Recombinant Fc-binding protein extraction reagent

Abstract

To provide an extraction reagent for efficiently extracting Fc-binding proteins expressed within the cells of Escherichia coli capable of expressing such proteins. [Solution] An extraction reagent for extracting an Fc-binding protein expressed in the cells of Escherichia coli capable of expressing the Fc-binding protein, wherein the Fc-binding protein is a polypeptide selected from a specific sequence, and the extraction reagent does not contain sodium chloride or contains 120 mmol / L or less of sodium chloride.

Description

Technical Field

[0001] The present disclosure relates to an extraction reagent for extracting the Fc-binding protein expressed in the cells of Escherichia coli capable of expressing the Fc-binding protein, and a method for producing the Fc-binding protein using the extraction reagent. One aspect of the present disclosure relates to an extraction reagent for extracting human neonatal Fc receptor (human FcRn) expressed in the cells of Escherichia coli capable of expressing human FcRn, and a method for producing FcRn using the extraction reagent.

Background Art

[0002] Fc-binding proteins such as Fc receptors (FcR) are receptor proteins that bind to the Fc region of immunoglobulin molecules, and bind to immune complexes of antigens and immunoglobulins to transmit signals into cells (Non-Patent Document 1). Each individual molecule recognizes a single immunoglobulin isotype or an immunoglobulin isotype of the same group via the recognition domain belonging to the immunoglobulin superfamily by the recognition domain on FcR. This determines which accessory cells are recruited in the immune response.

[0003] FcR can be further classified into several subtypes. In addition to Fcγ receptors (FcγR), which are receptors for immunoglobulin G (IgG), there are Fcα receptors (FcαR), Fcε receptors (FcεR), and the like. Each receptor is further classified in detail, and in the case of FcγR, it can be classified into subtypes FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIc (CD32c), FcγRIIIa (CD16a) and FcγRIIIb (CD16b) (Non-Patent Documents 1 and 2).

[0004] On the other hand, human neonatal FcR (human FcRn) is a major histocompatibility complex (MHC) class I related molecule, composed of a heavy chain (α chain) and β2 microglobulin (β chain) (Non-Patent Literature 3). The amino acid sequence of the α chain of human FcRn (SEQ ID NO: 1) is published in public databases such as UniProt (Accession number: P55899). The amino acid sequence of the β chain (SEQ ID NO: 2) is published in UniProt (Accession number: P61769).

[0005] The binding of human FcRn to IgG (Fc region) is pH-dependent, binding between pH 6.0 and 6.5 and dissociation at pH 7.4 or higher. This pH dependence is involved in the recycling and transport mechanisms of IgG in the body, and IgG and Fc fusion proteins that bind to and dissociate from human FcRn in a pH-dependent manner are known to have a long lifespan in the body (Non-Patent Literature 4). Taking advantage of this characteristic of human FcRn, a method for evaluating the lifespan of human IgG in the body using an affinity column with recombinant human FcRn as a ligand is known (Non-Patent Literature 5).

[0006] As mentioned above, recombinant human FcRn has the property of functioning as a ligand for affinity columns, so there is a need for an efficient method for producing recombinant human FcRn. Patent Document 1 discloses that human FcRn can be efficiently extracted by adjusting the pH of the reagent used to extract the human FcRn expressed in the cells of Escherichia coli capable of expressing human FcRn to between pH 6.8 and pH 10.5. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2024-123994 [Non-patent literature]

[0008] [Non-Patent Document 1] Takai T.,Jpn.J.Clin.Immunol.,28,318-326,2005 [Non-Patent Document 2] J.Galon et al.,Eur.J.Immunol.,27,1928-1932,1997 [Non-Patent Document 3] NESimister et al., Nature, 337, 184-187, 1989 [Non-Patent Document 4] M. Raghavan et al., Biochemistry, 34, 14649-14657, 1995 [Non-Patent Document 5] F. Cymer et al., Bioanalysis, 9, 1305-1317, 2017 [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of this disclosure is to provide an extraction reagent for efficiently extracting Fc-binding proteins expressed in the cells of Escherichia coli capable of expressing such proteins. Another object of this disclosure is to provide a method for producing Fc-binding proteins, which includes a step of extracting the Fc-binding proteins using the extraction reagent.

[0010] One object of this disclosure is to provide an extraction reagent for efficiently extracting human neonatal FcR (human FcRn) expressed within the cells of Escherichia coli capable of expressing the human FcRn. Another object of this disclosure is to provide a method for producing human FcRn, which includes a step of extracting human FcRn using the extraction reagent. [Means for solving the problem]

[0011] This disclosure includes the following aspects: [1] An extraction reagent for extracting the Fc-binding protein expressed in the cells of Escherichia coli capable of expressing the Fc-binding protein, The Fc-binding protein is a polypeptide selected from any of the following (i) to (iii): The extraction reagent is one that does not contain sodium chloride, or contains sodium chloride at a concentration of 120 mmol / L or less: (i) Polypeptides comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 2; (ii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from isoleucine at position 21 to methionine at position 119 of the amino acid sequence described in SEQ ID NO: 2, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within these amino acid residues; (iii) A polypeptide having 70% or more identity to the entire amino acid sequence and possessing antibody-binding activity, comprising at least the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in SEQ ID NO: 1 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence described in SEQ ID NO: 2. [2] A step of culturing Escherichia coli capable of expressing Fc-binding protein, [1] A step of extracting the Fc-binding protein expressed in the cells of the cultured Escherichia coli using the extraction reagent described in [1] (extraction step), A method for producing an Fc-binding protein, comprising the step of isolating the Fc-binding protein from the extract obtained in the extraction step, The Fc-binding protein is a polypeptide selected from any of (i) to (iii) below, Method: (i) Polypeptides comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 2; (ii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from isoleucine at position 21 to methionine at position 119 of the amino acid sequence described in SEQ ID NO: 2, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within these amino acid residues; (iii) A polypeptide having 70% or more identity to the entire amino acid sequence and possessing antibody-binding activity, comprising at least the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in SEQ ID NO: 1 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence described in SEQ ID NO: 2. [Effects of the Invention]

[0012] This disclosure provides an extraction reagent for efficiently extracting Fc-binding proteins expressed within the cells of Escherichia coli capable of expressing such proteins. Furthermore, this disclosure provides a method for producing Fc-binding proteins, comprising the step of extracting the Fc-binding proteins using the extraction reagent.

[0013] According to one aspect of this disclosure, an extraction reagent can be provided for efficiently extracting human neonatal FcR (human FcRn) expressed within the cells of Escherichia coli capable of expressing such human FcRn. Furthermore, according to one aspect of this disclosure, a method for producing human FcRn can be provided, comprising the step of extracting human FcRn using the extraction reagent.

[0014] According to the present disclosure, the Fc-binding protein expressed in the cells of Escherichia coli capable of expressing the Fc-binding protein can be efficiently extracted, and as a result, the Fc-binding protein can be efficiently produced. According to one aspect of the present disclosure, the human FcRn expressed in the cells of Escherichia coli capable of expressing human FcRn can be efficiently extracted, and as a result, human FcRn can be efficiently produced. [BRIEF DESCRIPTION OF DRAWINGS]

[0015] [Figure 1] It is a schematic diagram of the α chain of human FcRn. The numbers in the figure indicate the numbers of the amino acid sequence set forth in SEQ ID NO: 1. In the figure, S represents a signal sequence, EC represents an extracellular region, TM represents a transmembrane region, and C represents an intracellular region, respectively. [Figure 2] It is a schematic diagram of the β chain of human FcRn. The numbers in the figure indicate the numbers of the amino acid sequence set forth in SEQ ID NO: 2. In the figure, S represents a signal sequence, and B2M represents β2-microglobulin, respectively. [MODES FOR CARRYING OUT THE INVENTION]

[0016] Hereinafter, the present disclosure is described in detail.

[0017] The Fc-binding protein extracted in the present disclosure is a polypeptide selected from any one of the following (i) to (iii). (i) a polypeptide comprising at least amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 1, and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2; (ii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from isoleucine at position 21 to methionine at position 119 of the amino acid sequence described in SEQ ID NO: 2, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within these amino acid residues; (iii) A polypeptide having 70% or more identity to the entire amino acid sequence and possessing antibody-binding activity, comprising at least the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in SEQ ID NO: 1 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence described in SEQ ID NO: 2.

[0018] The amino acid residues from alanine position 24 to serine position 297 in the amino acid sequence described in Sequence ID No. 1, or the amino acid sequence from alanine position 24 to serine position 297 in the amino acid sequence described in Sequence ID No. 1, correspond to the extracellular region of the human FcRnα chain (the EC region in Figure 1). The amino acid residues from isoleucine position 21 to methionine position 119 in the amino acid sequence described in Sequence ID No. 2, or the amino acid sequence from isoleucine position 21 to methionine position 119 in the amino acid sequence described in Sequence ID No. 2, correspond to the β2 microglobulin region of the human FcRnβ chain (the B2M region in Figure 2).

[0019] The Fc-binding protein extracted in this disclosure may include all or part of the signal peptide region (region S in Figures 1 and 2) located at the N-terminal end of the extracellular region (EC region in Figure 1) of the human FcRnα chain or the β2 microglobulin region (region B2M in Figure 2) of the human FcRnβ chain, or it may include all or part of the transmembrane region (region TM in Figure 1) and extracellular region (region C in Figure 1) located at the C-terminal end of the extracellular region (EC region in Figure 1) of the human FcRnα chain.

[0020] The Fc-binding protein extracted in this disclosure does not depend on the order of the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 (also referred to as the extracellular region of the human FcRnα chain) and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2 (also referred to as the β2 microglobulin region of the human FcRnβ chain). That is, the β2 microglobulin region of the human FcRnβ chain may be located on the N-terminal side or the C-terminal side of the extracellular region of the human FcRnα chain. Furthermore, the extracellular region of the human FcRnα chain and the β2 microglobulin region of the human FcRnβ chain may be directly linked, or they may be linked via a known linker such as a GS linker (a linker consisting of repeating units of 4 glycine residues and 1 serine residue). Furthermore, the Fc-binding protein extracted in this disclosure may contain two or more extracellular regions of the human FcRnα chain and / or β2 microglobulin regions of the human FcRnβ chain.

[0021] The Fc-binding protein extracted in this disclosure may be a polypeptide selected from any of (iv) to (vi) below. (iv) Fc-binding proteins that include amino acid residues from alanine position 24 to serine position 297 in the amino acid sequence described in SEQ ID NO: 1 and amino acid residues from isoleucine position 21 to methionine position 119 in the amino acid sequence described in SEQ ID NO: 2, wherein at least one of the amino acid substitutions shown in (1) to (10) below occurs in the said amino acid residues: (1) The 71st cysteine ​​(C) in sequence number 1 is replaced with arginine (R). (2) The asparagine (N) at position 78 of SEQ ID NO: 1 is replaced with aspartic acid (D). (3) The valine (V) at position 80 of SEQ ID NO: 1 is replaced with aspartic acid (D). (4) The 96th lysine (K) in sequence number 1 is replaced with glutamic acid (E). (5) The asparagine (N) at position 172 of sequence number 1 is replaced with aspartic acid (D). (6) The 192nd arginine (R) in sequence number 1 is replaced with leucine (L). (7) The asparagine (N) at position 196 of SEQ ID NO: 1 is replaced with aspartic acid (D). (8) The glutamine (Q) at position 232 of sequence number 1 is replaced with leucine (L). (9) The 274th cysteine ​​(C) in sequence number 1 is replaced with serine (S). (10) The lysine (K) at position 295 of sequence number 1 is replaced with glutamic acid (E). (v) An Fc-binding protein having antibody-binding activity, comprising amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence described in Sequence ID No. 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 of the amino acid sequence described in Sequence ID No. 2, wherein the amino acid substitutions described in (1) to (10) above occur at said amino acid residues, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions described in (1) to (10) above; (vi) An Fc-binding protein having antibody-binding activity, wherein the amino acid sequence from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2 has an amino acid sequence that is 70% or more identical to the entire amino acid sequence in which the amino acid substitutions described in (1) to (10) above occur, and which includes an amino acid sequence in which the amino acid substitutions described in (1) to (10) above remain.

[0022] In (ii) and (v) above, "one or several" means one of the following, although this can vary depending on the position and type of amino acid residues in the three-dimensional structure of the protein: for example, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Furthermore, modifications of "one or several" amino acid residues may occur in positions other than those disclosed in Japanese Patent Publication No. 2018-183087, Japanese Patent Publication No. 2021-073883, Japanese Patent Publication No. 2021-136967, and Japanese Patent Publication No. 2022-076998, as long as antibody-binding activity is maintained. Furthermore, "one or more of the substitutions, deletions, insertions, and additions" also include naturally occurring mutations (mutants or variants) based on individual differences in the microorganisms from which the genes originate, differences in species, etc. In addition, the Fc-binding proteins of this disclosure may further have conservative substitutions between amino acids that are similar in physical and / or chemical properties. It is known to those skilled in the art that, not limited to Fc-binding proteins, conservative substitutions maintain the function of a protein between a substituted and an unsubstituted sample. Examples of conservative substitutions include those occurring between glycine and alanine, aspartic acid and glutamic acid, serine and proline, or glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005).

[0023] The amino acid sequence identity in (iii) and (vi) above only needs to be 70% or more, and may have a higher degree of identity, for example, 80% or more, 85% or more, 90% or more, or 95% or more.

[0024] In this specification, "identity" of amino acid sequences is expressed as a percentage obtained by aligning the two amino acid sequences to be compared so that as many amino acid residues as possible match, and then dividing the number of matching amino acid residues by the total number of amino acid residues. When aligning the sequences as necessary, gaps are inserted into one or both of the two sequences to be compared as appropriate. The method of aligning sequences is not particularly limited, but it can be done using well-known sequence comparison programs such as BLAST (Basic Local Alignment Search Tool), FASTA, or CLUSTALW. When gaps are inserted, the total number of amino acid residues is calculated by counting each gap as one amino acid residue. If the total number of amino acid residues counted in this way differs between the two sequences to be compared, the sequence identity [%] is calculated by dividing the number of matching amino acid residues by the total number of amino acid residues of the longer sequence.

[0025] In (ii), (iii), (v), and (vi) above, "antibody binding activity" may also mean the activity of binding an antibody to the Fc region.

[0026] One method for measuring the antibody binding activity of Fc-binding proteins is to measure their binding activity to human IgG using, for example, the Enzyme-Linked ImmunoSorbent Assay (ELISA) method.

[0027] The Fc-binding protein of this disclosure may have an oligopeptide added to its N-terminus or C-terminus that is useful for separation from solutions in the presence of contaminants. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid.

[0028] Furthermore, a cysteine-containing oligopeptide, useful for immobilizing the Fc-binding protein of this disclosure onto an insoluble support for chromatography, may be further added to the N-terminal or C-terminal side of the Fc-binding protein. The length of the oligopeptide added to the N-terminal or C-terminal side of the Fc-binding protein is not particularly limited, as long as it does not impair the antibody binding ability or stability of the Fc-binding protein.

[0029] When attaching the oligopeptide to the Fc-binding protein, the polynucleotide encoding the oligopeptide may be prepared and then genetically engineered to attach it to the N-terminus or C-terminus of the Fc-binding protein using methods well known to those skilled in the art. Alternatively, the oligopeptide may be chemically synthesized and attached to the N-terminus or C-terminus of the Fc-binding protein.

[0030] Furthermore, a signal peptide may be added to the N-terminus of the Fc-binding protein to promote efficient expression in the host. Examples of such signal peptides when the host is E. coli include signal peptides that induce protein secretion into the periplasm, such as PelB, DsbA, MalE (the region from amino acid 1 to 26 in the amino acid sequence described in UniProt No. P0AEX9, SEQ ID NO: 3), and TorT (Japanese Patent Publication No. 2011-097898). In particular, (A) A polynucleotide encoding the native OmpA signal peptide (regions 1 to 21 of UniProt No. P0A910, SEQ ID NO. 27), or (B) Oligonucleotides encoding a polypeptide that has been modified by substituting, deleting, inserting, or adding one or more residues of the signal peptide described in (A) above. Using this method allows for more efficient production, which is preferable.

[0031] Escherichia coli capable of expressing the Fc-binding protein of this disclosure may be obtained by transforming Escherichia coli with a polynucleotide encoding the Fc-binding protein. Alternatively, Escherichia coli capable of expressing the Fc-binding protein of this disclosure may be obtained by transforming Escherichia coli with an expression vector containing a polynucleotide encoding the Fc-binding protein, such as a bacteriophage, cosmid, or plasmid commonly used for the transformation of prokaryotic or eukaryotic cells.

[0032] The expression vector is not particularly limited as long as it can be stably present and replicated in E. coli, and may be, for example, a pET plasmid vector, a pUC plasmid vector, a pTrc plasmid vector, a pCDF plasmid vector, or a pBBR plasmid vector.

[0033] When inserting a polynucleotide encoding an Fc-binding protein into the expression vector, it is preferable to insert it in a state where it is linked to a functional polynucleotide such as a promoter necessary for expression. Examples of such promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and also the λPL promoter and λPR promoter of λ phage.

[0034] Transforming Escherichia coli with the aforementioned expression vector can be done using methods commonly used by those skilled in the art, such as the method described in known literature (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992). Examples of Escherichia coli strains used may include JM109, BL21(DE3), and W3110.

[0035] To prepare the expression vector of this disclosure from the transformant of this disclosure, the transformant can be cultured and the resulting culture can be prepared using alkaline extraction or a commercially available extraction kit such as the QIAprep Spin Miniprep kit (Qiagen).

[0036] The method for producing Fc-binding proteins according to this disclosure comprises the following steps (I) to (III): (I) A step of culturing E. coli capable of expressing Fc-binding protein (culturing step) (II) A step of extracting the Fc-binding protein expressed in the cells of the cultured Escherichia coli (extraction step) (III) A step of isolating the Fc-binding protein from the extract obtained in the extraction step (isolation step). <Extraction Reagents> The extraction reagent of this disclosure is used in the extraction step (II) described above, and Fc-binding proteins are extracted by reacting the extraction reagent of this disclosure with the cells of Escherichia coli.

[0037] The extraction reagents of this disclosure are characterized by being sodium chloride-free or containing sodium chloride at a concentration of 120 mmol / L or less. If the extraction reagents of this disclosure contain sodium chloride, the concentration is preferably 90 mmol / L or less, and more preferably 60 mmol / L or less.

[0038] The extraction reagents of this disclosure may further contain additives such as nonionic surfactants, carbohydrate-degrading enzymes, and nucleolytic enzymes. These additives may preferably contain one or more of them, and may also contain two or all three of them.

[0039] If the extraction reagent of this disclosure further contains a nonionic surfactant, it can be appropriately selected from among those commonly used as membrane protein solubilizers, such as Triton X-100 (trade name), Triton X-114 (trade name), Brij 58 (trade name), Brij 35 (trade name), Tween 20 (trade name), and Tween Examples include 80 (trade name), 1-On-octyl-β-D-glucopyranoside, n-octyl-β-D-thioglucopyranoside, n-dodecyl-β-D-maltopyranoside, n-dodecyl-α-D-maltopyranoside, n-dodecyl-N,N-dimethylamine-N-oxide, isopropyl-β-D-thiogalactoside, sucrose monododecanoic acid, n-octyl-β-D-glucopyranoside, n-dodecyl-β-D-maltopyranoside, n-tridecyl-β-D-maltopyranoside, etc. The concentration of the nonionic surfactant to be included in the extraction reagent of this disclosure should be set appropriately, taking into consideration the critical micelle concentration of the surfactant used, the properties and concentration of impurities in the culture medium such as cell disruptors, etc. For example, when using Triton X-100 as a nonionic surfactant, it is preferable to set the final concentration in the range of 0.2% (w / v) to 2% (w / v), and more preferable to set it in the range of 0.3% (w / v) to 1% (w / v).

[0040] Further inclusion of a carbohydrate-degrading enzyme in the extraction reagent of this disclosure is preferable because it promotes the lysis of the transformant (E. coli). The carbohydrate-degrading enzyme that may be included in the extraction reagent of this disclosure can be appropriately selected from those commonly used as membrane protein solubilizers, such as lysozyme, cellulase, pectinase, and their salts. The concentration of the carbohydrate-degrading enzyme should be appropriately set considering the properties and concentrations of impurities in the culture medium, such as cell lysates. As an example, when using human-derived lysozyme as the carbohydrate-degrading enzyme, it is preferable to set the final concentration in the range of 0.0001% (w / v) to 0.01% (w / v), and more preferably in the range of 0.003% (w / v) to 0.008% (w / v).

[0041] It is preferable to further include a nuclease, particularly an endonuclease (nuclease-degrading enzyme) such as benzonase (Merck), in the extraction reagent of this disclosure, because this suppresses the increase in viscosity caused by nucleic acids, which are extracted from the transformed tissue along with the protein by the aforementioned extraction reagent. When further including a nuclease in the extraction reagent of this disclosure, it is preferable to include magnesium sulfate at a final concentration of about 2 mmol / L as an auxiliary agent.

[0042] The pH of the extraction reagent in this disclosure is not particularly limited, but may be, for example, pH 5.5 or higher and 10.5 or lower.

[0043] Other components that may be further included in the extraction reagent of this disclosure include buffer components, such as citrate-phosphate buffer, phosphate buffer, glycine-NaOH buffer, MES (2-Morpholinoethanesulfonic acid) buffer, ADA (N-(2-Acetamido)iminodiacetic acid) buffer, PIPES (Piperazine-1,4-bis(2-ethanesulfonic acid)) buffer, ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid) buffer, cholamine-HCl buffer, BES (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer, TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffer, HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic The buffers may include acid buffer, acetamidoglycine buffer, tricinamide buffer, tricinamide buffer, bicinamide buffer, tris(2-Amino-2-hydroxymethyl-1,3-propanediol) buffer, CAPS(N-Cyclohexyl-3-aminopropanesulfonic acid) buffer, etc.

[0044] Furthermore, the extraction reagent of this disclosure may further contain salts other than NaCl. These salts may be, for example, sulfates, phosphates, acetates, citrates, carbonates, and the like. <Culture process> E. coli capable of expressing Fc-binding proteins can be cultured according to standard methods. The culture medium can be, for example, LB (Luria-Bertani) medium, 2×YT medium, TB (Terrific Broth) medium, etc.

[0045] Furthermore, in order to selectively grow the transformants of this disclosure depending on whether or not the expression vector is introduced, it is preferable to add a drug corresponding to the drug resistance gene contained in the expression vector to the culture medium. For example, if the expression vector contains a kanamycin resistance gene, kanamycin should be added to the culture medium. In addition to carbon, nitrogen, and inorganic salt sources, the culture medium may also contain a suitable nutrient source, and optionally, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol. Further reagents that promote protein secretion from the transformants into the culture medium, such as glycine, may also be added.

[0046] The culture temperature may be, for example, 10°C to 40°C, preferably 20°C to 37°C, or around 25°C. The pH of the culture medium may be pH 6.8 to pH 7.4, preferably around pH 7.0. If the expression vector disclosed herein contains an inducible promoter, it is preferable to induce it under conditions that allow for good expression of the Fc-binding protein. IPTG (isopropyl-β-D-thiogalactopyranoside) can be used as an example of an inducer. The expression of the Fc-binding protein may be induced by measuring the turbidity of the culture medium (absorbance at 600 nm), adding an appropriate amount of IPTG when it reaches approximately 0.5 to 1.0, and then continuing the culture. The concentration of IPTG added may be appropriately selected from the range of 0.005 mmol / L to 1.0 mmol / L, but the range of 0.01 mmol / L to 0.5 mmol / L is preferred. Various conditions for IPTG induction may be carried out under conditions well known in the art. <Extraction process> Prior to the extraction step, the procedure may include a step of recovering the culture obtained in the cultivation step and recovering the E. coli cells by centrifugation.

[0047] Fc-binding proteins are extracted by reacting E. coli cells with the extraction reagent of this disclosure. For example, the extraction reagent may be added to E. coli cells and left for a predetermined time.

[0048] The aforementioned predetermined time may be, for example, 10 minutes or more, 30 minutes or more, or 1 hour or more.

[0049] The aforementioned "standing" may include, for example, letting it stand, stirring, or vortexing.

[0050] Furthermore, while the extraction reagent of this disclosure is used to extract Fc-binding proteins from E. coli cells, the E. coli cells to which the extraction reagent of this disclosure has been added may also be subjected to physical disruption such as ultrasonic disruption or French press treatment.

[0051] The extract obtained in the extraction step can be centrifuged to obtain a soluble fraction, which may be used in the subsequent isolation step. <Isolation Process> The isolation step may also refer to the step of purifying Fc-binding proteins from the extract. Purification of Fc-binding proteins from the extract can be carried out using known methods, and as an example, purification using liquid chromatography may be performed. Liquid chromatography may be, for example, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc., and these chromatography methods may be combined to perform the purification operation.

[0052] Purification using liquid chromatography can be performed, for example, by equilibrating a column packed with a predetermined adsorbent, applying the extract, and eluting the Fc-binding protein with a predetermined eluent.

[0053] The extract applied to the column may be the soluble fraction of the extract. Alternatively, the extract applied to the column may be the soluble fraction of the extract, and that soluble fraction may be adjusted to the pH at which the column was equilibrated. [Examples]

[0054] The present disclosure will be described in more detail below using examples and comparative examples, but the present disclosure is not limited to these examples.

[0055] Example 1: Production of recombinant Fc-binding protein (examination of sodium chloride content in extraction reagent) (1) Recombinant E. coli was obtained by transforming E. coli BL21 strain (DE3) with an expression vector containing a polynucleotide (SEQ ID NO: 4) encoding an Fc-binding protein (OmpA-FcRn(B2M-GS-EC_m10)-6HC) consisting of the amino acid sequence described in SEQ ID NO: 3.

[0056] Of the sequence number 3, the first methionine (M) to the 21st alanine (A) is the OmpA signal peptide (the region from the 1st to the 21st in UniProt No. P0A910), the 22nd isoleucine (I) to the 120th methionine (M) is the β2 microglobulin region of the human FcRn β chain (the region from the 21st to the 119th in sequence number 2, B2M), the 121st glycine (G) to the 145th serine (S) is the GS linker sequence, the 146th alanine (A) to the 419th serine (S) is the amino acid substitution EC_m10 (sequence number 5) region of the extracellular region of the human FcRn α chain, the 420th to the 425th histidine (H) is the histidine tag (6H) sequence, and the 426th cysteine ​​(C) to the 432nd glycine (G) is the cysteine ​​tag (C) sequence. Furthermore, EC_m10 is a polypeptide obtained by introducing the following 10 amino acid substitutions into the extracellular region of the human FcRnα chain (the region from position 24 to 297 of SEQ ID NO: 1, EC); The cysteine ​​(C) at position 71 in sequence number 1 (position 48 in sequence number 5) is replaced with arginine (R). The asparagine (N) at position 78 in SEQ ID NO: 1 (position 55 in SEQ ID NO: 5) is replaced with aspartic acid (D). The valine (V) at position 80 in SEQ ID NO: 1 (position 57 in SEQ ID NO: 5) is replaced with aspartic acid (D). The lysine (K) at position 96 in sequence number 1 (and position 73 in sequence number 5) is replaced with glutamic acid (E). The asparagine (N) at position 172 in SEQ ID NO: 1 (and position 149 in SEQ ID NO: 5) is replaced with aspartic acid (D). The arginine (R) at position 192 in sequence number 1 (position 169 in sequence number 5) is replaced with leucine (L). The asparagine (N) at position 196 in SEQ ID NO: 1 (position 173 in SEQ ID NO: 5) is replaced with aspartic acid (D). The glutamine (Q) at position 232 in sequence number 1 (position 209 in sequence number 5) is replaced with leucine (L). The cysteine ​​(C) at position 274 in sequence number 1 (position 251 in sequence number 5) is replaced with serine (S). The lysine (K) at position 295 in sequence number 1 (position 272 in sequence number 5) is replaced with glutamic acid (E). (2) Genetically modified Escherichia coli capable of expressing the Fc-binding protein obtained in (1) was inoculated into 2×YT medium (tryptone: 16 g / L, yeast extract: 10 g / L, sodium chloride: 5 g / L, kanamycin sulfate: 50 mg / L) and pre-cultured at 30°C for 16 hours.

[0057] (3) The main culture was performed by adding 36 mL of the pre-culture solution from (1) to 1.2 L of the initial culture medium (18 g / L disodium hydrogen phosphate dodecahydrate, 6 g / L trisodium phosphate dodecahydrate, 40 g / L Difco Soytone (Thermo Fisher Scientific), 1 g / L ammonium chloride, 10 g / L glucose, 1 g / L magnesium sulfate heptahydrate, 0.01 g / L iron(II) sulfate heptahydrate, 0.005 g / L manganese(II) chloride tetrahydrate, 200 μL / L adecanol (ADEKA), 50 mg / L kanamycin sulfate).

[0058] The culture apparatus used was an Able BMS-03PI, with a stirring speed of 400 to 700 rpm, an airflow rate of 1.5 L / min, a culture temperature of 30°C, and a pH of 6.8 to 7.2. pH fluctuations during culture were controlled within the aforementioned range by adding 14% (w / v) aqueous ammonia or 50% (w / v) phosphoric acid.

[0059] (4) When the DO (dissolved oxygen concentration) measured by the DO electrode attached to the BMS-03PI exceeded 40% saturation, the fed-batch pump was activated, and the operation of supplying fed-batch medium (425 g / L D(+)-glucose, 124 g / L yeast extract red label (Oriental Yeast Co., Ltd.), 12.5 g / L magnesium sulfate heptahydrate, 50 mg / L kanamycin sulfate) was continued until the DO fell below 40% saturation again, until the end of the culture.

[0060] (5) Between 19 and 21 hours after the start of culture, the culture temperature was changed to 25°C and the stirring speed to 600 rpm, and IPTG (Isopropyl β-D-thiogalactopyranoside) was added to a final concentration of 0.1 mmol / L to induce the expression of Fc-binding proteins.

[0061] (6) 48 hours after the start of cultivation, the cultivation was terminated and the cultured cells were recovered by centrifugation of the culture medium.

[0062] (7) The collected cultured cells were mixed with one of the extraction reagents shown in Table 1 (A) to (F), and stirred at room temperature for 2 hours to extract the Fc-binding protein expressed within the cells.

[0063] [Table 1]

[0064] (8) After the extraction procedure, the mixture was centrifuged at 15,000 rpm for 20 minutes at 4°C, and the supernatant was collected to obtain an extract containing Fc-binding protein.

[0065] If the extraction procedure in (9)(7) was performed using any of the extraction reagents shown in (A) to (C) of Table 1, the extract containing the Fc-binding protein obtained in (8) was adjusted to pH 6.0 using 1 mol / L hydrochloric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0066] The extracts containing Fc-binding proteins obtained in (10)(8) (each extract extracted using any of the extraction reagents shown in (D) to (F)), or each extract containing Fc-binding proteins after pH adjustment in (9), were applied to a polyprep column (Bio-Rad) packed with 3 mL of IgG-Sepharose (Cytiva) that had been pre-equalized with 50 mmol / L bis-trispropane buffer (pH 6.0) containing 150 mmol / L sodium chloride.

[0067] After washing with the buffer used for equilibration, Fc-binding proteins were obtained by eluting with 50 mmol / L bis-trispropane buffer (pH 8.5) containing 150 mmol / L sodium chloride.

[0068] The purified protein yield was calculated by measuring the absorbance at a wavelength of 280 nm for the protein purified solution obtained in (11)(10).

[0069] Comparative Example 1 Except for using one of the extraction reagents shown in (G) to (J) of Table 2 as the extraction reagent added in Example 1(7), Fc-binding proteins were obtained in the same manner as in Example 1, and the purified yield was calculated. Note that the procedure in Example 1(9) was performed when the extraction reagent shown in (G) or (H) of Table 2 was added.

[0070] [Table 2]

[0071] Table 3 shows the results from Example 1 and Comparative Example 1 when using pH 10.0 extraction reagents (i.e., (A), (B), (C), (G), and (H)), and Table 4 shows the results when using pH 6.0 extraction reagents (i.e., (D), (E), (F), (I), and (J)). In Tables 3 and 4, the purified yield is expressed as a relative value with the purified yield using an extraction reagent containing 150 mmol / L sodium chloride (G in Table 3, and I in Table 4) set to 1.

[0072] [Table 3]

[0073] [Table 4]

[0074] Regardless of the pH of the extraction reagent, the purified yield improved when the extraction reagent did not contain sodium chloride, or when the sodium chloride content in the extraction reagent was 120 mmol / L or less. This result indicates that when extracting the Fc-binding protein expressed within the cells of Escherichia coli capable of expressing the protein, using an extraction reagent that does not contain sodium chloride, or has a sodium chloride concentration of 120 mmol / L or less, improves the extraction efficiency of the protein and increases the purified yield.

Claims

1. An extraction reagent for extracting the Fc-binding protein expressed in the cells of Escherichia coli capable of expressing the Fc-binding protein, The Fc-binding protein is a polypeptide selected from any of the following (i) to (iii): The extraction reagent is one which does not contain sodium chloride, or contains sodium chloride at a concentration of 120 mmol / L or less: (i) Polypeptides comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2; (ii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within these amino acid residues; (iii) A polypeptide having 70% or more identity to the entire amino acid sequence and possessing antibody-binding activity, comprising at least the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in SEQ ID NO: 1 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence described in SEQ ID NO:

2.

2. A step of culturing E. coli capable of expressing Fc-binding protein, A step of extracting the Fc-binding protein expressed in the cells of the cultured Escherichia coli using the extraction reagent described in claim 1 (extraction step), A method for producing an Fc-binding protein, comprising the step of isolating the Fc-binding protein from the extract obtained in the extraction step, The Fc-binding protein is a polypeptide selected from any of (i) to (iii) below, Method: (i) Polypeptides comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2; (ii) A polypeptide having antibody-binding activity, comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within these amino acid residues; (iii) A polypeptide having 70% or more identity to the entire amino acid sequence and possessing antibody-binding activity, comprising at least the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in SEQ ID NO: 1 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence described in SEQ ID NO: 2.

Images