Method for producing immunoglobulin-binding protein

Abstract

To provide a method for efficiently producing Protein L, derived from Finegoldia bacteria, employing recombinant Escherichia coli capable of expressing the Protein L.SOLUTION: The foregoing problem is solved by a production method that includes the steps of: culturing the recombinant Escherichia coli in a medium containing at least one of magnesium salt, calcium salt, and manganese salt, as well as peptone, and either containing no glucose or containing glucose at 3.8 g / L or less, thereby inducing the expression of the Protein L; and recovering the expressed Protein L.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method for efficiently producing an immunoglobulin-binding protein by using a recombinant Escherichia coli obtained by genetic engineering techniques and capable of expressing the protein, and in particular to a method for efficiently producing the protein from the Escherichia coli by optimizing the culture conditions of the Escherichia coli and the expression conditions of the protein. [Background technology]

[0002] Antibody drugs are medicines that use antibodies (immunoglobulins), molecules that play an important role in the body's immune system. Due to the diversity of the variable regions of antibodies, antibody drugs bind to target molecules with high specificity and affinity. For this reason, antibody drugs have few side effects, and in recent years, the range of diseases for which they can be used has been expanding, leading to a rapid expansion of the market.

[0003] The production of antibody drugs involves a culture process and a purification process, during which antibody-producing cells are modified and culture conditions are optimized to improve productivity. In the purification process, affinity chromatography is used for crude purification, followed by intermediate purification, final purification, and virus removal before being formulated.

[0004] In the purification process, an affinity carrier that specifically recognizes antibody molecules is used. Staphylococcus-derived Protein A, which has the property of binding to antibodies (immunoglobulins), is often used as a ligand protein used in the carrier (Patent Document 1). However, since Protein A is a protein that specifically binds to the Fc region of an antibody, it cannot be applied to the purification of antibodies that do not have an Fc region, such as single chain Fv (scFv), Fab, F(ab')2, IgA, and bispecific T cell-inducing (BiTE) antibodies. On the other hand, Finegoldia-derived Protein L (hereinafter also referred to as "FpL") is a protein that binds to the κ light chain of immunoglobulins, and by using FpL as a ligand protein, it is possible to purify antibodies that do not have an Fc region, which cannot be purified with the above-mentioned Protein A (Patent Document 2).

[0005] In order to produce FpL inexpensively, production methods using genetic recombinants capable of expressing FpL have been investigated. For example, a method for producing FpL using recombinant Escherichia coli has been reported (Patent Documents 3 and 4), but there is a demand for further improvement in the expression level. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2010-504754 [Patent Document 2] WO2017 / 191748 issue [Patent Document 3] WO2016 / 121701 [Patent Document 4] WO2017 / 069158 issue Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for efficiently producing Protein L derived from bacteria of the genus Finegoldia using a recombinant Escherichia coli capable of expressing said Protein L. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors have conducted extensive investigations into the culture conditions for recombinant Escherichia coli capable of expressing Protein L derived from bacteria of the genus Finegoldia (hereinafter also referred to as "FpL"), and the expression conditions for said FpL, and as a result have completed the present invention.

[0009] That is, the present invention includes the following aspects: [1] a step of culturing a recombinant Escherichia coli containing a polynucleotide encoding an immunoglobulin-binding protein to express the protein; recovering the expressed protein; A method for producing an immunoglobulin-binding protein, comprising: the immunoglobulin-binding protein is a polypeptide comprising at least the immunoglobulin-binding domain of FpL; The medium used for culturing the recombinant E. coli contains at least peptone, and the medium does not contain glucose or contains 3.8 g / L or less of glucose.

[0010] [2] The method according to [1], wherein the medium used for culturing the recombinant E. coli further contains yeast extract.

[0011] [3] The method according to either [1] or [2], wherein the medium used for culturing the recombinant E. coli contains one or more of a magnesium salt, a calcium salt, and a manganese salt.

[0012] [4] The method according to either [1] or [2], wherein the medium used for culturing the recombinant E. coli contains all of a magnesium salt, a calcium salt, and a manganese salt. Effect of the Invention

[0013] The present invention is characterized in that, when producing a polypeptide using a recombinant Escherichia coli containing a polynucleotide encoding the polypeptide containing at least an immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia, the recombinant Escherichia coli is cultured in a medium containing at least one of a magnesium salt, a calcium salt, and a manganese salt and peptone, and containing no glucose or 3.8 g / L or less of glucose. The production method of the present invention significantly improves the expression amount compared to conventional production methods for immunoglobulin-binding proteins, and therefore the protein can be produced at lower cost. [Brief description of the drawings]

[0014] [Figure 1] This figure shows the results of evaluating the expression of (FpL_C3KX 9a)5 due to differences in glucose concentrations contained in the medium used to culture recombinant Escherichia coli capable of expressing immunoglobulin-binding protein (FpL_C3KX 9a)5 (hereinafter also referred to as (FpL_C3KX 9a)5-expressing Escherichia coli). The rightmost lane is the band of the standard product used to evaluate purified (FpL_C3KX 9a)5. [Diagram 2] FIG. 1 shows the results of evaluating the expression of (FpL_C3KX 9a) 5 due to differences in the concentration of yeast extract contained in the medium used for culturing (FpL_C3KX 9a) 5-expressing Escherichia coli. [Diagram 3] 1 shows the results of evaluating the expression of (FpL_C3KX 9a) 5 due to differences in metal salts added to the medium used for culturing (FpL_C3KX 9a) 5-expressing Escherichia coli. Lane 12 shows the results of culturing in a medium without the addition of metal salts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in detail below.

[0016] An example of the immunoglobulin-binding domain of FpL, which is an immunoglobulin-binding protein produced in the present invention, is the immunoglobulin-binding domain of Protein L derived from Finegoldia magna. Domain B1 (amino acid residues 104 to 173 of GenBank No. AAA25612), Domain B2 (amino acid residues 176 to 245 of GenBank No. AAA25612), Domain B3 (amino acid residues 248 to 317 of GenBank No. AAA25612), Domain B4 (amino acid residues 320 to 389 of GenBank No. AAA25612), Domain B5 (amino acid residues 393 to 462 of GenBank No. AAA25612), Domain C1 (amino acid residues 249 to 317 of SEQ ID NO: 1 (GenBank No. AAA67503)), Domain C2 (amino acid residues 320 to 389 of SEQ ID NO: 1), Domain C3 (amino acid residues 394 to 463 of SEQ ID NO: 1: SEQ ID NO: 2), and Domain C4 (amino acid residues 468 to 537 of SEQ ID NO: 1) is an example.

[0017] When the immunoglobulin-binding domain of FpL is the domain C3 (SEQ ID NO: 2), an example thereof includes a polypeptide shown in any one of (I) to (III) below: (I) a polypeptide comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2; (II) A polypeptide having an amino acid sequence that contains at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO:2, with the proviso that the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, and having antibody binding activity; (III) A polypeptide comprising at least the amino acid residues consisting of the amino acid sequence set forth in SEQ ID NO:2, and having a homology of 70% or more to the amino acid sequence consisting of said amino acid residues, and having antibody binding activity.

[0018] In (II), "one or several" will vary depending on the position of amino acid substitution in the three-dimensional structure of the antibody and the type of amino acid residue, but as an example, it means any of the following: 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0019] In addition, the "substitution of one or several amino acid residues" in (II) may include, in addition to the amino acid substitution at the specific position described above, conservative substitution in which a substitution occurs between amino acids having similar physical and / or chemical properties. Conservative substitution is generally known to those skilled in the art to maintain the function of a protein between a substituted and unsubstituted protein. Examples of conservative substitution include substitution between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). In addition, the "substitution, deletion, insertion, or addition of one or several amino acid residues" in (II) also includes naturally occurring mutations (mutants or variants) based on differences in the origin or species of the antibody.

[0020] An example of the above-mentioned (II) is a protein that contains at least amino acid residues consisting of the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that the amino acid residues have at least one amino acid substitution selected from the following (1) to (53), and has immunoglobulin-binding activity: (1) Substitution of asparagine at position 50 of SEQ ID NO:2 with tyrosine (2) The first glutamic acid in SEQ ID NO:2 is replaced with glycine or valine (3) The third proline in SEQ ID NO:2 is replaced with serine (4) The fourth glutamic acid in SEQ ID NO:2 is replaced with lysine or glycine (5) The fifth glutamic acid in SEQ ID NO:2 is replaced with valine (6) Substitution of lysine at position 7 of SEQ ID NO:2 with alanine (7) Substitution of glutamic acid at position 8 of SEQ ID NO:2 with glycine (8) Substitution of glutamic acid at position 9 of SEQ ID NO:2 with valine (9) Substitution of isoleucine at position 17 of SEQ ID NO:2 with phenylalanine (10) Substitution of glycine at position 21 of SEQ ID NO:2 with arginine (11) Substitution of glutamic acid at position 27 of SEQ ID NO:2 with glycine or arginine (12) Substitution of lysine at position 29 of SEQ ID NO:2 with proline (13) Substitution of threonine at position 31 of SEQ ID NO:2 with leucine (14) Substitution of threonine at position 36 of SEQ ID NO:2 with alanine (15) Substitution of alanine at position 41 of SEQ ID NO:2 with threonine (16) Substitution of asparagine at position 44 of SEQ ID NO:2 with serine or glycine (17) Substitution of glutamic acid at position 49 of SEQ ID NO:2 with threonine or isoleucine (18) Substitution of asparagine at position 50 of SEQ ID NO:2 with serine, lysine, or aspartic acid (19) Glycine at position 51 of SEQ ID NO:2 is replaced with valine (20) Glutamic acid at position 52 of SEQ ID NO:2 is substituted with any one of valine, glycine, and aspartic acid (21) Tyrosine at position 53 of SEQ ID NO:2 is replaced with phenylalanine (22) Substitution of threonine at position 54 of SEQ ID NO:2 with isoleucine or methionine (23) Asparagine at position 62 of SEQ ID NO:2 is substituted with any one of histidine, arginine, leucine, methionine, and tryptophan (24) Asparagine at position 65 of SEQ ID NO:2 is substituted with tyrosine (25) Substitution of alanine at position 69 of SEQ ID NO:2 with threonine (26) The second threonine in SEQ ID NO:2 is substituted with serine or proline (27) The third proline in SEQ ID NO:2 is replaced with arginine (28) Substitution of lysine or arginine for glutamic acid at position 5 of SEQ ID NO:2 (29) Glutamic acid at position 27 of SEQ ID NO:2 is substituted with any one of valine, lysine, and isoleucine (30) Substitution of phenylalanine at position 32 of SEQ ID NO:2 with leucine (31) Substitution of lysine at position 38 of SEQ ID NO:2 with alanine (32) The 44th asparagine in SEQ ID NO: 2 is replaced with isoleucine, (33) Substitution of alanine at position 47 of SEQ ID NO:2 with threonine or arginine (34) Substitution of glutamic acid at position 52 of SEQ ID NO:2 with asparagine (35) The second threonine in SEQ ID NO:2 is replaced with alanine (36) Substitution of the 5th glutamic acid in SEQ ID NO:2 with aspartic acid (37) The sixth proline in SEQ ID NO:2 is replaced with leucine or threonine (38) Substitution of lysine at position 7 of SEQ ID NO:2 with proline (39) Substitution of valine at position 14 of SEQ ID NO:2 with alanine (40) Isoleucine at position 23 of SEQ ID NO:2 is substituted with arginine or valine (41) Substitution of lysine at position 29 of SEQ ID NO:2 with phenylalanine (42) Substitution of threonine at position 31 of SEQ ID NO:2 with methionine (43) Glutamic acid at position 33 of SEQ ID NO:2 is substituted with aspartic acid, glycine, or valine. (44) Substitution of alanine at position 35 of SEQ ID NO:2 with threonine (45) Substitution of threonine at position 36 of SEQ ID NO:2 with serine (46) Substitution of alanine at position 37 of SEQ ID NO:2 with threonine (47) Substitution of alanine at position 41 of SEQ ID NO:2 with isoleucine or tyrosine (48) Asparagine at position 44 of SEQ ID NO:2 is substituted with histidine or arginine (49) Glutamic acid at position 52 of SEQ ID NO:2 is substituted with leucine or tyrosine (50) Glycine at position 60 of SEQ ID NO:2 is replaced with arginine (51) Isoleucine at position 64 of SEQ ID NO:2 is replaced with leucine (52) Isoleucine at position 66 of SEQ ID NO:2 is replaced with valine (53) Alanine at position 69 of SEQ ID NO: 2 is replaced with leucine or valine.

[0021] The homology of the amino acid sequence in (III) may be 70% or more, and may be higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). In this specification, "homology" may mean similarity or identity, and may particularly mean identity. "Homology of amino acid sequence" means homology with respect to the entire amino acid sequence. "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). "Similarity" between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, 31(3), Yodosha). The homology of amino acid sequences can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0022] The immunoglobulin-binding protein produced by the present invention may further have an oligopeptide attached to its N-terminus or C-terminus, which is useful for accelerating analysis and purification from a solution containing contaminants, stabilizing the protein, etc. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, C-myc tag, etc.

[0023] Furthermore, a signal peptide may be added to the N-terminus of the immunoglobulin-binding protein produced by the present invention to promote efficient expression in a host. Examples of signal peptides that cause secretion of proteins into the periplasm include PelB, OmpA, DsbA, DsbC, MalE, and TorT (JP Patent Publication No. 2011-097898).

[0024] In the present invention, the above-mentioned immunoglobulin-binding protein is produced by expressing the protein by culturing recombinant Escherichia coli containing a polynucleotide encoding the protein.

[0025] In the present invention, the Escherichia coli (scientific name: Escherichia coli) strain is not particularly limited, and representative examples include the E. coli JM109 strain, the E. coli W3110 strain, the E. coli HB101 strain, the E. coli MG1655 strain, the E. coli BL21 strain, and the E. coli BL21(DE3) strain. Furthermore, the recombinant E. coli may contain at least a polynucleotide encoding an immunoglobulin-binding protein in an expressible state, and the polynucleotide may be present on an expression vector that autonomously replicates outside of the genomic DNA. The expression vector may be any vector that can express a heterologous protein in E. coli, and examples include pUC plasmid vector, pCDF plasmid vector, pTrc plasmid vector, and pET plasmid vector.

[0026] A polynucleotide encoding an immunoglobulin-binding protein can be, for example, (α) a method of converting the amino acid sequence of an immunoglobulin-binding protein into a nucleotide sequence and artificially synthesizing a polynucleotide containing the nucleotide sequence; (β) A method in which a polynucleotide containing the entire or partial sequence of an immunoglobulin-binding protein is prepared artificially directly or from a cDNA of the immunoglobulin-binding protein by a DNA amplification method such as PCR, and the prepared polynucleotide is ligated by an appropriate method; It can be made with.

[0027] In the above method (α), when converting an amino acid sequence to a nucleotide sequence, it is preferable to convert the sequence taking into consideration the frequency of codon usage in the E. coli to be transformed. Specifically, the following codons are used infrequently (so-called rare codons): AGA / AGG / CGG / CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro), so conversion should be performed to avoid these codons.

[0028] The present invention relates to a medium for culturing a recombinant Escherichia coli containing a polynucleotide encoding an immunoglobulin-binding protein, comprising: One or more of magnesium salts, calcium salts, and manganese salts, and Contains at least peptone, and It is characterized by containing no glucose or less than 3.8 g / L.

[0029] Examples of magnesium salts to be contained in the culture medium include magnesium chloride, magnesium sulfate, magnesium phosphate, magnesium acetate, magnesium nitrate, magnesium oxide, magnesium hydroxide, magnesium citrate, magnesium aspartate, magnesium stearate, magnesium fluoride, magnesium bromide, magnesium iodide, magnesium perchlorate, magnesium sulfide, magnesium hydrogen sulfate, magnesium sulfite, magnesium thiosulfate, magnesium nitrite, magnesium arsenate, magnesium carbonate, magnesium metasilicate, magnesium tetraborate, and magnesium permanganate.

[0030] Examples of calcium salts that may be contained in the culture medium include calcium hydride, calcium hydroxide, calcium oxide, calcium fluoride, calcium chloride, calcium bromide, calcium iodide, calcium perchlorate, calcium chlorate, calcium hypochlorite, calcium sulfide, calcium sulfate, calcium hydrogen sulfate, calcium thiosulfate, calcium sulfite, calcium selenide, calcium selenate, calcium selenate, calcium nitrate, calcium nitrite, calcium phosphate, calcium arsenate, calcium carbonate, calcium hydrogen carbonate, calcium metasilicate, calcium cyanide, calcium tetraborate, calcium permanganate, calcium chromate, calcium dichromate, and calcium acetate.

[0031] Examples of manganese salts that may be included in the medium include manganese chloride, manganese sulfide, manganese sulfate, manganese nitrate, manganese phosphate, manganese carbonate, manganese metasilicate, manganese acetate, manganese bromide, manganese oxalate, manganese fluoride, and manganese iodide.

[0032] In the present invention, it is sufficient that the medium contains at least one of a magnesium salt, a calcium salt, and a manganese salt. It may contain only one of the above salts, i.e. only magnesium salts, only calcium salts, or only manganese salts; It may comprise two of the aforementioned salts, i.e. a magnesium salt and a calcium salt, a magnesium salt and a manganese salt, or a calcium salt and a manganese salt; All three of the above mentioned salts may be included, namely magnesium salts, calcium salts and manganese salts.

[0033] Among these, a medium containing at least a magnesium salt is preferred in that it allows good expression of the immunoglobulin-binding protein.

[0034] Furthermore, it is preferable to further contain yeast extract in the medium used for culturing the recombinant Escherichia coli, since this improves the expression amount of the immunoglobulin-binding protein.

[0035] In the present invention, the method of culturing recombinant E. coli is not particularly limited, and the recombinant E. coli may be cultured by any of batch culture, semi-batch culture (also called fed-batch culture), and perfusion culture, or a combination of these. However, if nutrient sources such as a carbon source and a nitrogen source are added to the medium at once at the start of the culture, the growth of E. coli and the expression of the immunoglobulin-binding protein by the E. coli are inhibited, and by-products such as organic acids are also produced, which may adversely affect the expression efficiency of the protein and the quality of the obtained protein. Therefore, it is preferable to culture the recombinant E. coli by fed-batch culture, in which the amount of nutrient sources added at the start of the culture is minimized and nutrient sources are appropriately supplied (fed-batch) during the culture.

[0036] In the present invention, the culture conditions for the recombinant E. coli are not particularly limited as long as the E. coli can grow and express the immunoglobulin-binding protein, but the culture temperature is preferably from 15° C. to 50° C., and particularly preferably from 20° C. to 33° C. The pH is preferably from 6 to 8. The culture time can be set arbitrarily, but is usually set to from several hours to 100 hours.

[0037] In addition, when the recombinant E. coli contains an inducible promoter and expresses an immunoglobulin-binding protein under the control of the promoter, it is preferable to induce the protein so that it can be well expressed. Examples of inducible promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter, which are promoters that function in E. coli. For example, an inducer according to the type of promoter can be used for expression induction. An example of the inducer is IPTG (isopropyl-β-D-thiogalactopyranoside). Specifically, when the turbidity (absorbance at 600 nm) of the culture solution becomes 0.03 to 2, an appropriate amount of IPTG is added and the culture is continued, whereby the expression of the protein of the present invention can be induced. The concentration of IPTG added is, for example, 0.005 mM to 1.0 mM, preferably 0.01 mM to 0.5 mM, in final concentration. It is preferable that the absorbance of the culture medium at 600 nm upon addition of IPTG is 0.03 to 2, preferably 0.05 to 1, in terms of favorable expression of the immunoglobulin-binding protein.

[0038] To recover the immunoglobulin-binding protein expressed by the above-mentioned method, the protein may be recovered by isolating / purifying it from the culture using a method suitable for the expression form of the protein in recombinant E. coli. For example, when the protein is expressed in the culture supernatant, the cells may be separated by centrifugation, and the immunoglobulin-binding protein may be purified from the resulting culture supernatant. When the protein is expressed intracellularly (including periplasm), the cells may be collected by centrifugation, disrupted by adding an enzyme treatment agent, a surfactant, or the like, and the immunoglobulin-binding protein may be extracted and purified.

[0039] The recovered immunoglobulin-binding protein can be purified by any method known in the art, such as separation / purification using liquid chromatography, which includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc., and the protein can be prepared to a high purity by performing a purification procedure using a combination of these chromatographies.

[0040] The method for measuring the binding activity of the obtained immunoglobulin-binding protein to an antibody includes, for example, a method in which the immunoglobulin-binding protein is separated using a general SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), and then stained with a dye or an immunological method and colorimetrically quantified. As another example, the binding activity to an antibody may be measured using an Enzyme-Linked ImmunoSorbent Assay method (hereinafter referred to as ELISA method) or a surface plasmon resonance method. The activity may be determined by activity quantification using the ELISA method, but the latter method is simple and preferable. The combination of the antibody reacted with the immunoglobulin-binding protein in the ELISA method is not particularly limited as long as the method can quantify the protein. The detection method of ELISA is also not particularly limited, and for example, a commercially available color-developing reagent, fluorescent reagent, or chemiluminescent reagent specific to the enzyme used for labeling may be used for detection. Specifically, when horseradish peroxidase (HRP) is used as the labeling enzyme, a colorimetric quantification method is used after oxidation of a color-developing substrate such as TMB (3,3',5,5'-TetraMethylBenzidine) with HRP and hydrogen peroxide. The antibody used to measure the binding activity is not limited as long as it has binding affinity with the immunoglobulin-binding protein. EXAMPLES

[0041] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0042] Example 1: Examination of glucose concentration in culture medium (1) A recombinant E. coli capable of expressing the immunoglobulin-binding protein (FpL_C3KX 9a)5 consisting of the amino acid sequence set forth in SEQ ID NO:4 (hereinafter, also referred to as (FpL_C3KX 9a)5-expressing E. coli) obtained by transforming E. coli W3110 strain with an expression vector containing a polynucleotide (SEQ ID NO:5) encoding the immunoglobulin-binding protein (FpL_C3KX 9a)5 consisting of the amino acid sequence set forth in SEQ ID NO:4 and an inducible promoter was inoculated into SOB medium (tryptone: 20 g / L, yeast extract: 5 g / L, magnesium sulfate (MgSO4) heptahydrate: 2.4 g / L, sodium chloride (NaCl): 0.5 g / L, potassium chloride (KCl): 0.186 g / L, kanamycin sulfate: 50 mg / L) and precultured at 30°C for 16 hours at 130 rpm. (FpL_C3KX 9a)5 is a polypeptide comprising a pentamer (amino acid residues from the second glutamic acid to the 351st glycine in SEQ ID NO:4) of five linked polypeptides FpL_C3KX 9a (SEQ ID NO:3) in which the following amino acid substitutions have been made in the immunoglobulin-binding domain C of Protein L derived from Finegoldia bacteria (amino acid residues from the 394th to 463rd in GenBank No. AAA67503 (SEQ ID NO:1), SEQ ID NO:2): (i) The fourth glutamic acid in SEQ ID NO:2 (the 397th glutamic acid in SEQ ID NO:1) is replaced with glycine (ii) Proline at position 6 of SEQ ID NO:2 (position 399 of SEQ ID NO:1) is replaced with serine (iii) The lysine at position 7 of SEQ ID NO:2 (position 400 of SEQ ID NO:1) is substituted with alanine (iv) Lysine at position 13 of SEQ ID NO:2 (position 406 of SEQ ID NO:1) is replaced with arginine (v) Lysine at position 22 of SEQ ID NO:2 (position 415 of SEQ ID NO:1) is replaced with arginine (vi) Isoleucine at position 23 of SEQ ID NO:2 (position 416 of SEQ ID NO:1) is substituted with arginine (vii) Lysine at position 29 of SEQ ID NO:2 (position 422 of SEQ ID NO:1) is substituted with isoleucine (viii) Lysine at position 38 of SEQ ID NO:2 (position 431 of SEQ ID NO:1) is substituted with glutamic acid (ix) Asparagine at position 44 of SEQ ID NO:2 (position 437 of SEQ ID NO:1) is substituted with arginine (x) Lysine at position 48 of SEQ ID NO:2 (position 441 of SEQ ID NO:1) is substituted with arginine (xi) Substitution of glutamic acid at position 49 of SEQ ID NO:2 (position 442 of SEQ ID NO:1) with aspartic acid (xii) Asparagine at position 50 of SEQ ID NO: 2 (position 443 of SEQ ID NO: 1) is substituted with tyrosine (xiii) Tyrosine at position 53 of SEQ ID NO:2 (position 446 of SEQ ID NO:1) is replaced with phenylalanine (xiv) Asparagine at position 62 of SEQ ID NO: 2 (position 455 of SEQ ID NO: 1) is substituted with tyrosine (xv) Lysine at position 67 of SEQ ID NO:2 (position 460 of SEQ ID NO:1) is substituted with arginine (xvi) Alanine at position 69 of SEQ ID NO:2 (position 462 of SEQ ID NO:1) is substituted with valine (2) Glucose was added to the SOB medium to a final concentration of 0 g / L (no addition), 0.5 g / L, 1 g / L, 2 g / L, 4 g / L, 10 g / L, 20 g / L, or 30 g / L, and the final OD of the preculture solution in (1) was measured. 600nm was added so that the amount was 0.02.

[0043] (3) After culturing for 4 hours at 30°C and 130 rpm, IPTG (Isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.1 mM, and the culture was continued for another 24 hours at 25°C and 130 rpm. After the culture was completed, 2 mL of the culture medium was centrifuged to collect the cells, and a pellet was obtained.

[0044] (4) 1 mL of BugBuster Protein Extraction Reagent (Merck) was added to the bacterial cell pellet obtained in (3) and vortexed for 20 minutes, followed by centrifugation at 15,000 rpm for 20 minutes, and the supernatant was collected as a bacterial cell extract.

[0045] (5) 10 μL of the bacterial extract was mixed with 10 μL of 2× sample buffer, and then heated at 98° C. for 3 minutes to prepare a sample for electrophoresis.

[0046] (6) 10 μL of the sample prepared in (5) was applied to an acrylamide gel and a current of 20 mA was applied for 90 minutes. After the current was applied, the gel was stained using a gel staining device (eStain, manufactured by GenScript).

[0047] Figure 1 shows the electrophoretic results of the band corresponding to (FpL_C3KX 9a)5. Table 1 shows the results of evaluating the presence or absence of (FpL_C3KX 9a)5 expression based on the band corresponding to purified (FpL_C3KX 9a)5 in the rightmost lane in Figure 1. It can be seen that when cultured in a medium containing glucose at a final concentration of 4 g / L or more, the immunoglobulin-binding protein (FpL_C3KX 9a)5 is no longer expressed. The samples in lanes 5 to 8 have bands at a higher position than the band corresponding to (FpL_C3KX 9a)5, and these bands are thought to correspond to impurity proteins derived from E. coli.

[0048] [Table 1]

[0049] Example 2: Examination of yeast extract added to culture medium (1)(1)(FpL_C3KX 9a)5-expressing E. coli was inoculated into 2xYT medium (phytone peptone: 16 g / L, yeast extract: 10 g / L, NaCl: 5 g / L, kanamycin sulfate: 50 mg / L) and pre-cultured for 16 hours at 30°C and 130 rpm.

[0050] (2) The preculture solution of (1) was added to a medium having one of the compositions shown in Table 2 to obtain a final OD 600nm was added so that the amount was 0.02.

[0051] [Table 2]

[0052] (3) After culturing for 4 hours at 30° C. and 130 rpm, IPTG was added to a final concentration of 0.1 mM, and the culture was continued for another 24 hours at 25° C. and 130 rpm. After the culture was completed, 2 mL of the culture medium was centrifuged to collect the bacteria and obtain a pellet.

[0053] (4) A cell extract was recovered from the cell pellet obtained in (3) by the method described in Example 1(4), and electrophoresis was performed by the method described in Example 1(5) to (6).

[0054] The electrophoretic results of the band corresponding to (FpL_C3KX 9a)5 are shown in Figure 2. It can be seen that the expression of (FpL_C3KX 9a)5 increases as the amount of yeast extract added to the medium increases. Therefore, the expression level of FpL can be improved by adding yeast extract to the medium in addition to peptone.

[0055] Example 3: Study of metal salts (1) (FpL_C3KX 9a)5-expressing E. coli was precultured in the same manner as in Example 2(1).

[0056] (2) The following <d1>from <d12>The preculture solution of (1) was added to the medium described in any one of the above to obtain a final OD 600nm was added so that the amount was 0.02. <d1>20g / L tryptone, 5g / L yeast extract, 1.2g / L sodium chloride <d2>20g / L tryptone, 5g / L yeast extract, 2.8g / L sodium sulfate (Na2SO4) <d3>20g / L tryptone, 5g / L yeast extract, 4.1g / L magnesium chloride (MgCl2) <d4>20g / L tryptone, 5g / L yeast extract, 1.1g / L ammonium chloride (NH4Cl) <d5>20g / L tryptone, 5g / L yeast extract, 2.6g / L ammonium sulfate ((NH4)2SO4) <d6>20g / L tryptone, 5g / L yeast extract, 2.9g / L calcium chloride (CaCl2) <d7>20g / L tryptone, 5g / L yeast extract, 5.5g / L iron sulfate (II) (FeSO4) <d8>20g / L tryptone, 5g / L yeast extract, 5.4g / L iron(III) chloride (FeCl3) <d9>20g / L tryptone, 5g / L yeast extract, 5.7g / L zinc sulfate (ZnSO4) <d10>20g / L tryptone, 5g / L yeast extract, 3.9g / L manganese chloride (MnCl2) <d11>20g / L tryptone, 5g / L yeast extract, 2.4g / L magnesium sulfate <d12>20g / L tryptone, 5g / L yeast extract (no salt added) (3) After culturing for 4 hours at 30° C. and 130 rpm, IPTG was added to a final concentration of 0.1 mM, and the culture was continued for another 24 hours at 25° C. and 130 rpm. After the culture was completed, 2 mL of the culture medium was centrifuged to collect the bacteria and obtain a pellet.

[0057] (4) A cell extract was recovered from the cell pellet obtained in (3) by the method described in Example 1(4), and electrophoresis was performed by the method described in Example 1(5) to (6).

[0058] The electrophoretic results of the band portion corresponding to (FpL_C3KX 9a)5 are shown in FIG. 3. The results of evaluating the presence or absence of expression of (FpL_C3KX 9a)5 are shown in Table 3. It was confirmed that the expression level of (FpL_C3KX 9a)5 was improved when magnesium sulfate, magnesium chloride, calcium chloride, and manganese chloride were added compared to when no salt was added. On the other hand, even when any of sodium salt, ammonium salt, iron salt, and zinc salt was added to the medium, the expression level of (FpL_C3KX 9a)5 was equal to or lower than the condition in which no salt was added, and it was confirmed that these metal salts did not contribute to improving the expression level of (FpL_C3KX 9a)5. From the above results, it can be seen that immunoglobulin-binding proteins can be efficiently expressed by adding at least one or all of magnesium salt, calcium salt, and manganese salt to the medium.

[0059] [Table 3]

Claims

1. Cultivating a recombinant Escherichia coli containing a polynucleotide encoding an immunoglobulin-binding protein to express said protein; recovering the expressed protein; A method for producing an immunoglobulin-binding protein, comprising: the immunoglobulin-binding protein is a polypeptide comprising at least the immunoglobulin-binding domain of FpL; The medium used for culturing the recombinant E. coli contains at least peptone, and the medium does not contain glucose or contains 3.8 g / L or less of glucose.

2. The method according to any one of claims 1 to 6, wherein the medium used for culturing the recombinant Escherichia coli further contains yeast extract.

3. 3. The method according to claim 1, wherein the medium used for culturing the recombinant Escherichia coli contains at least one of a magnesium salt, a calcium salt, and a manganese salt.

4. 3. The method according to claim 1, wherein the medium used for culturing the recombinant Escherichia coli contains all of a magnesium salt, a calcium salt and a manganese salt.

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