing E28G+N60E mutations (SEQ ID NO: 12) an amino acid sequence modified by introducing the E28L mutation (SEQ ID NO: 23); an amino acid sequence modified by introducing the E28P mutation (SEQ ID NO: 24); an amino acid sequence modified by introducing the E28W mutation (SEQ ID NO: 25); an amino acid sequence modified by introducing E28G+K43E mutations (SEQ ID NO: 26); an amino acid sequence modified by introducing E28G+D51T mutations (SEQ ID NO: 27); an amino acid sequence modified by introducing E28G+N60F mutations (SEQ ID NO: 28); or Amino acid sequence modified by introducing Y13F+E28G+K43P mutations (SEQ ID NO: 47); It may include:

[0055] The amino acid sequences of the polypeptides (protein L B3 domain mutants) are summarized in Table 1 below.

[0056] [Table 1]

[0057] Another example provides a polypeptide multimer comprising two or more of the above polypeptides as repeating units (monomers). In one specific example, the polypeptide multimer may comprise two or more repeating units comprising (or consisting of) the above polypeptides, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units. For example, the polypeptide multimer may be represented by SEQ ID NO: 49 (containing four consecutive copies of SEQ ID NO: 47), but is not limited thereto.

[0058] Another example provides an immunoglobulin-binding protein comprising the above polypeptide or polypeptide multimer.

[0059] In one example, the immunoglobulin-binding protein may further comprise, in addition to the polypeptide or polypeptide multimer, one or more domains selected from the group consisting of the A, B1, B2, B4, C, W, and M domains of protein L.

[0060] Another example provides a protein L variant comprising the polypeptide or polypeptide multimer.

[0061] The polypeptides (Protein L B3 domain mutants), polypeptide multimers, immunoglobulin-binding proteins, and / or Protein L mutants provided in the present application may have increased alkaline tolerance compared to the wild-type B3 domain (SEQ ID NO: 4) or polypeptide multimers, immunoglobulin-binding proteins, and / or Protein L containing the same. For example, the polypeptide, polypeptide multimer, immunoglobulin-binding protein, and / or Protein L variant provided herein may have an increased ability to bind to an immunoglobulin (e.g., IgG (IgG1, IgG2, IgG3, or IgG4)) or a fragment comprising its light chain portion (e.g., scFv, Fab, etc.) under basic conditions (e.g., pH 9 or higher, pH 9.5 or higher, pH 10 or higher, pH 10.5 or higher, pH 11 or higher, pH 11.5 or higher, pH 12 or higher, pH 12.5 or higher, or pH 13 or higher; the upper limit may be pH 14) by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, or about 30% or more, compared to the wild-type B3 domain (SEQ ID NO: 4) or a polypeptide multimer, immunoglobulin-binding protein, and / or Protein L comprising the same, but is not limited thereto. In particular, the polypeptide multimer may have an ability to bind to an immunoglobulin or a fragment thereof under basic conditions that is increased by about 1.5-fold or more, about 1.7-fold or more, about 2-fold or more, about 2.3-fold or more, about 2.5-fold or more, or about 2.7-fold or more compared to the wild-type B3 domain, but is not limited thereto.

[0062] As used herein, immunoglobulins may be derived from primates such as humans, monkeys, or rodents such as mice or rats, and may be selected from various subtypes, such as IgG (IgG1, IgG2, IgG3, or IgG4), IgA, IgD, IgE, and IgM. The polypeptides (Protein L B3 domain mutants) and / or polypeptide multimers, immunoglobulin-binding proteins, and / or Protein L variants provided herein may bind to the light chain portion of immunoglobulins. Therefore, immunoglobulins that can be bound by the polypeptides (Protein L B3 domain mutants), polypeptide multimers, immunoglobulin-binding proteins, and / or Protein L variants provided herein may be in the intact form or in the form of fragments containing the light chain, such as, but not limited to, scFv, Fab, (scFv)2, Fab, Fab', or F(ab')2.

[0063] In one example, the polypeptides, polypeptide multimers, immunoglobulin-binding proteins, and / or Protein L variants provided herein may be non-naturally occurring, for example, but not limited to, produced by recombinant methods or chemical synthesis.

[0064] Nucleic acid molecules, recombinant expression vectors, and recombinant cells Other examples provide nucleic acid molecules encoding the aforementioned polypeptides (B3 domain variants), polypeptide multimers, immunoglobulin-binding proteins, or Protein L variants.

[0065] Another example provides a recombinant vector containing the nucleic acid molecule. The recombinant vector may be an expression vector that can express the nucleic acid molecule as a protein in a suitable host cell. The recombinant vector can be used as an expression vector for expressing the polypeptide, polypeptide multimer, and / or protein L variant in a suitable host cell.

[0066] Another example provides a recombinant cell containing the nucleic acid molecule or recombinant vector. The recombinant cell can be a suitable host cell that has been transformed with the nucleic acid molecule or recombinant vector.

[0067] The polypeptide, polypeptide multimer, immunoglobulin-binding protein, or protein L variant may be used in an immobilized state rather than in a free state. The immobilization can be performed by conventional methods known in the art. Examples of carriers for immobilization include natural polymers such as cellulose, starch, dextran, and agarose; synthetic polymers such as polyacrylamide, polyacrylate, polymethacylate, and Eupergit C; and minerals such as silica, bentonite, and metals. The polypeptide, polypeptide multimer, immunoglobulin-binding protein, or protein L variant can be bound to these carriers by covalent bonding, ionic bonding, hydrophobic bonding, physical adsorption, microencapsulation, and the like. Furthermore, the polypeptide, polypeptide multimer, immunoglobulin-binding protein, or protein L variant can be immobilized by forming a covalent bond with these carrier-enzyme conjugates through the action of glutaraldehyde, cyanogen bromide, etc. Furthermore, microbial cells containing the polypeptide, polypeptide multimer containing the polypeptide, immunoglobulin-binding protein, or protein L variant can be directly immobilized and used without the need for separate purification of the polypeptide, polypeptide multimer, immunoglobulin-binding protein, or protein L variant. During whole cell immobilization, techniques such as cell entrapment or surface display can be applied to enhance the reactivity of the polypeptide, polypeptide multimer, immunoglobulin-binding protein, or protein L variant contained in the microorganism.

[0068] The nucleic acid sequences described herein may be modified in various ways in the coding region, taking into account the codons preferred in the microorganism in which the polypeptide, polypeptide multimer, immunoglobulin-binding protein, or Protein L variant is to be expressed, as long as the amino acid sequence and / or function of the protein expressed from the coding region is not altered, due to codon degeneracy.

[0069] Introduction of the nucleic acid molecule or vector can be carried out by a known transformation method appropriately selected by those skilled in the art. As used herein, the term "transformation" refers to the introduction of a vector containing a nucleic acid molecule encoding a target protein (foreign protein) into a host cell so that the protein encoded by the nucleic acid molecule can be expressed in the host cell. The transformed nucleic acid molecule can be inserted into the host cell's chromosome and / or extrachromosomally located, as long as it can be expressed in the host cell. The nucleic acid molecule can also include DNA and / or RNA encoding the target protein (the polypeptide, polypeptide multimer, immunoglobulin-binding protein, or protein L variant). There are no limitations on the form in which the nucleic acid molecule is introduced, as long as it can be introduced and expressed in the host cell. For example, the nucleic acid molecule can be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically includes expression regulatory elements, such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, operably linked to the nucleic acid molecule. The expression cassette can be in the form of a self-replicating expression vector. Alternatively, the nucleic acid molecule may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell. The term "operably linked" as used herein may mean that the nucleic acid molecule is functionally linked to an expression regulatory element (e.g., a promoter) so that the expression regulatory element can regulate transcription (e.g., initiate transcription) of the nucleic acid molecule encoding the target protein (foreign protein). Operable linkage can be achieved using recombinant DNA techniques known in the art, such as, but not limited to, conventional site-specific DNA cleavage and ligation.

[0070] The nucleic acid molecule can be transformed into a host cell by any method for introducing nucleic acid into a cell (microorganism), and can be transformed by appropriately selecting a transformation technique known in the art depending on the host cell. Examples of known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation (polyethylene glycol-mediated uptake), DEAE-dextran, cationic liposome, lipofection, lithium acetate-DMSO, heat shock, particle gun bombardment, silicon carbide whiskers, and sonication.

[0071] The introduction (insertion) of the nucleic acid molecule into the host cell chromosome can be carried out by a known method appropriately selected by those skilled in the art. For example, the introduction (insertion) can be carried out using an RNA-guided endonuclease system (RNA-guided endonuclease system or CRISPR system; for example, at least one selected from the group consisting of (a) an RNA-guided endonuclease (e.g., Cas9 protein, etc.), a gene encoding the same, or a vector containing the gene; and (b) a guide RNA (e.g., single guide RNA (sgRNA)), a DNA encoding the same, or a vector containing the DNA (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA), a complex (e.g., a ribonucleic acid fusion protein (RNP)), a recombinant vector (e.g., a vector containing both an RNA-guided endonuclease-encoding gene and a guide RNA-encoding DNA), etc., but is not limited thereto.

[0072] As used herein, the term "vector" refers to a DNA construct containing a base sequence of a nucleic acid molecule encoding a target protein operably linked to a suitable regulatory sequence to enable expression of the target protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and / or a sequence regulating the termination of transcription and / or translation. After being transformed into a suitable host microorganism, the vector may be expressed independently of the host microorganism's genetic code or may be integrated into the host microorganism's genetic code.

[0073] The vectors that can be used herein are not particularly limited as long as they are replicable in host cells, and can be selected from any commonly used vector, including naturally occurring or recombinant plasmids, cosmids, viruses, bacteriophages, and the like. For example, the vector may be a phage vector or cosmid vector such as pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, or Charon21A. Plasmid vectors that may be used include, but are not limited to, pBC series (e.g., pBC-KS(+)), pBR series (e.g., pBR322, pBR325), pUC series (e.g., pUC118 and pUC119), pBluescriptII series, pGEM series, pTZ series, pCL series, pET series (e.g., pET-22b(+)), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5), plasmids derived from animal viruses such as retrovirus, adenovirus, or vaccinia virus, and plasmids derived from insect viruses such as baculovirus.

[0074] The host cell may be selected from the group consisting of all types of commonly used unicellular organisms, for example, prokaryotic microorganisms such as various bacteria (e.g., Escherichia, Clostridia, etc.) and eukaryotic microorganisms such as yeast, for example, Clostridia microorganisms (e.g., Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, or Clostridium saccharobutylicum) and Escherichia microorganisms (e.g., Escherichia coli, etc.), but is not limited thereto.

[0075] The vectors usable herein may be known expression vectors and / or vectors for inserting nucleic acid molecules into host cell chromosomes. Insertion of the nucleic acid molecules into host cell chromosomes can be achieved by any method known in the art, including, but not limited to, homologous recombination or the CRISPR system. The vector may additionally contain a selection marker for confirming the presence or absence of the chromosomal insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the polynucleotide insertion. It can be used to select from genes that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing for the selection of transformed cells.

[0076] Another example provides a method for producing the aforementioned polypeptide (B3 domain variant), polypeptide multimer, immunoglobulin-binding protein, or Protein L variant, comprising expressing the nucleic acid molecule in a suitable host cell. The production method may include culturing the aforementioned recombinant cell, and may optionally further include isolating and / or purifying the polypeptide, polypeptide multimer, immunoglobulin-binding protein, or Protein L variant from the culture.

[0077] Matrix (resin) containing B3 domain mutants Another example provides a matrix (resin) in which the aforementioned polypeptide (B3 domain mutant), polypeptide multimer, immunoglobulin-binding protein, or Protein L mutant is coupled to a solid support. The matrix may be, but is not limited to, a chromatographic matrix for use in techniques for separating and / or purifying proteins by adsorption, such as (affinity) chromatography.

[0078] In the matrix, the polypeptide, polypeptide multimer, immunoglobulin-binding protein, or protein L variant is used as a ligand coupled to a solid support, and two or more of the polypeptide, polypeptide multimer, immunoglobulin-binding protein, or protein L variant may be used. In one embodiment, the chromatography matrix may be, but is not limited to, a solid support to which multiple polypeptide multimers are coupled.

[0079] The solid support may be suitably selected from all known types of suitable solid supports to which polypeptides can be coupled, such as those used in conventional affinity separation matrices. For example, the solid support may be an organic or inorganic material.

[0080] In one example, the solid support may be organic and based on a polymer that exposes a hydrophilic surface to aqueous media and / or exposes hydroxy (-OH), carboxy (-COOH), carboxyamide (-CONH, possibly in N-substituted form), amino (-NH, possibly in substituted form), oligo-, or polyethyleneoxy groups on its internal surface. The polymer may be based on a polysaccharide such as dextran, starch, cellulose, pullulan, or agarose, e.g., a polysaccharide cross-linked with a bisepoxide, epihalohydrin, or 1,2,3-trihalosubstituted lower hydrocarbon, to provide suitable porosity and strength. In one specific example, the solid support may be, but is not limited to, porous agarose beads. The solid supports used herein may be prepared by standard methods such as inverse suspension gelatinization (S. Hjerten: Biochem Biophys Acta 79 (2), 393-398 (1964)), or may be commercially available products such as, but not limited to, Sepharose™ FF (Amersham Biosciences, Uppsala, Sweden).

[0081] In other examples, the solid support can be based on synthetic polymers such as polyvinyl alcohol, polyhydroxyalkylacrylate, polyhydroxyalkylmethacrylate, polyacrylamide, and polymethacrylamide. In the case of hydrophobic polymers, such as divinyl- and monovinyl-substituted benzene-based matrices, the matrix surface is sometimes hydrophilized to expose these hydrophilic groups to the surrounding aqueous liquid. The polymers can be prepared by standard methods ("Styrene-based polymer supports developed by suspension polymerization," R. Arshady: Chimica eL'Industria 70 (9), 70-75 (1988)) or commercially available products, such as, but not limited to, Source™ (Amersham Biosciences, Uppsala, Sweden).

[0082] In yet another example, the solid support may comprise a support of inorganic nature, such as silica, zirconium oxide, or the solid support may be in other forms, such as a surface, a chip, a capillary, or a filter.

[0083] In one example, the matrix may be in the form of a porous monolith, or in the form of porous or non-porous beads or particles. Bead or particle-type matrices may be used as packed beds or in suspended form. Suspended forms include those known as expanded beds and pure suspensions, in which the particles or beads are free to move. For monoliths, packed beds, and expanded beds, separation is typically performed using conventional chromatography through a concentration gradient.

[0084] The ligand (the polypeptide, polypeptide multimer, immunoglobulin-binding protein, or Protein L variant) can be attached to the support through conventional coupling techniques, for example, using amino and / or carboxy groups present on the ligand. Well-known coupling agents include bisepoxide, epichlorohydrin, CNBr, and N-hydroxysuccinimide (NHS). A molecule known as a spacer can be introduced between the support and the ligand, which improves the availability of the ligand and facilitates chemical coupling of the ligand to the support. Alternatively, the ligand can be attached to the support by non-covalent bonding, such as physical adsorption or biospecific adsorption.

[0085] The matrices provided herein may have a binding ability to immunoglobulins (e.g., IgG (IgG1, IgG2, IgG3, or IgG4)) or fragments containing their light chain portions (e.g., scFv, Fab, etc.) increased by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, or about 30% or more under basic conditions (e.g., pH 9 or higher, pH 9.5 or higher, pH 10 or higher, pH 10.5 or higher, pH 11 or higher, pH 11.5 or higher, pH 12 or higher, pH 12.5 or higher, or pH 13 or higher); more specifically, after 1 to 20 (e.g., 15) washes using an alkaline solution (e.g., 0.3 M NaOH solution, about pH 13.48), compared to existing matrices containing protein L used in affinity chromatography, but are not limited thereto.

[0086] Immunoglobulin binding, separation, and purification applications One or more target compounds (e.g., proteins such as immunoglobulins) can be separated and / or purified from a liquid by adsorption to the polypeptides (B3 domain variants), polypeptide multimers, immunoglobulin-binding proteins, Protein L variants, and / or chromatographic matrices provided herein.

[0087] Thus, other examples provide uses of the aforementioned polypeptides (B3 domain variants), polypeptide multimers, immunoglobulin-binding proteins, Protein L variants, and / or chromatographic matrices for immunoglobulin binding and / or immunoglobulin separation and / or purification.

[0088] More specifically, one example provides a composition for immunoglobulin binding comprising one or more selected from the group consisting of the aforementioned polypeptide (B3 domain mutant), polypeptide multimer, immunoglobulin-binding protein, protein L mutant, nucleic acid molecules encoding these, recombinant vectors and recombinant cells, and chromatography matrices.

[0089] Another example provides a composition for separating and / or purifying immunoglobulins, comprising one or more selected from the group consisting of the aforementioned polypeptide (B3 domain mutant), polypeptide multimer, immunoglobulin-binding protein, protein L mutant, and chromatographic matrix.

[0090] Another example provides a method for separating and / or purifying immunoglobulins, which includes a step of contacting a sample containing immunoglobulins with one or more selected from the group consisting of the aforementioned polypeptides (B3 domain mutants), polypeptide multimers, immunoglobulin-binding proteins, protein L mutants, and chromatography matrices, thereby adsorbing the immunoglobulins.

[0091] Another example provides a method for separating and / or purifying one or more target compounds from a liquid, comprising the step of contacting a liquid sample containing the target compounds with one or more selected from the group consisting of the aforementioned polypeptide (B3 domain mutant), polypeptide multimer, immunoglobulin-binding protein, protein L mutant, and chromatographic matrix to adsorb the target compounds.

[0092] As used herein, immunoglobulins may be derived from primates such as humans, monkeys, or rodents such as mice or rats, and may be selected from various subtypes such as IgG (IgG1, IgG2, IgG3, or IgG4), IgA, IgD, IgE, and IgM. The immunoglobulin to be purified and / or separated may be in an intact form or in the form of a fragment containing a light chain, such as, but not limited to, scFv, Fab, (scFv)2, Fab, Fab', or F(ab')2.

[0093] The method for separating and / or purifying said immunoglobulin or target compound may be chromatography, for example affinity chromatography.

[0094] Briefly, affinity chromatography involves passing a solution containing a target compound, e.g., an immunoglobulin or a fragment thereof (e.g., a cell culture medium expressing the same immunoglobulin or fragment thereof), through a separation matrix under conditions that allow the target compound to be adsorbed to the ligand present on the matrix. These conditions are controlled, for example, by adjusting the pH and / or salt concentration, i.e., the ionic strength of the solution. Care must be taken not to exceed the capacity of the matrix, i.e., the flow must be slow enough to allow satisfactory adsorption. During this stage, other components of the solution essentially pass through unobstructed. Although not essential, the matrix is ​​subsequently washed, for example, with an aqueous solution, to remove retained and / or loosely bound substances. The matrix provided herein has the advantage of maintaining its binding capacity for the target compound (e.g., an immunoglobulin or a fragment thereof) even after a washing step using an alkaline preparation, as described above. In the next step, a second solution, referred to as an eluent, is passed over the matrix under conditions that allow the target compound to be desorbed, i.e., released. These conditions are typically achieved by changing the pH, salt concentration, i.e., the ionic strength, or hydrophobicity. A variety of elution methods are known, including gradient elution and step elution. Elution can also be achieved by a second solution containing a competitor that displaces the desired antibody on the matrix. [Effects of the Invention]

[0095] By providing an immunoglobulin-binding protein with improved alkali resistance and stability under alkaline conditions, it can be advantageously used as an immunoglobulin-binding ligand in immunoglobulin purification techniques that necessarily involve a washing step using an alkaline agent, such as chromatography. [Brief explanation of the drawings]

[0096] [Figure 1]This is a graph showing the results of comparing the scFv binding ability (measured by absorbance) of B3 domain mutants EP1 (E28A) and EP2 (E28G) after alkaline treatment with the wild type (wB3) in one embodiment (first error prone PCR results). [Figure 2] This is a graph showing the results of one embodiment in which the scFv binding ability (measured by absorbance) of four B3 domain mutants, BEP1 (E28G / Y13F), BEP2 (E28G / K43P), BEP3 (E28G / D51V), and BEP4 (E28G / N60E), after alkaline treatment was compared with that of mutant EP2 (E28G) (100%) (secondary error-prone PCR results). [Figure 3] This is a graph showing the results of one embodiment in which the scFv binding ability (measured by absorbance) of six B3 domain mutants, EPS1 (E28L), EPS2 (E28P), EPS3 (E28W), EPS4 (E28G / K43E), EPS5 (E28G / D51T), and EPS6 (E28G / N60F), after alkaline treatment was compared with that of mutant EP2 (E28G) (third-order error-prone PCR results). [Figure 4] FIG. 1 is a schematic diagram showing an example of a process for constructing a site-directed mutation library for screening mutants with increased alkaline tolerance. [Figure 5] 1 is a graph showing the change in scFv binding ability (measured by absorbance) over time after alkali treatment of a B3 domain mutant mBF4 (Y13F / E28G / K43P) according to one example, compared to the wild-type B3 domain. [Figure 6] This is a graph showing the change in scFv binding ability (measured by absorbance) of tetramer 4mBF containing four B3 domain mutant mBF4 (Y13F / E28G / K43P) molecules according to one embodiment, following alkaline treatment (washing) and recovery (cycle), in comparison with commercially available products (Cytiva 17-5478-01, Capto L). DETAILED DESCRIPTION OF THE INVENTION

[0097] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It will be obvious to those skilled in the art that the examples described below can be modified within the scope of the essential gist of the invention.

[0098] Example 1: Preparation of recombinant immunoglobulin G fragment (IgG(f)) protein for improvement

[0099] <1-1> Synthesis of immunoglobulin G IgG(f) gene The nucleic acid sequence (IgG(f) gene; SEQ ID NO: 1) encoding the human IgG1-based scFv region polypeptide (IgG(f); Herceptin scFv; SEQ ID NO: 2) was searched for through Blast on the NCBI site (GenBank accession no. AWW43726) and synthesized by Cosmogenetech Co., Ltd. (Daejeon, Korea).

[0100] <1-2> Preparation of pET-IgG(f) plasmid The IgG(f) gene obtained in Example <1-1> was inserted into the NdeI and XhoI restriction enzyme recognition sites of the pET29a(+) vector (Stratagene, USA) to prepare the pET-IgG(f) plasmid. The details are as follows.

[0101] The IgG(f) gene DNA product (SEQ ID NO: 1) synthesized in Example <1-1> above was cleaved with restriction enzymes NdeI and XhoI and purified using a purification kit (QIAEX Gel Extraction Kit; Qiagen, Germany). This was used as the insert DNA. Additionally, pET29a(+) vector DNA was cleaved with restriction enzymes NdeI and XhoI and dephosphorylated with antarctic phosphatase (Engenomics, Korea), resulting in a DNA fragment used as the vector DNA. The insert DNA and vector DNA were ligated using T4 DNA ligase (Engenomics, Korea) at 16°C for 16 hours, and the ligation solution was then used to transform E. coli BL21(DE3) strain (Engenomics) for expression by electroporation. The strain was smeared on LB agar medium containing 50 μg / mL of kanamycin antibiotic and cultured at 37°C for 16 to 18 hours to select transformants. Plasmids were isolated from the transformants and the base sequence of the inserted DNA was determined to produce the pET-IgG(f) plasmid containing the IgG(f) gene having the nucleic acid sequence of SEQ ID NO: 1. The pET-IgG(f) plasmid expresses the wild-type IgG(f) protein (Herceptin scFv) represented by SEQ ID NO: 2.

[0102] <1-3> Purification of IgG(f) protein using nickel affinity resin To seed culture the E. coli BL21(DE3) transformant containing the IgG(f) gene, 5 mL of LB liquid medium (BD (Becton, Dickinson and Company)) containing kanamycin antibiotic was dispensed into a 50 mL conical tube, and the selected transformant was inoculated and cultured with shaking at 37°C and 200 rpm for 16 hours. A 500 mL Erlenmeyer flask containing 200 mL of LB liquid medium was inoculated with 1% (v / v) of the seed culture and cultured with shaking at 37°C and 200 rpm. After culturing with shaking until the OD600 reached approximately 0.6, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 1 mM, and the culture was further cultured with shaking at 37°C and 200 rpm for 18 hours. The flask culture was centrifuged (4°C, 8,000 rpm, 20 minutes) to collect the bacterial cells, which were then suspended in 10 mL of PBS buffer (pH 7.4) (Intron Biotechnology, Korea). The suspension was disrupted in an ultrasonic homogenizer at 4°C for 15 minutes and centrifuged (4°C, 20,000 rpm, 20 minutes) to collect the supernatant. A column was filled with 1 mL of WorkBeads™ 40 Ni-NTA (bioworks, Sweden), a nickel affinity resin, and 5 mL of binding buffer (20 mM NaHPO, 300 mM NaCl, 10 mM imidazole, pH 8.0) was poured through it. Then, 2.5 mL of the supernatant and 2.5 mL of binding buffer were mixed and poured through the column. After 5 mL of wash buffer (20 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole pH 8.0) was applied, 2.5 mL of elution buffer (20 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole pH 8.0) was applied, and the flow-through was collected in a 15 mL conical tube. The purified protein thus collected was desalted using a HiPrep 26 / 10 desalting column (Cytiva, Sweden).

[0103] Example 2: Preparation of a template

[0104] <2-1> Synthesis of the B3 domain gene The B3 domain gene (SEQ ID NO: 3) of protein L derived from Peptostreptococcus magnus was synthesized by Cosmogenec Co., Ltd. (Daejeon, Korea) with a 6xHis tag added, taking into consideration the subsequent protein purification.

[0105] <2-2> Construction of pBC-wB3 plasmid The wild-type B3 domain (wB3) gene of protein L (SEQ ID NO: 3) obtained in Example <2-1> above was inserted into the NdeI and NotI restriction enzyme recognition sites of the pBC KS(+) vector (Stratagene, USA) to prepare the pBC-wB3 plasmid. The procedure was as follows.

[0106] The wB3 gene DNA product (SEQ ID NO: 3) synthesized in Example <2-1> above was cleaved with restriction enzymes NdeI and NotI and purified using a purification kit (QIAEX Gel Extraction Kit; Qiagen, Germany) and used as the insert DNA. Additionally, pBC KS(+) vector DNA was cleaved with restriction enzymes NdeI and NotI and dephosphorylated with antarctic phosphatase (Engenomics, Korea) to prepare a DNA fragment, which was used as the vector DNA. The insert DNA and vector DNA were ligated using T4 DNA ligase (Engenomics, Korea) at 16°C for 16 hours, and the ligation solution was then used to transform E. coli DH5α (Engenomics) by electroporation. The strain was smeared on LB agar medium (BD (Becton, Dickinson and Company)) containing 20 μg / mL of chloramphenicol antibiotic and statically cultured at 37°C for 16 to 18 hours to select transformants. Plasmids were isolated from the selected transformants, and the base sequence of the inserted DNA was determined to prepare pBC-wB3 plasmid containing the wild-type B3 domain gene having the nucleic acid sequence of SEQ ID NO: 3. The pBC-wB3 plasmid expresses the wild-type protein L B3 domain protein represented by SEQ ID NO: 4.

[0107] Example 3: Improvement of wB3 using error prone PCR method

[0108] <3-1> Construction of wB3 mutant library by error-prone polymerase chain reaction (error-prone PCR) To artificially induce random mutations in the nucleic acid sequence of the wB3 gene (SEQ ID NO: 3) synthesized in Example 2, error-prone polymerase chain reaction (error prone PCR) was performed to construct a mutation library.

[0109] The specific process for constructing the mutation library is as follows. Error-prone polymerase chain reaction was performed using the Diversity® PCR Random Mutagenesis kit (Clontech, USA) to induce 1-2 mutations per 1,000 bp. The PCR reaction mixture consisted of 1 ng of pBC-wB3 plasmid (Example 2-2) as template DNA, 10 pmol each of EP-F primer (SEQ ID NO: 5) and T7 primer (SEQ ID NO: 6), 40 μM dGTP, Diversity dNTP mix, and TITANIUM™ Taq DNA polymerase, adjusted to a final volume of 100 μL. PCR was performed using a C1000 Touch thermal cycler (BIO-RAD, USA) under the following conditions: The reaction mixture was pre-denatured at 94°C for 30 seconds, and the cycle of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 68°C for 3 minutes was repeated 16 times, followed by post-polymerization at 68°C for 1 minute.

[0110] Each wB3 mutant gene PCR product obtained through the error-prone polymerase chain reaction (PCR) performed under the above conditions was digested with the restriction enzymes NdeI and NotI and purified using a QIAEX Gel Extraction Kit (Qiagen, Germany) before being used as insert DNA. Additionally, the pBC-KS(+) plasmid was digested with the restriction enzymes NdeI and NotI and dephosphorylated with antarctic phosphatase (Engenomics, Korea) to obtain a 3.4 kb DNA fragment, which was used as vector DNA. The insert DNA and vector DNA were ligated using T4 DNA ligase (Engenomics, Korea) at 16°C for 16 hours, and the ligation solution was then transformed into E. coli DH5α by electroporation. The strain was smeared on LB agar medium containing 20 μg / mL of chloramphenicol antibiotic and cultured at 37° C. for 16 to 18 hours to prepare a random mutation library.

[0111] <3-2> Selection of mutants with increased alkaline tolerance To cultivate the E. coli DH5α transformant containing the wB3 mutant gene induced by the above method, 500 μL of LB liquid medium containing chloramphenicol antibiotic was dispensed into a 96-deep well plate (Bioneer, Korea), and the transformant was inoculated and cultured with shaking at 37°C and 280 rpm for 18 hours.

[0112] The specific protein purification process is as follows. Protein purification was performed using the Promega HisLink™ 96 Protein Purification System (Promega, USA). 10x FastBreak™ Cell Lysis Reagent and DNase I solution were mixed to prepare the FastBreak™ Reagent / DNase I solution. 600 μL of culture medium and 60 μL of FastBreak™ Reagent / DNase I solution were added to each well, followed by 65 μL of HisLink™ Resin. This was mixed on a shaker at 100 rpm for 30 minutes to allow the culture medium and resin to react. The reaction mixture and resin were transferred to a filtration plate and filtered using a Vac-Man™ 96 Vacuum Manifold (Promega, USA) by applying vacuum for 10 seconds. Next, 250 μL of binding / wash buffer (100 mM HEPES, 10 mM imidazole, pH 7.5) was added to each well, followed by washing with vacuum for 10 seconds. This procedure was repeated three times. 200 μL of elution buffer (100 mM HEPES, 500 mM imidazole, pH 7.5) was added to the plate, and the plate was incubated for 10 minutes. After incubation, the purified protein was collected in a new 96-well plate by vacuum application for 1 minute. The purified wB3 mutant was coupled to NHS (N-hydroxysuccinimide)-Activated Sepharose 4 Fast flow (Cytiva, Sweden) in the 96-well plate and then transferred to a filtration plate. 150 μL (71.5 μg / mL) of the IgG(f) protein purified in Example <1-3> was added to the filtration plate and incubated at room temperature for 1 hour on a shaker at 100 rpm. Unbound IgG(f) protein was removed by vacuum application, and then 150 μL of PBS buffer (pH 7.4) was added to each well and washed by vacuum application, followed by washing three times in the same manner.After adding 150 μL of elution buffer (0.1 M glycine-HCl, pH 2.5) and incubating at room temperature for 30 seconds, the proteins were collected in a new 96-well plate by applying a vacuum for 1 minute. The filtration plate treated with the elution buffer was washed three times in the same manner by adding 150 μL of PBS buffer to each well and applying a vacuum.

[0113] The recovered protein was transferred to a new 96-well plate, and the amount of IgG(f) protein was measured at OD280 using a Synergy HTX multi-mode reader (BioTek, USA). After the initial binding assay, to confirm the alkaline resistance of the wB3 mutant, 200 μL of 0.3 M NaOH (pH 13.48) was added to the wB3 mutant resin in the filtration plate and incubated at 100 rpm for 1 hour at room temperature. The plate was then washed three times with 200 μL of PBS buffer, and the IgG(f) protein was allowed to bind in the same manner as in the initial binding assay. The antibody was then recovered and measured. The remaining IgG(f) protein was measured by repeating the same procedure and comparing the amount of remaining IgG(f) protein.

[0114] Two wB3 mutants with enhanced alkaline tolerance were selected by comparing the absorbance of the wB3 mutants before and after treatment with 0.3 M NaOH. The two selected mutants were confirmed to have been mutated from wild-type wB3 (SEQ ID NO: 4) through nucleic acid sequencing to mutant EP1 (SEQ ID NO: 7) with the E28A mutation (meaning that the E at the 28th amino acid residue (i.e., the 29th amino acid residue in SEQ ID NO: 4) counting from the amino acid residue next to the methionine (M) encoded by the initiation codon during recombinant synthesis (i.e., the second amino acid residue, P, in SEQ ID NO: 4) is replaced with A; amino acid mutations are interpreted in the same manner throughout this specification). The absorbance of the two selected mutants, EP1 (E28A) and EP2 (E28G), compared with that of the wild-type (wB3) is shown in Figure 1 (relative values ​​to the absorbance of wB3 (100%)).

[0115] <3-3> Construction of an EP2 mutant library by error-prone polymerase chain reaction (error-prone PCR) Among the wB3 mutants selected in Example 3-2, the more alkaline-tolerant mutant EP2 (E28G) (SEQ ID NO: 8) was further modified by the coding gene. EP2 inserted into the pBC KS(+) vector was used as a new template for another error-prone PCR.

[0116] After performing error-prone polymerase chain reaction in the same manner as in Examples <3-1> and <3-2>, proteins with increased alkaline tolerance compared to EP2 were selected. As a result, four mutants were additionally selected, and by sequencing the genes, it was confirmed that mutant EP2 had been mutated into mutant BEP1 (SEQ ID NO: 9) having E28G / Y13F mutations, mutant BEP2 (SEQ ID NO: 10) having E28G / K43P mutations, mutant BEP3 (SEQ ID NO: 11) having E28G / D51V mutations, and mutant BEP4 (SEQ ID NO: 12) having E28G / N60E mutations. The absorbance of the four mutants identified above, BEP1 (E28G / Y13F), BEP2 (E28G / K43P), BEP3 (E28G / D51V), and BEP4 (E28G / N60E), was compared with that of the mutant EP2 (E28G), and the results are shown in Figure 2 (shown as relative values ​​to the absorbance of EP2 (100%)).

[0117] <Example 4> Improvement of wB3 using site-saturation mutagenesis

[0118] <4-1> Construction of wB3 mutant library by site-saturation mutagenesis To further confer alkaline resistance to the EP2(E28G) gene improved with the wB3 gene, a site-saturation mutagenesis library was constructed for the five amino acid residues (Y13, E28, K43, D51, and N60) that were mutated in the mutant selected in Example 3. The specific method is as follows.

[0119] To prepare a library with a mutation at the 28th amino acid (corresponding to the 29th amino acid of SEQ ID NO: 4, including the methionine (M) at the N-terminus due to the initiation codon; amino acid positions are interpreted in the same manner throughout this specification), a reaction mixture was prepared using the wB3 gene (SEQ ID NO: 3) inserted into the pBC KS(+) vector as a template. The reaction mixture was adjusted to a final volume of 50 μL with the 28-F primer (SEQ ID NO: 13), 28-R primer (SEQ ID NO: 14), Pfu-X DNA polymerase, 10× Pfu-X Reaction buffer, and 10 mM dNTPs. The reaction mixture was reacted under the following conditions: initial denaturation at 95°C for 2 minutes, followed by 25 cycles of denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and polymerization at 72°C for 1 minute, followed by post-polymerization at 72°C for 5 minutes. The PCR product obtained under these conditions was treated with the restriction enzyme DpnI for 18 hours, purified using a PCR purification kit (Cosmogenetech, Korea), and immediately transformed into E. coli DH5α by electrophoresis. The transformed strain was plated on LB agar medium containing 20 μg / mL of chloramphenicol antibiotic and incubated at 37°C for 16 to 18 hours to prepare a site-saturation mutation library.

[0120] Then, in the same manner as described above, a library was prepared using the BEP1 (SEQ ID NO: 9)-encoding gene, BEP2 (SEQ ID NO: 10)-encoding gene, BEP3 (SEQ ID NO: 11)-encoding gene, or BEP4 (SEQ ID NO: 12)-encoding gene inserted into the pBC KS(+) vector as a template, and the 13-F primer (SEQ ID NO: 15) and 13-R primer (SEQ ID NO: 16), 43-F primer (SEQ ID NO: 17) and 43-R primer (SEQ ID NO: 18), 51-F primer (SEQ ID NO: 19) and 51-R primer (SEQ ID NO: 20), and 60-F primer (SEQ ID NO: 21) and 60-R primer (SEQ ID NO: 22), respectively.

[0121] <4-2> Selection of mutants with increased alkaline tolerance The library prepared in Example <4-1> was screened using the same method as in Example <3-2>. As a result, six additional mutants, EPS1 (E28L) (SEQ ID NO: 23), EPS2 (E28P) (SEQ ID NO: 24), EPS3 (E28W) (SEQ ID NO: 25), EPS4 (E28G / K43E) (SEQ ID NO: 26), EPS5 (E28G / D51T) (SEQ ID NO: 27), and EPS6 (E28G / N60F) (SEQ ID NO: 28), were selected as mutants with equivalent or increased alkaline tolerance compared to mutant EP2 (E28G). The absorbance of the six additionally selected mutants was compared with that of mutant EP2 (E28G), and the results are shown in Figure 3 (expressed as relative values ​​to the absorbance of EP2 (100%)).

[0122] <Example 5> Improvement of wB3 using site-directed mutagenesis (DNA shuffling) Site-directed mutagenesis (DNA shuffling) was used to further confer alkaline tolerance to the mutants with increased alkaline tolerance confirmed in Examples 3 and 4. To this end, a site-directed mutagenesis library was constructed for the five selected amino acid mutations (Y13F, E28G / W, K43E / P, D51T / V, and N60E / F), and the specific construction process was as follows (see Figure 4).

[0123] To prepare a library with a mutation at position 13, PCR was performed using the EP2 (E28G) (SEQ ID NO: 8)-encoding gene as a template with the EP-F primer (SEQ ID NO: 5), SH13-R1 primer (SEQ ID NO: 31), and SH13-R2 primer (SEQ ID NO: 32), yielding a PCR product of approximately 0.1 kb. To prepare a library with a mutation at position 28, PCR was performed using the wB3 (SEQ ID NO: 4)-encoding gene, the EP2 (E28G) (SEQ ID NO: 8)-encoding gene, and the EPS3 (SEQ ID NO: 25)-encoding gene as templates with the SH13-F1 primer (SEQ ID NO: 29), SH13-F2 primer (SEQ ID NO: 30), SH43-R1 primer (SEQ ID NO: 35), and SH43-R2 primer (SEQ ID NO: 36), yielding a PCR product of approximately 0.14 kb. To prepare a library with a mutation at position 43, PCR was performed using the EP2 (E28G) (SEQ ID NO: 8)-encoding gene as a template with the SH43-F1 primer (SEQ ID NO: 33), SH43-F2 primer (SEQ ID NO: 34), SH51-R1 primer (SEQ ID NO: 39), and SH51-R2 primer (SEQ ID NO: 40) to recover a PCR product of approximately 0.07 kb. To prepare a library with a mutation at position 51, PCR was performed using the EP2 (E28G) (SEQ ID NO: 8)-encoding gene as a template with the SH51-F1 primer (SEQ ID NO: 37), SH51-F2 primer (SEQ ID NO: 38), SH60-R1 primer (SEQ ID NO: 44), SH60-R2 primer (SEQ ID NO: 45), and SH60-R3 primer (SEQ ID NO: 46) to recover a PCR product of approximately 0.08 kb. To prepare a library in which the 60th amino acid was mutated, PCR was performed using the EP2 (E28G) (SEQ ID NO: 8) coding gene as a template with the SH60-F1 primer (SEQ ID NO: 41), SH60-F2 primer (SEQ ID NO: 42), SH60-F3 primer (SEQ ID NO: 43) and T7 primer (SEQ ID NO: 6), and a PCR product of approximately 0.32 kb in size was recovered.The PCR reaction mixture consisted of the template DNA, primers, Pfu-X DNA polymerase, 10x Pfu-X Reaction buffer, and 10 mM dNTPs, adjusted to a final volume of 100 μL. The reaction mixture was reacted under the following conditions: initial denaturation at 95°C for 2 minutes, followed by 25 cycles of denaturation at 95°C for 50 seconds, annealing at 58°C for 40 seconds, and polymerization at 72°C for 1 minute, followed by post-polymerization at 72°C for 5 minutes.

[0124] The 102 bp PCR product, 141 bp PCR product, 67 bp PCR product, 78 bp PCR product, and 320 bp PCR product obtained under these conditions were mixed and PCR was performed without adding primers to recover a PCR product of approximately 0.7 kb in size in which the five PCR products were ligated into one. Using the approximately 0.7 kb PCR product as a template, PCR was performed using the EP-F primer (SEQ ID NO: 5) and the T7 primer (SEQ ID NO: 6) to amplify a multi-mutant DNA fragment of approximately 0.7 kb. The resulting approximately 0.7 kb PCR product was inserted into the pBC KS(+) vector DNA as described in Example 2-2 and transformed into E. coli DH5α to prepare a site-directed mutant library.

[0125] <Example 6> Development of mBF mutants with increased alkaline tolerance: Comparison of alkaline tolerance of mutant protein mBF monomers The library prepared in Example 5 was purified as in Examples <1-3>, and then the absorbance was measured over time in the same manner as in <Example 3> to compare the alkaline resistance. Figure 5 shows the absorbance over time as a relative value to the absorbance at the start (0 h) (100%).

[0126] As shown in Figure 5, the library of Example 5 was screened using the same method as in Example <3-2>, and mBF4 (Y13F / E28G / K43P) (SEQ ID NO: 47) was selected as a mutant with increased alkaline tolerance compared to mutant EP2. As shown in Figure 5, when the activity of the mBF4 protein was compared after treatment with 0.3 M NaOH for 5 hours, it was confirmed that the alkaline tolerance of the mBF4 protein was increased by about 2.7-fold compared to the wB3 protein having the wild-type amino acid sequence.

[0127] The amino acid sequences of the protein L B3 domain mutants screened in Examples 1 to 6 are summarized in Table 2 below.

[0128] [Table 2]

[0129] Example 7: Investigation of alkaline resistance of mBF tetramer

[0130] <7-1>4mBF gene synthesis To further confirm the alkaline tolerance of the mBF4 protein confirmed in Example 6-1, a tetramer encoding the mBF4 mutant was synthesized and named 4mBF. The gene sequence was subjected to codon optimization (SEQ ID NO: 48) and then synthesized by Cosmogenetech Co., Ltd. (Daejeon, Korea).

[0131] <7-2> Construction of pET-4mBF plasmid The 4mBF gene (SEQ ID NO: 48) obtained in Example <7-1> was cloned into the pET29a(+) vector (Stratagene, USA) in the same manner as in Example <1-2> to prepare the pET-4mBF plasmid, which expresses the mutant protein 4mBF represented by SEQ ID NO: 49.

[0132] <7-3> Purification of mutant protein mBF tetramer using nickel affinity resin To cultivate the E. coli DH5α transformants transformed with the pET-4mBF plasmid and containing the 4mBF gene, 5 mL of LB liquid medium containing kanamycin antibiotic was dispensed into a 50 mL conical tube, inoculated with the selected transformants, and cultured with shaking at 37°C and 200 rpm for 16 hours. 500 mL of TB liquid medium containing kanamycin antibiotic was dispensed into a 2000 mL Erlenmeyer flask, and the culture medium was inoculated with 1% (v / v) of the seed culture medium. Cultured with shaking at 37°C and 200 rpm. After culturing with shaking until the OD600 reached approximately 0.6, IPTG (isopropyl-β-D-thio-galactopyranoside) was added to a final concentration of 1 mM, and cultured with shaking at 37°C and 200 rpm for an additional 18 hours. The flask culture was centrifuged (4°C, 8,000 rpm, 20 minutes) to recover the bacterial cells, which were then suspended in 30 mL of PBS buffer (pH 7.4) (Intron Biotechnology, Korea). The suspension was disrupted using an ultrasonic homogenizer at 4°C for 30 minutes, then centrifuged (4°C, 20,000 rpm, 20 minutes) to remove the supernatant. A column was filled with 7 mL of WorkBeads™ 40 Ni-NTA (bioworks, Sweden), and 35 mL of binding buffer (20 mM NaHPO, 300 mM NaCl, 10 mM imidazole, pH 8.0) was poured through. 30 mL of the supernatant and 30 mL of binding buffer were mixed and then poured through the column. After 35 mL of wash buffer (20 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole pH 8.0) was applied, 40 mL of elution buffer (20 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole pH 8.0) was applied, and the flow-through was collected in a 50 mL conical tube. The purified protein was desalted using a HiPrep 26 / 10 desalting column (Cytiva, USA).

[0133] <7-4> Comparison of alkaline resistance of mutant protein mBF tetramer 4mBF purified in Example 7-3 was coupled to NHS (N-hydroxysuccinimide)-Activated Sepharose 4 Fast flow (Cytiva, Sweden). The alkaline resistance of the prepared 4mBF resin (4mBF-agarose beads) was compared with that of a commercially available protein L-based resin from Cytiva (Cytiva 17-5478-01, Capto L), known for its high alkaline resistance. A Tricorn 5 / 50 column (Cytiva, USA) was filled with 1 ml of each resin, and 5 ml of PBS buffer (pH 7.4) (Intron Biotechnology, Korea) was run at 1 ml / min. 25 ml of approximately 1 mg / ml purified antibody (KBIO, Korea) was diluted with 25 ml of PBS buffer and run at 1 ml / min. After 5 ml of PBS buffer was run at 1 ml / min to remove unbound antibody protein, 5 ml of elution buffer (0.1 M glycine-HCl, pH 2.5) was run at 0.5 ml / min and the flow-through was collected to isolate the bound antibody. The isolated antibody was neutralized and collected in a tube containing 0.5 ml of neutralization buffer (1 M Tris-HCl, pH 8.5), and the amount of recovered antibody was measured. After the initial binding assay, 5 ml of PBS buffer was run at 1 ml / min for alkaline treatment, followed by 7.5 ml of 0.3 M NaOH at 0.5 ml / min, allowing the NaOH to contact the resin for 15 minutes. The remaining NaOH was removed by running 5 ml of PBS buffer at 1 ml / min, which constituted one alkaline treatment. The alkaline treatment was repeated in the same manner (1 to 15 cycles). After alkaline treatment, the purified antibody was bound and collected in the same manner as in the initial binding assay, and the antibody amount was measured. The alkaline resistance of each resin was analyzed by repeating the above procedure and comparing the amount of antibody recovered.

[0134] The results are shown in Figure 6. As shown in Figure 6, 4mBF resin was confirmed to have superior alkali resistance compared to Cytiva's product, which is already widely used in industrial applications. In particular, after 15 alkali treatments, the residual activity of 4mBF resin was 43.46%, approximately 1.4 times higher than Cytiva's product.

Claims

1. A polypeptide comprising an amino acid mutation in the amino acid sequence of SEQ ID NO: 4, the amino acid mutation is a substitution of glutamic acid (Glu, E), which is the 28th amino acid, counting from the proline (Pro, P), which is the second amino acid in the amino acid sequence of SEQ ID NO: 4, with glycine (Gly, G), leucine (Leu, L), proline (Pro, P), or tryptophan (Trp, W); A polypeptide having a sequence identity of 93% or more with the amino acid sequence of SEQ ID NO: 4 and having immunoglobulin-binding ability.

2. The polypeptide of claim 1, further comprising one or more substitutions selected from the following, counting from the second amino acid, proline (Pro, P), in the amino acid sequence of SEQ ID NO: 4: Substitution of the 43rd amino acid, lysine (Lys, K), with proline (Pro, P) or glutamic acid (Glu, E); Substitution of the 13th amino acid, tyrosine (Tyr, Y), with phenylalanine (Phe, F); Substitution of the 51st amino acid, aspartic acid (Asp, D), with valine (Val, V) or threonine (Thr, T); and Substitution of the 60th amino acid, asparagine (Asn, N), with phenylalanine (Phe, F) or glutamic acid (Glu, E).

3. The polypeptide of claim 1, further comprising one substitution selected from the following, counting from the second amino acid, proline (Pro, P), in the amino acid sequence of SEQ ID NO: 4: Substitution of the 43rd amino acid, lysine (Lys, K), with proline (Pro, P) or glutamic acid (Glu, E); Substitution of the 13th amino acid, tyrosine (Tyr, Y), with phenylalanine (Phe, F); Substitution of the 51st amino acid, aspartic acid (Asp, D), with valine (Val, V) or threonine (Thr, T); and Substitution of the 60th amino acid, asparagine (Asn, N), with phenylalanine (Phe, F) or glutamic acid (Glu, E).

4. Counting from the second amino acid, proline (Pro, P), in the amino acid sequence of SEQ ID NO: 4, Substitution of the 43rd amino acid, lysine (Lys, K), with proline (Pro, P); and 2. The polypeptide of claim 1, further comprising: a substitution of the 13th amino acid, tyrosine (Tyr, Y), with phenylalanine (Phe, F).

5. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 4, and comprising the following amino acid mutations: Starting from the second amino acid, proline (Pro, P), in the amino acid sequence of SEQ ID NO: 4, the amino acids are E28G, E28L, E28P, E28W, E28G+Y13F, E28G+K43P, E28G+D51V, E28G+N60E, E28G+K43E, E28G+D51T, E28G+N60F, or Y13F+E28G+K43P.

6. The polypeptide of claim 1, represented by the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 11, 12, 23, 24, 25, 26, 27, 28, or 47.

7. 7. A polypeptide according to any one of claims 1 to 6, further comprising one or more selected from the group consisting of the A, B1, B2, B4, C, W, and M domains of protein L derived from Peptostreptococcus magnus, as represented by the amino acid sequence of SEQ ID NO:

50.

8. A polypeptide multimer comprising two or more repeating units each consisting of the polypeptide according to any one of claims 1 to 6.

9. The polypeptide multimer of claim 8, represented by the amino acid sequence of SEQ ID NO:

49.

10. The polypeptide multimer of claim 8, further comprising one or more selected from the group consisting of the A, B1, B2, B4, C, W, and M domains of protein L derived from Peptostreptococcus magnus, represented by the amino acid sequence of SEQ ID NO:

50.

11. A protein L mutant comprising, as a B3 domain, a polypeptide multimer containing two or more repeating units of a polypeptide described in any one of claims 1 to 6 or said polypeptide, wherein said protein L is derived from Peptostreptococcus magnus and is represented by the amino acid sequence of SEQ ID NO:

50.

12. A nucleic acid molecule encoding a polypeptide described in any one of claims 1 to 6, a polypeptide multimer containing two or more repeating units consisting of said polypeptide, or a protein L mutant containing said polypeptide or polypeptide multimer as a B3 domain, wherein said protein L is derived from Peptostreptococcus magnus and is represented by the amino acid sequence of SEQ ID NO:

50.

13. A recombinant vector comprising the nucleic acid molecule of claim 12.

14. A recombinant cell comprising the nucleic acid molecule of claim 12 or a recombinant vector comprising the same.

15. A chromatography matrix comprising a polypeptide described in any one of claims 1 to 6, a polypeptide multimer containing two or more repeating units consisting of said polypeptide, or a protein L mutant containing said polypeptide or polypeptide multimer as a B3 domain coupled to a solid support, wherein said protein L is derived from Peptostreptococcus magnus and is represented by the amino acid sequence of SEQ ID NO:

50.

16. (1) A polypeptide according to any one of claims 1 to 6. (2) A polypeptide multimer containing two or more repeating units consisting of the polypeptide. (3) A protein L mutant containing the polypeptide or polypeptide multimer as a B3 domain. (4) a nucleic acid molecule encoding the polypeptide, polypeptide multimer, or protein L variant; (5) A recombinant vector containing the nucleic acid molecule. (6) A recombinant cell containing the nucleic acid molecule or recombinant vector, and (7) A composition for immunoglobulin binding, comprising one or more selected from the group consisting of a chromatography matrix in which the polypeptide, polypeptide multimer, or protein L variant is coupled to a solid support, wherein the protein L is derived from Peptostreptococcus magnus and is represented by the amino acid sequence of SEQ ID NO:

50.

17. (a) a polypeptide according to any one of claims 1 to 6; (b) a polypeptide multimer containing two or more repeating units consisting of the polypeptide; (c) a protein L mutant comprising the polypeptide or polypeptide multimer as a B3 domain; or (d) A composition for separating or purifying immunoglobulins, comprising a chromatographic matrix having a plurality of ligands, including the polypeptide, polypeptide multimer, or protein L variant, coupled to a solid support, wherein the protein L is derived from Peptostreptococcus magnus and is represented by the amino acid sequence of SEQ ID NO:

50.

18. A sample containing immunoglobulins is (a) a polypeptide according to any one of claims 1 to 6; (b) a polypeptide multimer containing two or more repeating units consisting of the polypeptide; (c) a protein L mutant comprising the polypeptide or polypeptide multimer as a B3 domain; and (d) a chromatography matrix in which a plurality of ligands, including the polypeptide, polypeptide multimer, or protein L variant, are coupled to a solid support, thereby adsorbing the immunoglobulin, wherein the protein L is derived from Peptostreptococcus magnus and is represented by the amino acid sequence of SEQ ID NO: 50.

Images