Method for producing fucose-binding protein

Abstract

To provide a method for efficiently producing fucose-binding protein using E. coli.SOLUTION: The above problems are solved by a method comprising the following steps (1) to (3). Step (1): adding a medium containing a carbon source and a nitrogen source to the culture tank, and stirring and culturing Escherichia coli obtained by transformation with an expression vector containing a DNA encoding a fucose-binding protein to obtain a culture; Step (2): adding at least one or more surfactants to the culture obtained in step (1) and disrupting E. coli without ultrasonic irradiation to obtain an aqueous solution containing a fucose-binding protein; and Step (3): purifying the fucose-binding protein from the solution containing the fucose-binding protein obtained in step (2) by cation exchange chromatography.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for efficiently producing a fucose-binding protein, particularly a recombinant BC2LCN lectin that has the ability to bind to sugar chains containing fucose, using Escherichia coli. [Background technology]

[0002] Recombinant BC2LCN lectin is a recombinant protein derived from the N-terminal domain of BC2L-C lectin produced by Burkholderia cenocepacia (Non-Patent Document 1). Recombinant BC2LCN lectin has been reported to bind to fucose-containing glycans such as H-type 1 glycans (Fucα1-2Galβ1-3GlcNAc) and H-type 3 glycans (Fucα1-2Galβ1-3GalNAc), which are known as undifferentiated glycan markers (Patent Documents 1 and 2, Non-Patent Document 2). Because undifferentiated human ES cells and human iPS cells are known to highly express the undifferentiated glycan markers, recombinant BC2LCN lectin has been used to detect glycoconjugates containing undifferentiated glycan markers and undifferentiated cells such as human iPS cells, as well as to remove tumorigenic undifferentiated cells (Patent Documents 1 to 3). In recent years, the development of regenerative medicine products using human iPS cells as a raw material has progressed significantly, and the detection and removal of undifferentiated cells such as human iPS cells during the production of regenerative medicine products is becoming increasingly important. Accordingly, the industrial applicability of recombinant BC2LCN lectin is also becoming increasingly important.

[0003] To use the recombinant BC2LCN lectin for the above-mentioned purposes, it is necessary to produce the recombinant BC2LCN lectin with high efficiency. Furthermore, in order for the recombinant BC2LCN lectin to bind to the above-mentioned glycoconjugates and undifferentiated cells with high selectivity without nonspecific binding, the recombinant BC2LCN lectin must be highly purified.

[0004] Known methods for producing recombinant BC2LCN lectin include, for example, a method in which Escherichia coli capable of expressing recombinant BC2LCN lectin is cultured, the Escherichia coli is disrupted by ultrasonic irradiation to obtain an aqueous solution containing a fucose-binding protein, and the aqueous solution is purified by affinity chromatography using a separation agent to which a sugar such as fucose is bound (Non-Patent Documents 3 and 4), and a method in which Escherichia coli capable of expressing recombinant BC2LCN lectin is cultured at high density, the Escherichia coli is disrupted by a surfactant to obtain an aqueous solution containing a fucose-binding protein, and the aqueous solution is purified by affinity chromatography using a nickel chelate gel (Patent Document 4). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2013 / 065302 issue [Patent Document 2] WO2013 / 128914 [Patent Document 3] Japanese Patent Application Publication No. 2018-134073 [Patent Document 4] Japanese Patent Publication No. 2020-058343 [Non-patent literature]

[0006] [Non-Patent Document 1] Sulak, O et al., Structure.2010, 18(1):59-72. [Non-patent document 2] Tateno, H et al., J. Stem Cells Transl Med.2013,2(4):265-273. [Non-patent document 3] Tateno, H et al., J Biol Chem. 2011, 286(23):20345-20353. [Non-patent document 4] Tateno, H et al., Stem Cell Reports 2015, 4:1-10. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for producing recombinant BC2LCN lectin, a fucose-binding protein, with high efficiency and high purity. Specifically, the present invention provides a method for efficiently culturing Escherichia coli capable of expressing recombinant BC2LCN lectin, a method for disrupting E. coli to efficiently obtain an aqueous solution containing recombinant BC2LCN lectin from the cultured E. coli, and a purification method for inexpensively obtaining highly pure recombinant BC2LCN lectin from the aqueous solution containing recombinant BC2LCN lectin obtained by disrupting E. coli. [Means for solving the problem]

[0008] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that recombinant BC2LCN lectin can be obtained with high efficiency and high purity by a method comprising the following three steps: (1) obtaining a culture by agitating E. coli obtained by transforming with an expression vector containing DNA encoding recombinant BC2LCN lectin, a fucose-binding protein; (2) obtaining an aqueous solution containing recombinant BC2LCN lectin by adding an aqueous solution containing a surfactant to the culture obtained in step (1) to disrupt the E. coli; and (3) recovering the recombinant BC2LCN lectin from the aqueous solution containing the recombinant BC2LCN lectin obtained in step (2) by cation exchange chromatography, thereby completing the present invention.

[0009] That is, the present invention encompasses the inventions described in the following [1] to

[14] : [1] A method for producing a fucose-binding protein using Escherichia coli obtained by transformation with an expression vector containing DNA encoding the fucose-binding protein, the method comprising the following steps (1) to (3): Step (1) adding a medium containing a carbon source and a nitrogen source to a culture vessel and culturing Escherichia coli obtained by transformation with an expression vector containing DNA encoding a fucose-binding protein with stirring to obtain a culture; Step (2) adding at least one surfactant to the culture obtained in Step (1) and disrupting the E. coli without ultrasonic irradiation to obtain an aqueous solution containing a fucose-binding protein; Step (3) A step of purifying the fucose-binding protein from the solution containing the fucose-binding protein obtained in step (2) by cation exchange chromatography. [2] The manufacturing method according to [1] above, wherein the step (3) includes the following steps (4) to (7): Step (4) is washing the cation exchange chromatography carrier with an equilibration buffer. Step (5) is a step of contacting a solution containing a fucose-binding protein with a cation exchange chromatography carrier that has been washed with an equilibration buffer, thereby adsorbing the fucose-binding protein onto the carrier. Step (6) is a step of washing the cation exchange chromatography carrier having the fucose-binding protein adsorbed thereon with a first washing solution. Step (7) A step of eluting the fucose-binding protein adsorbed to the cation exchange chromatography carrier using a second washing solution. [3] The method according to [1] or [2] above, wherein the equilibration buffer and the first washing solution are buffers containing less than 750 mmol / L of an alkali metal salt. [4] The manufacturing method according to any one of [1] to [3] above, wherein the second washing solution is a buffer solution containing an alkali metal salt at 750 mmol / L or more and 3000 mmol / L or less. [5] The method according to [3] or [4] above, wherein the alkali metal salt is sodium chloride. [6] The method according to any one of [1] to [5] above, wherein the buffer solution used in step (3) contains an organic acid at 200 mmol / L or less. [7] The method according to [6] above, wherein the organic acid is citric acid. [8] The method according to any one of [1] to [7] above, wherein the step (3) is carried out using a column packed with a carrier for cation exchange chromatography. [9] The method according to any one of [1] to [8] above, wherein one of the surfactants used in step (2) is a cationic surfactant.

[10] The method according to [9] above, wherein the cationic surfactant is hexadecyltrimethylammonium halide.

[11] The method according to any one of [1] to

[10] above, wherein the surfactant used in step (2) is not an animal-derived surfactant.

[12] The method for producing a plant according to any one of [1] to

[11] above, wherein the carbon source and nitrogen source used in step (1) are not derived from animals.

[13] The method according to any one of [1] to

[12] , wherein the fucose-binding protein is one of the following (a) to (d): (a) A fucose-binding protein comprising an amino acid sequence from the first proline residue to the Xth amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or greater. (b) A fucose-binding protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, and which has binding affinity to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, where X is an integer of 120 or more. (c) A fucose-binding protein comprising an amino acid sequence containing one or more of the amino acid substitutions listed in (1) to (3) below in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or greater: (1) Substitution of the glutamine residue at position 39 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (2) Substitution of the cysteine ​​residue at position 72 of the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from a glycine residue and an alanine residue. (3) Substitution of the glutamine residue at position 65 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (d) A fucose-binding protein comprising the amino acid sequence of the fucose-binding protein of (c) above, in which one or more amino acid residues are deleted, substituted, inserted or added in a region other than positions 39, 65 and 72 of SEQ ID NO: 1, and having binding affinity to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

[14] The method according to any one of [1] to

[12] , wherein the fucose-binding protein is any one of the following (e) to (h): (e) A fucose-binding protein consisting of an amino acid sequence comprising the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, to which an oligopeptide containing a polyhistidine sequence is further added at the N-terminus and an oligopeptide containing cysteine ​​is further added at the C-terminus, wherein X is an integer of 120 or greater. (f) A fucose-binding protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, and further comprising a polyhistidine sequence added to the N-terminus and an oligopeptide containing cysteine ​​added to the C-terminus, wherein the fucose-binding protein has binding affinity to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, and X is an integer of 120 or more. (g) A fucose-binding protein comprising an amino acid sequence comprising the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, to which an oligopeptide containing a polyhistidine sequence is added at the N-terminus and an oligopeptide containing cysteine ​​is added at the C-terminus, and which comprises one or more of the amino acid substitutions listed in (4) to (6) below, wherein X is an integer of 120 or greater: (4) Substitution of the glutamine residue at position 39 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (5) Substitution of the cysteine ​​residue at position 72 of the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from a glycine residue and an alanine residue. (6) Substitution of the glutamine residue at position 65 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (h) A fucose-binding protein having an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added in a region other than positions 39, 65 and 72 of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of (g) above, and further having an oligopeptide containing a polyhistidine sequence added to the N-terminus and an oligopeptide containing cysteine ​​added to the C-terminus, and having binding affinity for glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

[0010] The present invention will be described in further detail below.

[0011] The method for producing a fucose-binding protein of the present invention (hereinafter referred to as the production method of the present invention) is characterized by comprising the following steps (1) to (3). Step (1) A step of adding a medium containing a carbon source and a nitrogen source to a culture vessel, and culturing Escherichia coli obtained by transformation with an expression vector containing DNA encoding a fucose-binding protein under agitation to obtain a culture. Step (2) A step of adding at least one surfactant to the culture obtained in step (1) and disrupting the E. coli without ultrasonic irradiation to obtain an aqueous solution containing a fucose-binding protein. Step (3) A step of purifying the fucose-binding protein from the solution containing the fucose-binding protein obtained in step (2) by cation exchange chromatography. Steps (1) to (3) are described in detail below.

[0012] In step (1) of the production method of the present invention, a medium containing a carbon source and a nitrogen source is added to a culture vessel, and then Escherichia coli obtained by transformation with an expression vector containing DNA encoding a fucose-binding protein is cultured under agitation to obtain a culture. In this specification, the culture includes the cultured transformant Escherichia coli itself, E. coli secretions, as well as the medium used for the culture.

[0013] There are no particular limitations on the size or shape of the culture vessel used in step (1) of the production method of the present invention, and these may be small vessels such as flasks or large vessels such as jar fermentors, and may be selected appropriately depending on the amount of the desired fucose-binding protein to be produced, i.e., the amount of culture obtained in this step. Furthermore, there are no particular limitations on the method of agitation culture, and for example, when the culture vessel is a small vessel such as a flask, the culture solution can be agitated by rotating or shaking the entire culture vessel. When the culture vessel is a large vessel such as a jar fermentor, the culture solution can be agitated using a culture tank with rotating blades at the bottom of the vessel, or the culture tank with agitating blades installed from above, and the culture solution can be agitated while blowing air or oxygen into the vessel.

[0014] When the amount of the desired culture to be obtained is small, a method in which the entire culture vessel is rotated or shaken using a small vessel such as a flask is preferred because it allows for the miniaturization of the entire culture apparatus and facilitates simplification of pre-culture operations such as sterilization. On the other hand, when the amount of the desired culture to be obtained is large, a method in which the culture solution is stirred using a large vessel such as a jar fermenter equipped with stirring blades is preferred because it has high stirring efficiency for the culture solution, reduces physical damage to the E. coli being cultured, and allows for high-density cultivation of E. coli. When stirring the culture solution using stirring blades, there are no particular restrictions on the composition or aeration rate of the aeration gas, or the diameter and shape of the stirring blades, and these may be selected appropriately taking into account the size of the culture vessel and the stirring efficiency of the culture solution.

[0015] The medium that can be used in step (1) is not particularly limited, and examples thereof include Terrific Broth (TB) medium and Lysogeny Broth (LB) medium supplemented with necessary nutrients. In addition to a carbon source, a nitrogen source, and inorganic salts, the medium may also contain nutrients commonly used in the art. Examples of carbon sources include glucose, fructose, maltose, sucrose, raw sugar, molasses, and glycerin. Examples of nitrogen sources include yeast extract, soybean protein and its enzymatic digests, casein and its enzymatic digests, meat and its enzymatic digests, meat extract, and fish extract.

[0016] As mentioned above, recombinant BC2LCN lectin, a fucose-binding protein, is used in the production of regenerative medicine products, etc. Therefore, to avoid contamination of regenerative medicine products with pathogens, it is preferable to produce the recombinant BC2LCN lectin without using animal-derived carbon and nitrogen sources. Therefore, among the carbon and nitrogen sources mentioned above, non-animal-derived glucose and glycerin are preferred carbon sources, as they favorably promote E. coli growth and enable efficient production of fucose-binding proteins, while yeast extract, soy protein, and enzymatic digests thereof are preferred nitrogen sources. The concentrations of the carbon and nitrogen sources used in the production method of the present invention can be appropriately determined depending on the growth rate of E. coli and the type of carbon and nitrogen source. For example, when the carbon source is glucose, the concentration is preferably 10 g / L to 50 g / L, and when the nitrogen source is yeast extract, soy protein, or enzymatic digests thereof, the concentration is preferably 10 g / L to 50 g / L.

[0017] The culture temperature in step (1) is not particularly limited as long as it is a temperature common in the art, and is 10°C to 40°C, preferably 20°C to 37°C, and may be appropriately selected taking into consideration the productivity of the fucose-binding protein. The pH of the medium may be appropriately selected from within a range common in the art. For example, when the host is Escherichia coli, the pH is preferably in the range of pH 6.0 to pH 8.0, more preferably pH 6.5 to pH 7.5. The culture time in the production method of the present invention can be set arbitrarily, but is usually set between 1 and 200 hours. Longer culture times can weaken the activity of the host cells, eventually leading to cell death and bacteriolysis, which may result in a change in the structure of the target fucose-binding protein. Therefore, the culture time is preferably within 150 hours, and more preferably within 100 hours.

[0018] In the production method of the present invention, in order to selectively grow the E. coli obtained by the transformation depending on whether or not an expression vector containing DNA encoding a fucose-binding protein has been introduced, it is preferable to add a drug corresponding to the drug resistance gene contained in the expression vector to the medium and perform agitation culture; for example, if the vector contains a kanamycin resistance gene, kanamycin can be added to the medium. Furthermore, to promote secretion of the protein from the E. coli obtained by the transformation into the culture medium, a reagent such as glycine may be added to the medium; specifically, when the host is E. coli, it is preferable to add glycine to the medium at 2% (w / v) or less. Other ingredients that can be added to the medium include inorganic and metal salts such as phosphates such as sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate, as well as potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, iron (II) sulfate, iron (III) sulfate, iron (II) chloride, iron (III) chloride, iron citrate, ammonium iron sulfate, calcium chloride dihydrate, calcium sulfate, zinc sulfate, zinc chloride, copper sulfate, copper chloride, manganese sulfate, and manganese chloride, and vitamins such as biotin, nicotinic acid, thiamine, riboflavin, inositol, and pyridoxine.

[0019] Furthermore, when an inducible promoter is introduced into an expression vector containing DNA encoding a fucose-binding protein, expression can be induced by adding an inducer to the medium and culturing E. coli with agitation. There are no particular limitations on the inducer that can be used in the production method of the present invention, but isopropyl-β-thiogalactopyranoside (hereinafter referred to as IPTG) is preferred because it allows efficient expression of the fucose-binding protein. The IPTG concentration after addition can be appropriately selected from the range of 0.005 mM to 5.0 mM, preferably 0.01 mM to 2.0 mM. The conditions for inducing the expression of the fucose-binding protein by adding IPTG can be those well known in the art. For example, at any time when the OD600nm of the culture medium reaches 60 or higher, IPTG can be added to the culture medium so that the concentration after addition is 2.0 mM or less, and the culture can be continued to induce the expression of the fucose-binding protein. After IPTG is added to start induction, the temperature of the culture medium should be set to 10°C to 35°C, preferably 15°C to 30°C, and more preferably 20°C to 25°C, to suppress excessive growth of E. coli and promote protein expression more efficiently.

[0020] In step (1), the number of times that spinner culture of E. coli capable of expressing recombinant BC2LCN lectin is performed may be appropriately set depending on the amount of the desired culture to be obtained. For example, the desired culture may be obtained by a single spinner culture in a large culture tank, or a small amount of E. coli may be obtained by spinner culture in a small culture tank, and then this may be passaged and spinner cultured in a large culture tank to obtain a large amount of E. coli, thereby obtaining a large amount of the desired culture. The method for obtaining the desired culture may be appropriately selected from methods commonly used by those skilled in the art depending on the expression form of the fucose-binding protein. For example, because the fucose-binding protein produced by the production method of the present invention is expressed intracellularly in E. coli, E. coli containing the medium can be obtained as a culture by centrifuging the culture solution after spinner culture is completed.

[0021] Step (2) of the production method of the present invention is a step of adding a solution containing at least one surfactant to the culture obtained in step (1) and disrupting the E. coli without ultrasonic irradiation, thereby obtaining an aqueous solution containing a fucose-binding protein.

[0022] The surfactant used in step (2) is preferably in a solution state (hereinafter referred to as surfactant solution) for ease of operation. In order to efficiently disrupt the E. coli in the culture obtained in step (1) and obtain an aqueous solution containing a fucose-binding protein, the surfactant solution must contain at least one type of surfactant. As long as it can efficiently disrupt E. coli, there are no particular limitations on the type of surfactant. Examples of surfactants include cationic surfactants such as hexadecyltrimethylammonium halide, tetradecyltrimethylammonium halide, dodecylpyridinium halide, and hexadecylpyridinium halide; anionic surfactants such as sodium cholate, sodium deoxycholate, sodium glycocholate, sodium taurocholate, sodium taurodeoxycholate, sodium N-lauroylsarcosinate, sodium dodecyl sulfate, and sodium dodecylsulfonate; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS); 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS); and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Amphoteric surfactants such as [N,N-dimethyloctylammonio]-2-hydroxy-1-propanesulfonic acid (CHAPSO), 3-(N,N-dimethyloctylammonio)propanesulfonic acid, 3-(decyldimethylammonio)propanesulfonate, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, 3-(N,N-dimethylmyristylammonio)propanesulfonic acid, and 3-(N,N-dimethylpalmititylammonio)propanesulfonic acid, octyl glucoside, heptyl glucoside, decanoyl-N-methylglucamide, polyoxyethylene dodecyl ether (product name: Brij series), and polyoxyethylene heptamethylhexyl ether (product name: Nikkol Examples of nonionic surfactants include polyoxyethylene isooctylphenyl ether (product name: Triton-X series), polyoxyethylene nonylphenyl ether (product name: Triton-N series), polyoxyethylene fatty acid ester (product name: Span series), and polyoxyethylene sorbitol ester (product name: Tween series).

[0023] Among these, it is preferable to use a combination of at least one cationic surfactant and at least one nonionic surfactant, because it can disrupt E. coli efficiently. Specifically, as the cationic surfactant, hexadecyltrimethylammonium halide is more preferable, and as the nonionic surfactant, polyoxyethylene isooctylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitol ester are more preferable, with polyoxyethylene isooctylphenyl ether being particularly preferable. Furthermore, there are no particular restrictions on the concentration of the surfactant in the surfactant solution, as long as it can disrupt E. coli efficiently and the sugar chain binding ability of the fucose-binding protein is maintained. For both the cationic surfactant and the nonionic surfactant, the concentration is preferably 0.001 to 10% by weight, more preferably 0.01 to 5% by weight.

[0024] Furthermore, as mentioned above, it is preferable to produce the recombinant BC2LCN lectin, a fucose-binding protein, without using animal-derived surfactants to avoid contamination with pathogens. Therefore, among the surfactants mentioned above, chemically synthesized cationic and nonionic surfactants such as hexadecyltrimethylammonium halide and polyoxyethylene isooctylphenyl ether are preferred over animal-derived surfactants such as sodium cholate and sodium deoxycholate.

[0025] In order to efficiently obtain an aqueous solution containing a fucose-binding protein from disrupted E. coli while retaining its glycan-binding ability, the surfactant solution used in step (2) is preferably a buffer solution having a buffering capacity at pH 2 to pH 10, at which the fucose-binding protein can maintain its glycan-binding ability. There are no particular restrictions on the reagent (hereinafter referred to as a buffering agent) added to make the surfactant solution into a buffer solution, as long as it has a buffering capacity within the pH range. However, it is preferably the same as the buffering agent in the equilibration buffer used in the cation exchange chromatography in step (3) of the production method of the present invention, which will be described later. Specifically, citric acid and its salts, acetic acid and its salts, phosphoric acid and its salts, and morpholinoethanesulfonic acid and its salts are preferred, with citric acid and its salts being more preferred. The concentration and pH of the buffer in the surfactant solution may be appropriately set so that the electrical conductivity and pH of the aqueous solution containing the fucose-binding protein obtained in step (2) do not differ significantly from those of the equilibration buffer used in step (3). The concentration of the buffer to be added is preferably 5 mmol / L to 100 mmol / L, and the pH is preferably pH 3 to pH 7.

[0026] In order to efficiently obtain an aqueous solution containing a fucose-binding protein from disrupted E. coli while maintaining its glycan-binding ability, the surfactant solution used in step (2) may contain, in addition to the surfactant and buffer described above, inorganic and metal salts such as sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, and calcium sulfate; chelating agents such as ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) and its salts, and glycoletherdiaminetetraacetic acid (hereinafter referred to as EGTA) and its salts; reducing agents such as dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride; nucleases such as benzonase for degrading E. coli-derived nucleic acids; and polysaccharidases such as lysozyme for degrading E. coli-derived cell walls. The surfactant solution preferably contains sodium chloride, magnesium sulfate, EDTA, nucleases, and polysaccharidases, as this allows for highly efficient production of an aqueous solution containing a fucose-binding protein. The concentrations of these compounds and enzymes may be selected appropriately from ranges commonly used in the art.

[0027] In step (2), an aqueous solution containing a fucose-binding protein can be obtained from the culture obtained in step (1) of the production method of the present invention by performing the procedure described in the Examples below. Specifically, a citrate buffer solution, an EDTA aqueous solution, and sodium chloride or an aqueous solution thereof are sequentially added to the precipitate containing the bacterial cells and medium obtained by centrifuging the culture obtained in step (1), and the bacterial cell suspension is gently stirred to an extent that foaming does not occur. Next, magnesium sulfate or an aqueous solution thereof, a nuclease, and a polysaccharide-degrading enzyme are sequentially added to the bacterial cell suspension, and the bacterial cell suspension is gently stirred at a temperature of 10°C to 40°C for 5 to 60 minutes to an extent that foaming does not occur. Next, a nonionic surfactant or an aqueous solution thereof is added to the bacterial cell suspension, and the mixture is stirred at 10°C to 40°C for 5 to 60 minutes until the nonionic surfactant is uniformly dispersed throughout the bacterial cell suspension. Then, a cationic surfactant or an aqueous solution thereof is added, and the mixture is gently stirred at 0°C to 40°C for 1 to 24 hours, without foaming the bacterial cell suspension. Finally, the bacterial cell suspension is centrifuged to remove the precipitate, thereby obtaining an aqueous solution containing the desired fucose-binding protein. The fucose-binding protein in the aqueous solution can be detected, for example, by analyzing the aqueous solution using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In step (2), the conditions for centrifuging the culture and bacterial cell suspension, the stirring method and speed, and the stirring temperature may be appropriately selected from ranges commonly used in the art.

[0028] As will be shown in the Examples and Comparative Examples described later, in step (2), by using the surfactant solution having the buffering capacity described above, an aqueous solution containing a fucose-binding protein can be obtained with high efficiency by disrupting E. coli without irradiating it with ultrasound.

[0029] Step (3) of the production method of the present invention is a step of purifying the fucose-binding protein from the solution containing the fucose-binding protein obtained in step (2) by cation exchange chromatography, more specifically, a step of purifying the fucose-binding protein by a process including the following steps (4) to (7): Step (4) is washing the cation exchange chromatography carrier with an equilibration buffer. Step (5) is a step of contacting a solution containing a fucose-binding protein with a cation exchange chromatography carrier that has been washed with an equilibration buffer, thereby adsorbing the fucose-binding protein onto the carrier. Step (6) is a step of washing the cation exchange chromatography carrier having the fucose-binding protein adsorbed thereon with a first washing solution. Step (7) A step of eluting the fucose-binding protein adsorbed to the cation exchange chromatography carrier using a second washing solution.

[0030] The cation exchange chromatography support that can be used in step (3) of the production method of the present invention, i.e., steps (4) to (7) of the production method of the present invention, is not particularly limited as long as it is a support into which a cation exchange group such as a carboxymethyl group, a sulfopropyl group, or a sulfonic acid group has been introduced. For example, commercially available products include TOYOPEARL CM-650, TOYOPEARL GigaCap CM-650, TOYOPEARL SP-550, TOYOPEARL SP-650, TOYOPEARL GigaCap S-650 (all manufactured by Tosoh), CM Sepharose Fast Flow, SP Sepharose Fast Flow, Capto S (all manufactured by Cytiva), Cellufine C-500, and Cellufine S-500 (all manufactured by JNC). Alternatively, commercially available size-exclusion chromatography supports such as TOYOPEARL HW-65 (manufactured by Tosoh), Sepharose 6 Fast Flow (manufactured by Cytiva), and Cellufine GCL-2000HF (manufactured by JNC) can be used, to which cation exchange groups such as the aforementioned carboxymethyl group, sulfopropyl group, and sulfonic acid group have been introduced using methods commonly used in the art.

[0031] When the cation exchange chromatography carrier is used to carry out step (3) of the production method of the present invention, the carrier may be packed in an amount determined based on the amount of the fucose-binding protein-containing solution to be added to the carrier and the protein adsorption capacity of the carrier, and the carrier may be packed in a container of a size and shape determined taking into consideration the treatment time, etc. The container is not particularly limited, and examples thereof include an open column in which the top end is open and a closed column in which both ends are closed. Among these, a closed column in which both ends are closed is more preferred because it allows for highly efficient purification of the fucose-binding protein in a short time when connected to an HPLC device.

[0032] Step (4) of the production method of the present invention is a step of washing the cation exchange chromatography support with an equilibration buffer in order to adsorb the fucose-binding protein in the solution containing the fucose-binding protein obtained in step (2) onto the cation exchange chromatography support. More specifically, this is a step of packing the above-mentioned cation exchange chromatography support into a column, and then adding an equilibration buffer to the column to wash it.

[0033] In order to adsorb the fucose-binding protein onto the support for cation exchange chromatography, the equilibration buffer used in step (4) needs to be a buffer with a low salt concentration. Specifically, it is preferable that the buffer contains less than 750 mmol / L of an inorganic salt, particularly an alkali metal salt, more preferably less than 250 mmol / L of an alkali metal salt, and more preferably less than 100 mmol / L of an alkali metal salt.

[0034] The type of buffer solution is not particularly limited as long as it has buffering capacity at a pH that allows the fucose-binding protein to be adsorbed to the support for cation exchange chromatography. Specific examples include phosphate buffer solutions containing the inorganic acid phosphoric acid as a buffering agent, glycine buffer solutions containing the organic acid glycine as a buffering agent, citrate buffer solutions containing the organic acid citric acid as a buffering agent, acetate buffer solutions containing the organic acid acetic acid as a buffering agent, MES buffer solutions containing the organic acid 2-(N-morpholino)ethanesulfonic acid (MES) as a buffering agent, and HEPES buffer solutions containing the organic acid 4-(2-hydroxyethyl)-1-piperazinesulfonic acid (HEPES) as a buffering agent. Because the fucose-binding protein of the present invention can be adsorbed to the support for cation exchange chromatography with high efficiency in the pH range of 4 to 6, preferred buffer solutions are citrate buffer solutions, acetate buffer solutions, and MES buffer solutions, which are organic acids with buffering capacity in the pH range of 4 to 6, with citrate buffer solutions being more preferred. Furthermore, the concentration of the buffering agent in the buffer solution is preferably 5 mmol / L or more and 200 mmol / L or less, more preferably 10 mmol / L or more and 100 mmol / L or less, because a high concentration makes it difficult for the fucose-binding protein to be adsorbed to the cation exchange chromatography carrier, whereas a low concentration reduces the pH buffering capacity.

[0035] The equilibration buffer used in step (4) may contain, in addition to the alkali metal salt and buffering agent at the aforementioned concentrations, components for maintaining the activity and stability of the fucose-binding protein, such as its sugar chain-binding ability, and specifically may contain a nonionic surfactant such as Triton-X100 or Tween 20, or a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or glycoletherdiaminetetraacetic acid (EGTA). The concentrations of these components may be appropriately selected within a range that does not inhibit the adsorption of the fucose-binding protein to the cation exchange chromatography carrier. Specifically, the nonionic surfactant is preferably 0.00001 to 0.5%, and the chelating agent is preferably 0.01 mmol / L to 100 mmol / L. As described above, step (3) of the production method of the present invention is preferably carried out by packing the cation exchange chromatography support into a closed column in which both ends are closed, in order to purify the fucose-binding protein highly efficiently in a short time. In this case, the cation exchange chromatography support can be washed with the equilibration buffer by passing an equilibration buffer through the closed column in an amount at least twice, preferably 3 to 20 times the volume of the cation exchange chromatography support packed in the closed column. The flow rate at which the equilibration buffer is passed through the column may be appropriately selected depending on the mechanical strength of the cation exchange chromatography support packed in the column.

[0036] Step (5) of the production method of the present invention is a step of bringing a solution containing the fucose-binding protein produced in step (2) into contact with the cation exchange chromatography support washed with the equilibration buffer in step (4) above, thereby adsorbing the fucose-binding protein to the support. More specifically, this step involves washing the cation exchange chromatography support packed in a column with the equilibration buffer in step (4) above, and then adding to the column the solution containing the fucose-binding protein produced in step (2). There are no particular limitations on the amount of the solution containing the fucose-binding protein to be added to the column, as long as the fucose-binding protein is adsorbed to the cation exchange chromatography support, and the amount may be appropriately selected from the range of 0.001 to 100 times the volume of the support.

[0037] The linear velocity (flow rate divided by the cross-sectional area of ​​the column) when adding the solution containing the fucose-binding protein is not particularly limited, as long as the fucose-binding protein is adsorbed to the carrier without destroying the carrier. It may be appropriately selected from the range of 1 cm / h to 200 cm / h, taking the cross-sectional area of ​​the column into consideration. The amount of the solution containing the fucose-binding protein added is preferably 0.5 to 10 times, and more preferably 1 to 5 times, the column volume, in order to shorten the time required for steps (5) to (7). The linear velocity when adding the solution containing the fucose-binding protein is preferably 5 cm / h to 150 cm / h, and more preferably 5 cm / h to 100 cm / h. Furthermore, in order to efficiently adsorb the fucose-binding protein to the carrier for cation exchange chromatography, it is preferable to dilute the solution containing the fucose-binding protein in the above-mentioned step (2) with the equilibration buffer to make the pH and electrical conductivity of the solution approximately the same as those of the equilibration buffer.

[0038] Step (6) of the production method of the present invention is a step of washing the cation exchange chromatography support to which the fucose-binding protein has been adsorbed in step (5) with a first wash solution to remove culture-derived impurities such as proteins that do not adsorb to the support. More specifically, this is a step of adding a first wash solution to the column after the fucose-binding protein has been adsorbed to the cation exchange chromatography support packed in a column in step (5) to remove culture-derived impurities such as proteins that do not adsorb to the support.

[0039] As in step (4) above, the first wash solution used in step (6) must be a buffer with a low salt concentration so as not to elute the fucose-binding protein adsorbed to the cation exchange chromatography support in the column. Specifically, it is preferably a buffer containing 50 mmol / L to less than 750 mmol / L of inorganic salt, particularly an alkali metal salt, more preferably a buffer containing 100 mmol / L to 600 mmol / L of alkali metal salt, and even more preferably a buffer containing 300 mmol / L to 600 mmol / L of alkali metal salt. The type and concentration of the buffer, and the components and concentrations contained in the buffer for maintaining the activity and stability, such as the glycan-binding ability, of the fucose-binding protein, are as described above in step (4).

[0040] The type and concentration of the alkali metal salt and buffer contained in the first wash solution used in step (6) may be the same as or different from the equilibration buffer used in step (4), as long as the fucose-binding protein adsorbed to the cation exchange chromatography carrier in the column is not eluted. These may be selected appropriately depending on the impurities derived from the culture. Furthermore, two or more different first wash solutions may be used in step (6). For example, the column may be washed with a first wash solution containing 50 mmol / L of inorganic salt, followed by stepwise washing with first wash solutions containing 500 mmol / L of inorganic salt (stepwise washing). Additionally, two different first wash solutions each having an inorganic salt concentration of less than 750 mmol / L may be mixed before being added to the column, and the column may be washed under conditions in which the salt concentration in the first wash solution changes over time (linear gradient washing).

[0041] Among these washing methods, a stepwise washing method using two different first wash solutions, one containing 30 mmol / L to 200 mmol / L of inorganic salt and the other containing 300 mmol / L to 600 mmol / L of inorganic salt, is preferred because it can remove culture-derived impurities with high efficiency and can produce a highly purified fucose-binding protein in step (7) described below. In step (6), the first wash solution is added to the column, and the protein concentration in the eluate is calculated by measuring the absorbance at 280 nm of the eluate from the column. The first wash solution may be added until the protein concentration decreases to the point where the culture-derived impurities can be removed. In terms of shortening the time required to perform steps (6) and (7), the amount of the first wash solution added is preferably 2 to 30 times the column volume, and more preferably 5 to 20 times the column volume. The linear velocity at which the first wash solution is added to the column is preferably 5 cm / h to 150 cm / h, more preferably 5 cm / h to 100 cm / h. In step (6), the detection of the fucose-binding protein in the eluate from the column can be confirmed, for example, by analyzing the eluate by SDS-PAGE.

[0042] Step (7) of the production method of the present invention is a step of washing the cation exchange chromatography support from which culture-derived impurities have been removed in step (6) with a second wash buffer to elute the fucose-binding protein adsorbed to the support. More specifically, this step is a step of washing the cation exchange chromatography support packed in a column with the first wash buffer in step (6), and then adding the second wash buffer to the column to elute the fucose-binding protein. In order to elute the fucose-binding protein from the cation exchange chromatography support in the column, the second wash buffer used in step (7) must be a buffer with a high salt concentration. Specifically, it is preferable that the buffer contains an inorganic salt, particularly an alkali metal salt, at a concentration of 750 mmol / L to 3000 mmol / L, more preferably an alkali metal salt at a concentration of 900 mmol / L to 2000 mmol / L, and even more preferably an alkali metal salt at a concentration of 900 mmol / L to 1500 mmol / L. The type and concentration of the buffer solution, and the components and concentrations contained in the buffer solution for maintaining the activity and stability of the fucose-binding protein, such as its sugar chain-binding ability, are as described above in step (4).

[0043] The second wash solution used in step (7) may be one type, or two or more different types. When one type of second wash solution is used, the target fucose-binding protein can be eluted by washing the column with a second wash solution containing, for example, 750 mmol / L or more of inorganic salt. When two or more types of second wash solutions are used, the target fucose-binding protein can be eluted by, for example, washing the column with a second wash solution containing 750 mmol / L of inorganic salt, and then washing the column stepwise with second wash solutions containing 1000 mmol / L of inorganic salt (stepwise elution). In addition, two different first wash solutions each having an inorganic salt concentration of 750 mmol / L or more may be mixed before being added to the column, and the column may be washed under conditions in which the salt concentration in the first wash solution changes over time (linear gradient elution). Among these washing methods, it is preferable to wash the column with one type of second washing solution containing 750 mmol / L or more of an inorganic salt to elute the target fucose-binding protein, since this method allows the production of a highly pure fucose-binding protein.

[0044] In step (7), a second wash buffer is added to the column, and the protein concentration in the eluate is calculated by measuring the absorbance at 280 nm of the column eluate. The second wash buffer is added until the protein concentration decreases and the fucose-binding protein of interest is completely eluted. The amount of the second wash buffer added is preferably 1 to 10 times, and more preferably 2 to 5 times, the column volume, in order to shorten the time required to perform step (7). The linear velocity at which the second wash buffer is added to the column is preferably 5 cm / h to 150 cm / h, and more preferably 5 cm / h to 100 cm / h. In step (7), the purity and molecular weight of the fucose-binding protein in the column eluate can be determined by a method known in the art, such as SDS-PAGE or gel filtration chromatography.

[0045] According to one preferred embodiment of steps (4) to (7) of the production method of the present invention shown below, the desired fucose-binding protein can be obtained inexpensively and with high purity. Step (4): The cation exchange chromatography support packed in the column is washed with an equilibration buffer containing a buffer containing 5 mmol / L or more and 200 mmol / L or less of a buffer agent and less than 750 mmol / L of an alkali metal salt. Step (5): The pH and electrical conductivity of the solution containing the fucose-binding protein produced in step (2) are adjusted to be approximately the same as those of the equilibration buffer, and then the solution is applied to the column. Step (6): The column is washed with a first washing solution containing 30 mmol / L or more and 200 mmol / L or less of an inorganic salt. After washing with the first washing solution, the substrate is washed with a first washing solution containing an inorganic salt of 300 mmol / L or more and 600 mmol / L or less. Step (7): The column is washed with one type of second washing solution containing 750 mmol / L or more of an inorganic salt to elute the target fucose-binding protein.

[0046] The fucose-binding protein in the production method of the present invention is a protein that has the ability to bind to fucose-containing glycans such as H-type 1 glycans (Fucα1-2Galβ1-3GlcNAc), H-type 3 glycans (Fucα1-2Galβ1-3GalNAc), Lewis Y glycans (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc), and Lewis b glycans (Fucα1-2Galβ1-3(Fucα1-4)GlcNAc), and the aforementioned recombinant BC2LCN lectin is also included in the fucose-binding protein in the production method of the present invention.

[0047] Specifically, the fucose-binding protein in the production method of the present invention is (a) a protein comprising the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence of recombinant BC2LCN lectin shown in SEQ ID NO: 1 (which corresponds to the amino acid sequence from the second to the 156th amino acid in the amino acid sequence registered with GenPept under registration number WP_006490828), where X is an integer of 120 or more, or (b) a protein consisting of an amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1, in which one or more amino acids have been deleted, substituted, or added, and which has the ability to bind to H type 1 glycans and / or H type 3 glycans, where X is an integer of 120 or more, expressed as a recombinant protein in an Escherichia coli transformant. As long as the fucose-binding protein in the production method of the present invention has the ability to bind to the fucose-containing glycan, particularly a glycan containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, one or more amino acids may be deleted, substituted, or added in the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1. For example, 15 or fewer, preferably 10 or fewer, amino acids may be deleted, substituted, or added. Furthermore, X may be 120 to 155, or 125 to 155. As disclosed in JP 2020-25535 A, the fucose-binding protein in the production method of the present invention can be produced in an Escherichia coli transformant with improved productivity (expression level) by deleting multiple amino acid residues from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1, compared to when the amino acid residues are not deleted.

[0048] Furthermore, the fucose-binding protein in the production method of the present invention may contain one or more of the following to improve its thermal stability: (i) a substitution of a leucine residue for the glutamine residue at position 39 in the amino acid sequence shown in SEQ ID NO: 1; (ii) a substitution of an amino acid residue selected from a glycine residue and an alanine residue for the cysteine ​​residue at position 72 in the amino acid sequence shown in SEQ ID NO: 1; or (iii) a substitution of a leucine residue for the glutamine residue at position 65 in the amino acid sequence shown in SEQ ID NO: 1. As disclosed in JP 2020-25535 A, the thermal stability of the fucose-binding protein in the production method of the present invention can be improved by making the amino acid substitutions described in (i) to (iii) above. While the amino acid substitutions described in (i) to (iii) above, whether used alone or in combination, are effective in improving thermal stability, combining multiple amino acid substitutions described in (i) to (iii) above is preferable because it further improves thermal stability. Furthermore, as long as the fucose-binding protein in the production method of the present invention has the ability to bind to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, one or more amino acid residues may be deleted, substituted, or inserted in a region other than the positions substituted by the substitutions (i) to (iii) in the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1. For example, 15 or fewer, preferably 10 or fewer amino acid residues may be deleted, substituted, or inserted.

[0049] Specific examples of fucose-binding proteins in the production method of the present invention include SEQ ID NO: 1, SEQ ID NO: 2 (the amino acid sequence from positions 1 to 127 of the amino acid sequence shown in SEQ ID NO: 1), SEQ ID NO: 3 (the amino acid sequence in which the 72nd cysteine ​​residue of SEQ ID NO: 2 is substituted with a glycine residue), SEQ ID NO: 4 (the amino acid sequence in which the 39th glutamine residue of SEQ ID NO: 2 is substituted with a leucine residue and the 72nd cysteine ​​residue is substituted with a glycine residue), SEQ ID NO: 5 (the amino acid sequence in which the 39th glutamine residue of SEQ ID NO: 2 is substituted with a leucine residue, the 65th glutamine residue of SEQ ID NO: 2 is substituted with a leucine residue and the 72nd cysteine ​​residue is substituted with a glycine residue), and SEQ ID NO: 6 (the amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus). Examples of such fucose-binding proteins include those represented by SEQ ID NO: 7 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 2 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus), SEQ ID NO: 8 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 3 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus), SEQ ID NO: 9 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus), and SEQ ID NO: 10 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 5 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus).

[0050] The fucose-binding protein in the production method of the present invention may have an additional amino acid sequence at its N-terminus and / or C-terminus that is useful for detecting the fucose-binding protein, as long as it has the ability to bind to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

[0051] Examples of the additional amino acid sequence include an oligopeptide containing a polyhistidine sequence, glutathione S-transferase (hereinafter referred to as GST), maltose-binding protein, cellulose-binding domain, myc tag, FLAG tag, etc. Among these additional amino acid sequences, an oligopeptide containing a polyhistidine sequence or GST is preferred, and an oligopeptide containing a polyhistidine sequence is more preferred, because they have high productivity when produced using Escherichia coli and allow fucose-binding proteins to be easily detected using fluorescently labeled anti-polyhistidine antibodies or anti-GST antibodies. There are no particular restrictions on the number of histidine repeats in an oligopeptide containing a polyhistidine sequence, but if the histidine repeats are short, detection with an anti-polyhistidine antibody becomes difficult, and if they are long, the binding ability of the fucose-binding protein to the sugar chain may be impaired. Therefore, the length of the histidine repeat sequence in an oligopeptide containing a polyhistidine sequence is preferably a repeat sequence consisting of 5 to 15 histidines, and more preferably a repeat sequence consisting of 5 to 10. The position at which the oligopeptide containing a polyhistidine sequence is added to a fucose-binding protein is not particularly limited, and it may be added to both the N-terminus and the C-terminus, or to either the N-terminus or the C-terminus, but from the viewpoint of efficient detection with an anti-polyhistidine antibody, it is preferable that the oligopeptide containing a polyhistidine sequence be added to the N-terminus of the fucose-binding protein.

[0052] Furthermore, the fucose-binding protein in the production method of the present invention may have an additional amino acid sequence (hereinafter referred to as a tag for carrier immobilization) consisting of an oligopeptide containing a cysteine ​​residue or a lysine residue at its N-terminus and / or C-terminus, which is useful for immobilizing the fucose-binding protein on a carrier such as a support for chromatography. By immobilizing the fucose-binding protein on a carrier, it is possible to prepare, for example, an undifferentiated cell adsorbent for removing undifferentiated cells such as human iPS cells, as described in Patent Document 3. There are no particular limitations on the length of the tag for carrier immobilization, as long as the fucose-binding protein has the ability to bind to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc. As the tag for immobilization on a carrier, an oligopeptide consisting of 2 to 10 amino acid residues and containing one or more cysteine ​​residues is preferred, in that it allows for highly selective and efficient immobilization to an insoluble carrier. Specific examples include an oligopeptide consisting of the three amino acid residues "Gly-Gly-Cys," an oligopeptide consisting of the five amino acid residues "Ala-Ser-Gly-Gly-Cys," and an oligopeptide consisting of the seven amino acid residues "Gly-Gly-Gly-Ser-Gly-Gly-Cys." There are no particular restrictions on the position at which the oligopeptide containing one or more cysteines is added to the fucose-binding protein, and it may be added to both the N-terminus and C-terminus, or to either the N-terminus or C-terminus. However, in that it allows for efficient immobilization of the fucose-binding protein to a carrier, it is preferred that the oligopeptide containing one or more cysteine ​​residues be added to the C-terminus of the fucose-binding protein.

[0053] A signal peptide may be added to the N-terminus of the fucose-binding protein in the production method of the present invention to promote efficient expression in the host. When the host is Escherichia coli, examples of the signal peptide include signal peptides that cause protein secretion into the periplasm, such as PelB, DsbA, MalE, and TorT. DNA encoding the fucose-binding protein in the production method of the present invention can be prepared by known methods.

[0054] Examples of methods for preparing the DNA include a method in which the amino acid sequence of the fucose-binding protein in the production method of the present invention is converted into a base sequence and DNA containing the base sequence is artificially synthesized; a method in which DNA encoding the fucose-binding protein in the production method of the present invention is directly artificially prepared; and a method in which DNA is prepared from the genomic DNA of Burkholderia cenocepacia using a DNA amplification method such as PCR.

[0055] In this preparation method, when designing the base sequence, it is preferable to consider the codon usage frequency in the E. coli to be transformed. For example, AGA, AGG, CGG, or CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) are all rarely used codons (rare codons), so it is preferable to select and convert codons other than these codons. Analysis of codon usage frequency can also be done using public databases (for example, the Codon Usage Database on the Kazusa DNA Research Institute website, http: / / www.kazusa.or.jp / codon / , accessed May 7, 2020).

[0056] To transform Escherichia coli with DNA encoding a fucose-binding protein prepared by the above method, the DNA itself may be used for transformation. However, it is preferable to use an expression vector prepared by inserting the DNA into an appropriate position in a vector based on a bacteriophage, cosmid, or plasmid, which is commonly used for transforming prokaryotic and eukaryotic cells, to achieve stable transformation. Here, "appropriate position" refers to a position that does not disrupt the replication function of the expression vector, the desired antibiotic marker, or the region involved in transduction. Furthermore, when inserting the DNA into a vector, it is preferably inserted into the vector in a state linked to functional DNA, such as a promoter required for expression. There are no particular limitations on the vector used as the expression vector, as long as it is stable and replicable in the host. Examples of such vectors include pET vectors, pUC vectors, pTrc vectors, pCDF vectors, and pBBR vectors. Examples of such promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter, as well as the λPL promoter and λPR promoter of λ phage. Transformation of the host Escherichia coli with the expression vector may be carried out using a method commonly used by those skilled in the art. For example, when selecting Escherichia coli JM109 strain, Escherichia coli BL21(DE3) strain, Escherichia coli NiCo21(DE3) strain, Escherichia coli W3110 strain, or the like as the host, methods described in known literature (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) can be used.

[0057] The binding ability of the fucose-binding protein obtained by the production method of the present invention to glycans can be evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, or the like. Surface plasmon resonance will be described as an example. Binding evaluation by surface plasmon resonance can be performed, for example, using a Biacore T200 instrument (GE Healthcare) with a recombinant protein as the analyte and a glycan as the solid phase. A sensor chip with immobilized glycans can be prepared using a streptavidin-coated sensor chip (Sensor Chip SA, GE Healthcare) or a dextran-coated sensor chip (Sensor Chip CM5, GE Healthcare) with streptavidin immobilized in advance, using biotin-labeled glycans. Binding evaluation can also be performed using the kinetic analysis program provided with the instrument. [Effects of the Invention]

[0058] The production method of the present invention enables the efficient and highly pure production of recombinant BC2LCN lectin, a fucose-binding protein that can be used to detect and remove undifferentiated cells such as human iPS cells during the production of regenerative medicine products. In particular, the production method of the present invention enables the recombinant BC2LCN lectin to be purified in high yield and purity by a single-step cation exchange column chromatography from an aqueous solution containing the recombinant BC2LCN lectin obtained by highly efficient E. coli cultivation and E. coli lysis. This reduces the production cost of the recombinant BC2LCN lectin compared to conventional production methods. Furthermore, the production method of the present invention enables the production of recombinant BC2LCN lectin without using animal-derived components, thereby preventing pathogen contamination of regenerative medicine products, even when recombinant BC2LCN lectin is used during production. [Brief explanation of the drawings]

[0059] [Figure 1]These are the results of SDS-PAGE analysis of the "extract," "flow-through recovery liquid," "37% B recovery liquid," and "100% B recovery liquid" obtained in Example 1 (3) and (Example 1-4) to (Example 1-6). [Figure 2] These are the results of SDS-PAGE analysis of the "extract," "flow-through recovery liquid," "37% B recovery liquid," and "100% B recovery liquid" obtained in (1) and (comparison 1-3) to (comparison 1-5) of Comparative Example 1. [Figure 3] The results of SDS-PAGE analysis of the "extract," "flow-through recovery liquid," "gradient elution recovery liquid," "100% B recovery liquid," and "200% B recovery liquid" obtained in (1) and (comparison 2-3) to (comparison 2-7) of Comparative Example 2. [Figure 4] The results of SDS-PAGE analysis of the "extract," "flow-through recovery," "48% B recovery," and "100% B recovery" obtained in (1) and (2) of Example 2 are shown. [Figure 5] These are the results of SDS-PAGE analysis of the "extract," "flow-through recovery liquid," "45% B recovery liquid," and "100% B recovery liquid" obtained in Example 4 (3) and (Example 4-4) to (Example 4-6). [Figure 6] The results of SDS-PAGE analysis of the "extract," "flow-through recovery liquid," "gradient elution recovery liquid," "100% B recovery liquid," and "200% B recovery liquid" obtained in (1) and (comparison 3-3) to (comparison 3-7) of Comparative Example 3. [Example]

[0060] The present invention will be explained in more detail below with reference to Examples, Comparative Examples and Reference Examples, but the present invention is not limited to these.

[0061] Example 1 Production of rBC2LCN(A)-1 Example 1 relates to the production of a fucose-binding protein consisting of 148 amino acids and represented by SEQ ID NO: 9, i.e., a fucose-binding protein in which an oligopeptide sequence containing histidine has been added to the N-terminus and an oligopeptide sequence containing cysteine ​​has been added to the C-terminus of the amino acid sequence of the fucose-binding protein consisting of 127 amino acids and represented by SEQ ID NO: 4 (hereinafter referred to as rBC2LCN(A)). (1) Construction of expression vector pTrc-rBC2LCN(A) and recombinant E. coli W3110 / pTrc-rBC2LCN(A) The expression vector pTrc-rBC2LCN(A) is an expression vector for expressing rBC2LCN(A). In the amino acid sequence of rBC2LCN(A) shown in SEQ ID NO: 9, the 5th to 10th positions correspond to an oligopeptide sequence containing histidine, the 15th to 141st positions correspond to the amino acid sequence of SEQ ID NO: 4, and the 142nd to 148th positions correspond to an oligopeptide sequence containing cysteine.

[0062] The expression vector pTrc-rBC2LCN (A) and recombinant E. coli W3110 / pTrc-rBC2LCN (A) were produced according to the method described in Example 6 of JP 2020-58343 A. That is, using the expression vector pET-BC2LCN (127Q39L / C72G) cys described in Example 3 (1) of JP 2020-58343 A as a template, PCR was performed by the method disclosed in JP 2018-000038 A using the oligonucleotides consisting of the sequences described in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers.

[0063] The resulting PCR product was digested with restriction enzymes NcoI and HindIII and ligated with the expression vector pTrc-PelBV3Km (an expression vector in which a kanamycin resistance gene was introduced in place of the carbenicillin resistance gene in the expression vector pTrc-PelBV3 prepared by the method described in WO 2015 / 199154) similarly digested with restriction enzymes NcoI and HindIII. This ligation product was used to transform E. coli W3110, yielding recombinant E. coli W3110 / pTrc-BC2LCN(A)cys.

[0064] Using the method disclosed in JP 2018-000038 A, the resulting recombinant E. coli W3110 / pTrc-BC2LCN(A)cys was cultured and extracted from the cells to obtain the expression vector pTrc-BC2LCN(A)cys. Sequence analysis confirmed the base sequence, confirming that the expression vector pTrc-BC2LCN(A)cys contains the base sequence of SEQ ID NO: 14, which encodes the amino acid sequence of SEQ ID NO: 4. (2) Production of rBC2LCN(A) using recombinant E. coli The recombinant E. coli W3110 / pTrc-rBC2LCN(A) prepared in (1) above was inoculated into three 500 mL baffled Erlenmeyer flasks containing the preculture medium shown in Table 1 (autoclaved at 121°C for 20 minutes, and then cooled and kanamycin sulfate added), and preculture was carried out at 30°C for 20 hours with a shaking rate of 130 rpm (amplitude 25 mm, circular rotation).

[0065] [Table 1]

[0066] To prepare the main culture medium, first, all the components shown in Table 2 were dissolved, added to a 10 L fermenter, sterilized in an autoclave (121°C, 30 minutes), and cooled to room temperature.

[0067] [Table 2]

[0068] Next, aqueous solutions of each component shown in Table 3 were prepared separately, and all the aqueous solutions except the kanamycin sulfate aqueous solution were sterilized in an autoclave (121°C, 20 minutes) and then cooled to room temperature. The kanamycin sulfate aqueous solution was sterilized by filtration through a 0.22 μm sterilizing filter (manufactured by Advantec). These sterilized aqueous solutions of each component were added (in the amounts shown in Table 3) to a 10 L fermenter containing the sterilized solution containing the components shown in Table 2, to prepare the main culture medium.

[0069] [Table 3]

[0070] Next, the fed-batch medium shown in Table 4 was prepared. Specifically, a glucose aqueous solution, a yeast extract aqueous solution, and a magnesium sulfate heptahydrate aqueous solution, each prepared at a predetermined concentration, were separately autoclaved (121°C, 30 minutes), cooled to room temperature, and then the autoclaved aqueous solutions and a filter-sterilized kanamycin sulfate aqueous solution were mixed in a sterilized container in the ratios shown in Table 4 to prepare the medium.

[0071] [Table 4]

[0072] After confirming that the turbidity (OD600) of the preculture was 3.0 or higher, 300 mL of this preculture was added to a 10-L fermenter containing the main culture medium to initiate the main culture. The main culture was performed using an Able BMS-10 culture device, an Able impeller consisting of two 95 mm diameter four-blade impellers combined with a basket-type mesh, an Able SDOC-16 DO sensor, and a Mettler Tredo Inpro3030 pH sensor. The air flow rate after filter sterilization was 6.0 L / min, the culture temperature was 30°C, and the pH of the culture medium was adjusted to a range of 6.9 to 7.1.

[0073] The pH of the culture medium was adjusted using a 14% (by weight) aqueous ammonia solution and a 42% (by weight) aqueous phosphoric acid solution. The agitation speed was set at 400 rpm at the start of the main culture, and once the dissolved oxygen concentration fell below 2.2 mg / L, the agitation speed was gradually increased using a control method known as the agitation speed cascade mode. The DO concentration in the culture medium, i.e., the consumption of the carbon source introduced at the beginning of the main culture, was monitored by detecting the signal from the DO electrode using a computer with an Able culture control program installed.

[0074] The DO electrode was calibrated using a dissolved oxygen concentration of 7.5 mg / L (saturation concentration) after adding the aforementioned main culture medium to the fermenter, heating it to 30°C, and stirring it for 45 minutes at an aeration rate of 6.0 L / min and an impeller speed of 700 rpm. Samples were taken periodically during the culture, and the OD600 (turbidity at 600 nm) was measured using a spectrophotometer (Hitachi High-Tech Science Model U-2900) and the glucose concentration was measured using a glucose analyzer (YSI Model 2700).

[0075] Approximately 10 hours after the start of main cultivation, the DO concentration stopped decreasing (the agitation speed under cascade control stopped increasing), and the analytical values ​​of the sample solution at this time were OD600 = 24 and glucose concentration = 0.1 g / L. This suggests that the glucose added to the culture solution at the start of cultivation was metabolized and depleted by the E. coli, suppressing E. coli growth and resulting in a decrease in oxygen consumption and an increase in the DO concentration indicated by the DO sensor.

[0076] Eleven hours after the start of main cultivation, the agitation speed cascade control was terminated and the agitation speed was fixed at 800 rpm. When the DO concentration exceeded 3.0 mg / L, the feed medium (glucose, yeast extract, magnesium sulfate) shown in Table 3 was added dropwise in 2.0 g increments. A Watson-Marlowe 101U high-speed metering pump was used for feeding, and the flow rate was set to 6.0 mL / min.

[0077] After 27 hours from the start of the main culture, the OD600 was confirmed to have reached 130, and the set temperature of the culture medium was changed to 25°C. Note that the amount of fed-batch medium added at 27 hours was 1200 g (1000 mL). After confirming that the culture medium temperature had reached 25°C, the agitation speed was changed to 600 rpm, and 8.1 mL of a 0.5 M IPTG solution prepared by dissolving IPTG (Fujifilm Wako Pure Chemical Industries, Ltd.) in purified water and sterilizing it through a 0.22 μm filter (Advantec) was added to initiate induction (final IPTG concentration = 0.83 mM).

[0078] 50 hours after the start of main culture, the OD600 reached 160, and the rotation speed of the agitator blade was reduced to 400 rpm. 68 hours after the start of main culture, the OD600 reached 180, and the culture was terminated. At the end of the culture, the total amount of added feed medium was 2500 g (2083 mL), the total amount of added 14 wt% ammonia water was 220 mL, and the total amount of added 42 wt% phosphoric acid was 25 mL.

[0079] The feed medium was added dropwise using the DO-stat method, which involves adding carbon sources in response to increases in DO concentration. From the start of feed (11 hours after the start of main culture) to the end of culture (68 hours), the glucose concentration in the culture medium was maintained below 0.6 g / L, and the dissolved oxygen concentration during the same period ranged from 0.0 mg / L to 3.3 mg / L. This indicates that carbon and nitrogen sources were intermittently supplied, using the DO concentration, which increased in response to glucose shortages, as an indicator. The total carbon source (glucose) and total nitrogen source (yeast extract) inputs throughout the main culture were 951 g (including the initial input) and 419 g (including the initial input), respectively.

[0080] After the cultivation was completed, 5,500 mL of the resulting culture medium was dispensed into 900 mL centrifuge containers and centrifuged (4°C, 9,000 rpm, 120 minutes) using a centrifuge (main body: Koki Holdings CR20GII, rotor: Koki Holdings R9A) to recover 1,170 g of bacterial cells.

[0081] In addition, the culture medium was diluted 10-fold with BugBuster HT (Merck) and incubated at 25°C for 1 hour. After that, it was analyzed by SDS-PAGE (ATTO, staining solution: EzApply, gel: P-R16.5S, electrophoresis buffer: EzRunT, electrophoresis apparatus: WSE-1150P). The band concentration around 14 kDa was confirmed, and the productivity of rBC2LCN(A) per 1 L of culture medium was confirmed to be approximately 7.0 g / L of culture medium. (3) Detergent extraction of rBC2LCN(A) Approximately 30 g of the cells collected in Example 1(2) was added to the extraction solution shown in Table 5 and stirred at room temperature for 30 minutes. The additives shown in Table 6 were then added and stirred overnight. The treated solution was then dispensed into 50 mL centrifuge vessels (HimaC50TC tubes manufactured by Koki Holdings) and centrifuged (4°C, 15,000 rpm, 30 minutes) using a centrifuge (main body: Koki Holdings CR20GII, rotor: Koki Holdings R15A). 75 mL of supernatant was recovered as a soluble protein extract containing rBC2LCN(A). The recovered soluble protein extract containing rBC2LCN(A) was filtered through a 0.2 μm filter and then used for purification by cation exchange chromatography, as described below. The resulting soluble protein extract was used as the "extract" for analysis by SDS-PAGE, as described below.

[0082] [Table 5]

[0083] [Table 6]

[0084] (4) Purification of rBC2LCN(A) by cation exchange chromatography Purification of rBC2LCN(A) from the soluble protein extract recovered in Example 1(3) was performed by cation exchange chromatography using TOYOPEARL GigaCap CM-650M (Tosoh Corporation, cation exchange chromatography packing, hereinafter abbreviated as GigaCap CM-650M). Specifically, rBC2LCN(A) was purified by the methods described in (Example 1-1) to (Example 1-6) below.

[0085] (Example 1-1): ToyoScreen GigaCap CM-650M (manufactured by Tosoh Corporation, a column packed with the GigaCap CM-650M, volume 5 mL) was equilibrated by passing 20 mL of buffer A (20 mM sodium citrate buffer containing 50 mM sodium chloride, pH 5.0, described in Table 7) through the column at a rate of 2.0 mL / min.

[0086] (Example 1-2): The pH of the soluble protein extract containing the rBC2LCN(A) was adjusted to pH 5.0 by adding 1.0 M citric acid. Furthermore, pure water was added to adjust the electrical conductivity to 7.5 mS / cm.

[0087] (Example 1-3): The extract whose pH and electrical conductivity had been adjusted in (Example 1-1) was added to the column equilibrated with buffer A in (Example 1-1) above at a rate of 2.0 mL / min.

[0088] (Example 1-4): 50 mL of buffer A was added to the column to which the soluble protein extract had been added in (Example 1-3) at a rate of 2.0 mL / min, and substances that did not bind to GigaCap CM-650M were washed away. The collected wash solution was used as the "flow-through recovery solution" for analysis by the SDS-PAGE method described below.

[0089] (Example 1-5): After washing with buffer A in (Example 1-4), 25 mL of a mixed solution of buffer A and buffer B (20 mM sodium citrate buffer containing 1 M sodium chloride, pH 5.0, listed in Table 7) (buffer A:buffer B = 63:37, v / v, sodium chloride concentration 500 mM) was added to the column at a rate of 2.0 mL / min to wash the GigaCap CM-650M and collect the wash solution. The first 12.5 mL from the start of collection was designated "37% B collected solution-1," and the subsequent collected solution was designated "37% B collected solution-2," and these were used for analysis by the SDS-PAGE method described below.

[0090] (Example 1-6): After washing the column with a mixed solution of buffer A and buffer B in (Example 1-5), 29 mL of buffer B was added at a rate of 2.0 mL / min to wash the GigaCap CM-650M and collect the wash solution. The first 12 mL from the start of collection was designated "100% B recovery solution-1," the 12-24 mL was designated "100% B recovery solution-2," and the solution collected from 24 mL onward was designated "100% B recovery solution-3," and these were used for analysis by the SDS-PAGE method described below.

[0091] [Table 7]

[0092] (5)Analysis The "extract," "flow-through recovery solution," "37% B recovery solution," and "100% B recovery solution" obtained in Examples 1 (3) and (Examples 1-4) to (Examples 1-6) were analyzed by SDS-PAGE. The results are shown in Figure 1. In Figure 1, "M" indicates a molecular weight marker. "Flow-through," "37% B-1, 2," and "100% B-1, 2, 3" respectively indicate the "flow-through recovery solution," "37% B recovery solution," and "100% B recovery solution" obtained in Example 1 (4). For SDS-PAGE analysis, a commercially available acrylamide gel (ATTO, P-R16.5S) was used, and a sample solution for SDS-PAGE analysis prepared from the recovery solution by the method described in Examples 1-7 below was used.

[0093] (Examples 1-7): The protein concentrations of the 37% B recovery solution and the 100% B recovery solution were adjusted to 0.20 mg / mL using BugBuster HT (Merck). The extract was diluted 10-fold with BugBuster HT. 15 μL of the undiluted flow-through recovery solution and each of the prepared samples were mixed with 15 μL of commercially available SDS sample buffer (ATTO, EzApply) and heated at 94°C for 5 minutes. 10 μL of the resulting solution was loaded onto an SDS-PAGE lane and analyzed by SDS-PAGE (electrophoresis buffer: ATTO EzRunT, electrophoresis tank: ATTO WSE-1150, staining solution: ATTO AE-1340).

[0094] In Figure 1, a band was observed in the "extract" and "100% B recovered solution" near the molecular weight of the rBC2LCN(A) monomer (approximately 14 kDa), confirming that all "100% B recovered solutions" contained highly pure rBC2LCN(A) with almost no impurities. On the other hand, this band was not observed in the "flow-through recovered solution" or "37% B recovered solution-1."

[0095] These "100% B Recovery Solution-1," "100% B Recovery Solution-2," and "100% B Recovery Solution-3" were combined to form the purified rBC2LCN(A) solution, and the absorbance of the purified rBC2LCN(A) solution at 280 nm was measured using a microspectrophotometer. The protein concentration in the purified rBC2LCN(A) solution was calculated assuming a molar extinction coefficient of 1.0 for rBC2LCN(A). The productivity of purified rBC2LCN(A) per liter of culture medium was calculated based on the protein concentration obtained, resulting in a high productivity of 4.9 g / L of culture medium. These results demonstrate that the production method of the present invention can produce rBC2LCN(A) with high purity and high yield, even without the use of animal-derived raw materials.

[0096] Comparative Example 1: Production of rBC2LCN(A)-2 (1) Extraction of rBC2LCN(A) by ultrasonic disruption Approximately 30 g of the cells collected in Example 1(2) was added to the extraction solution shown in Table 8 and stirred at room temperature for 30 minutes. The cells were then disrupted by sonication at 150 W for 10 minutes using an ultrasonic generator (Insonator 201M, Kubota Shoji Co., Ltd.) and a 100 mL processing tank (Kubota Shoji Co., Ltd.). The disrupted solution was then dispensed into 50 mL centrifuge vessels (HimaC 50TC tubes, Koki Holdings Co., Ltd.) and centrifuged (4°C, 15,000 rpm, 30 minutes) using a centrifuge (main body: Koki Holdings Co., Ltd. CR20GII, rotor: Koki Holdings Co., Ltd. R15A) to recover 75 mL of soluble protein extract containing rBC2LCN(A) as the supernatant. The recovered soluble protein extract containing rBC2LCN(A) was filtered through a 0.2 μm filter and used for purification by cation exchange chromatography, as described below. The prepared soluble protein extract was used as the "extract" for analysis by the SDS-PAGE method described below.

[0097] [Table 8]

[0098] (2) Purification of rBC2LCN(A) by cation exchange chromatography Purification of rBC2LCN(A) from the soluble protein extract recovered in (1) of Comparative Example 1 was performed by cation exchange chromatography using a TOYOPEARL GigaCap CM-650M. Specifically, rBC2LCN(A) was purified by the methods described below in (Comparison 1-1) to (Comparison 1-5).

[0099] (Ratio 1:1): ToyoScreen GigaCap CM-650M (manufactured by Tosoh Corporation, a column packed with the GigaCap CM-650M, volume 5 mL) was equilibrated by passing 20 mL of buffer A (20 mM MES buffer containing 50 mM sodium chloride, pH 5.0, described in Table 9) through the column at a rate of 2.0 mL / min.

[0100] (Ratio 1-2): 5 mL of the soluble protein extract containing the rBC2LCN(A) was loaded onto the column equilibrated with buffer A in (Ratio 1-1) above at a rate of 2.0 mL / min.

[0101] (Ratio 1-3): 45 mL of Buffer A was added to the column containing the soluble protein extract in (Ratio 1-2) above at a rate of 2.0 mL / min to wash away any substances that did not bind to the packing material (GigaCap CM-650M). The collected wash solution was used as the "flow-through recovery solution" for SDS-PAGE analysis, as described below.

[0102] (Ratios 1-4): After washing with Buffer A in the above (Ratios 1-3), 20 mL of a mixture of Buffer A and Buffer B (20 mM MES buffer containing 1 M sodium chloride, pH 5.0, listed in Table 9) (Buffer A:Buffer B = 63:37, v / v) was added to the column at a rate of 2.0 mL / min to wash the packing (GigaCap CM-650M) and collect the wash solution. The first 11.5 mL of the collection was designated "37% B Collection Solution-1," and the subsequent collection solution was designated "37% B Collection Solution-2," and these were used for SDS-PAGE analysis described below.

[0103] (Ratio 1-5): After washing the column with a mixed solution of Buffer A and Buffer B in (Ratio 1-4), 16 mL of Buffer B was added at a rate of 2.0 mL / min to wash the packing material (GigaCap CM-650M) and collect the wash solution. The first 10 mL of the column was designated "100%B Collection Solution-1," and the subsequent collection solution was designated "100%B Collection Solution-2," and these were used for SDS-PAGE analysis, as described below.

[0104] [Table 9]

[0105] (3)Analysis The "extract," "flow-through recovery solution," "37% B recovery solution," and "100% B recovery solution" obtained in (1) and (Ratios 1-3) to (Ratios 1-5) above were analyzed by SDS-PAGE. The results are shown in Figure 2. In Figure 2, "M" indicates a molecular weight marker. "Flow-through," "37% B-1, 2," and "100% B-1, 2" respectively refer to the "flow-through recovery solution," "37% B recovery solution," and "100% B recovery solution" obtained in (2) of Comparative Example 1. For SDS-PAGE analysis, a commercially available acrylamide gel (ATTO, e-PAGEL P-R16.5S) was used, and the sample solution for SDS-PAGE analysis was prepared from the recovery solution by the method described in (Examples 1-7).

[0106] 2, a band was observed near the molecular weight of the rBC2LCN(A) monomer (approximately 14 kDa) not only in the "extract" but also in the "flow-through recovered solution," and bands other than rBC2LCN(A) were also observed in the "100% B recovered solution." Therefore, it was revealed that the method of Comparative Example 1 was unable to purify the target rBC2LCN(A) with high purity and high yield, even under the same conditions as the purification of rBC2LCN(A) by cation exchange chromatography described in Example 1(4).

[0107] Comparative Example 2: Production of rBC2LCN(A)-3 (1) Detergent extraction of rBC2LCN(A) Approximately 200 g of the cells collected in Example 1(2) was added to the extract solution shown in Table 10 and stirred at room temperature for 30 minutes. The additives shown in Table 11 were then added and stirred overnight. The treated solution was then dispensed into a 900 mL centrifuge vessel and centrifuged (4°C, 9,000 rpm, 30 minutes) using a centrifuge (main body: Koki Holdings CR20GII, rotor: Koki Holdings R9A) to recover 500 mL of soluble protein extract containing rBC2LCN(A) as the supernatant. The recovered soluble protein extract containing rBC2LCN(A) was filtered through a 0.2 μm filter and then used for purification by nickel chelate affinity chromatography, as described below. The resulting soluble protein extract was used as the "extract" for analysis by SDS-PAGE, as described below.

[0108] [Table 10]

[0109] [Table 11]

[0110] (2) Purification of rBC2LCN(A) by nickel chelate affinity chromatography Purification of rBC2LCN(A) from the soluble protein extract recovered in Comparative Example 2(1) was performed by nickel chelate affinity chromatography using Ni-NTA His·Bind Resin (Merck) as the packing material. Specifically, rBC2LCN(A) was purified by the methods described below in (Comparison 2-1) to (Comparison 2-7).

[0111] (Ratio 2:1): A column (Merck Millipore Vantage L Laboratory Column VL22 × 500) was packed with 70 mL of His·Bind Resin and equilibrated by passing 140 mL of Buffer A (20 mM Tris buffer containing 500 mM sodium chloride and 20 mM imidazole, pH 8.3, as described in Table 12) through the column at a rate of 7.0 mL / min.

[0112] (Ratio 2-2): To the column equilibrated with buffer A in (Ratio 2-1) above, about 450 mL of the soluble protein extract containing the rBC2LCN(A) was added at a rate of 7.0 mL / min.

[0113] (Ratio 2-3): 400 mL of Buffer A was added to the column containing the soluble protein extract in (Ratio 2-2) above at a rate of 7.0 mL / min to wash away any substances that did not bind to the His·Bind Resin. The collected wash solution was used as the "flow-through recovery solution" for SDS-PAGE analysis, as described below.

[0114] (Ratio 2-4): After washing with Buffer A in (Ratio 2-3), 184 mL of a mixture of Buffer A and Buffer B (20 mM Tris buffer containing 500 mM sodium chloride and 250 mM imidazole, pH 9.0, listed in Table 12) (Buffer A:Buffer B = 95:5, v / v) was added to the column at a rate of 7.0 mL / min to wash the His·Bind Resin, and the wash solution was collected. The collected wash solution was used as the "5% B recovery solution" for SDS-PAGE analysis, as described below.

[0115] (Ratio 2-5): After washing the column with a mixed solution of Buffer A and Buffer B in the above (Ratio 2-4), a linear gradient elution was performed by gradually changing the mixture ratio of Buffer A to Buffer B. The flow rate was 7.0 mL / min, and the mixture ratio of Buffer A to Buffer B was continuously changed from Buffer A:Buffer B = 95:5, v / v, to Buffer A:Buffer B = 0:100, v / v, over 35 minutes. The linear gradient wash solution was collected in 12 mL portions and used as the "gradient elution recovered solution" for analysis by SDS-PAGE, as described below.

[0116] (Ratio 2-6): After washing the column with a mixture of Buffer A and Buffer B in (Ratio 2-5), 204 mL of Buffer B was added at a rate of 7.0 mL / min to wash the His·Bind Resin, and the wash solution was collected in 12 mL aliquots. The collected wash solution was designated "100% B recovery solution" and used for SDS-PAGE analysis, as described below.

[0117] (Ratio 2-7): After washing with the mixed solution of Buffer B in (Ratio 2-6), 48 mL of Buffer C (20 mM Tris buffer containing 500 mM sodium chloride and 500 mM imidazole, pH 9.0, as described in Table 12) was added to the column at a rate of 7.0 mL / min to wash the His·Bind Resin, and the wash solution was collected in 12 mL aliquots. The collected wash solution was designated "200% B recovery solution" and used for SDS-PAGE analysis, as described below.

[0118] [Table 12]

[0119] (3)Analysis The "extract," "flow-through recovery solution," "gradient elution recovery solution," "100% B recovery solution," and "200% B recovery solution" obtained in (1) and (Ratio 2-3) to (Ratio 2-7) above were analyzed by SDS-PAGE. The results are shown in Figure 3. In Figure 3, "M" indicates a molecular weight marker. "Flow-through," "gradient," "100% B," and "200% B" refer to the "flow-through recovery solution," "gradient elution recovery solution," "100% B recovery solution," and "200% B recovery solution" obtained in Comparative Example 2(2), respectively. For SDS-PAGE analysis, a commercially available acrylamide gel (ATTO, e-PAGEL P-R16.5S) was used. The sample solution for SDS-PAGE analysis was prepared from the recovery solution by the method described in (Ratio 2-7) below.

[0120] (Ratios 2-7): The extract was diluted 10-fold with BugBuster HT (Merck). The protein concentrations of flow-through recovery solution-1, flow-through recovery solution-2, 5% B recovery solution, and gradient elution recovery solution were adjusted to 6.0 mg / mL, 8.0 mg / mL, and 0.20 mg / mL, respectively, using BugBuster HT. The 100% B recovery solution and 200% B recovery solution were both adjusted to 0.20 mg / mL. 15 μL of the prepared sample was mixed with 15 μL of commercially available SDS sample buffer (ATTO, EzApply) and heated at 94°C for 5 minutes. 10 μL of the resulting solution was loaded onto an SDS-PAGE lane and analyzed by SDS-PAGE (running buffer: ATTO, EzRunT; running tank: ATTO, WSE-1150; staining solution: ATTO, AE-1340).

[0121] In Figure 3, a band was confirmed in the extract and all recovered solutions near the molecular weight of the rBC2LCN(A) monomer (approximately 14 kDa). It can be seen that "Gradient Elution Recovery Solutions No. 17 to 20," "100% B Recovery Solution," and "200% B Recovery Solution" contain highly pure rBC2LCN(A), with almost no impurities. Furthermore, "Gradient Elution Recovery Solutions No. 17 to 20," "100% B Recovery Solution," and "200% B Recovery Solution" were combined to form the purified rBC2LCN(A) solution, and the absorbance of the purified rBC2LCN(A) solution at 280 nm was measured using a microspectrophotometer. The protein concentration in the purified rBC2LCN(A) solution was calculated assuming the molar extinction coefficient of rBC2LCN(A) to be 1.0, and the productivity of the purified rBC2LCN(A) solution per 1 L of culture medium was then calculated based on the obtained protein concentration.The productivity was found to be 1.0 g / L of culture medium, which was lower than the productivity in Example 1 (4.9 g / L of culture medium).

[0122] Example 2 Production of rBC2LCN(A)-4 (1) Detergent extraction of rBC2LCN(A) Approximately 30 g of the cells collected in Example 1(2) was added to the extraction solution shown in Table 13 and stirred at room temperature for 30 minutes. The additives shown in Table 14 were then added and stirred overnight. The treated solution was then dispensed into 50 mL centrifuge vessels (HimaC50TC tubes manufactured by Koki Holdings) and centrifuged (4°C, 15,000 rpm, 30 minutes) using a centrifuge (main body: Koki Holdings CR20GII, rotor: Koki Holdings R15A). 75 mL of soluble protein extract containing rBC2LCN(A) was recovered as the supernatant. The recovered soluble protein extract containing rBC2LCN(A) was filtered through a 0.2 μm filter and then used for purification by cation exchange chromatography, as described below. The resulting soluble protein extract was used as the "extract" for analysis by SDS-PAGE, as described below.

[0123] [Table 13]

[0124] [Table 14]

[0125] (2) Purification of rBC2LCN(A) by cation exchange chromatography Purification of rBC2LCN(A) from the soluble protein extract recovered in Example 2(1) was carried out by the method described in Comparative Example 1(2). However, in (Ratio 1-4), the column was washed with a mixed solution of Buffer A and Buffer B, with the ratio of Buffer A:Buffer B being 52:48, v / v. The recovered wash solution was then used as the "48% B recovered solution" for analysis by the SDS-PAGE method described below. (3)Analysis The "extract," "flow-through recovery solution," "48% B recovery solution," and "100% B recovery solution" obtained in (1) and (2) above were analyzed by SDS-PAGE. The results are shown in Figure 4. In Figure 4, "M" indicates a molecular weight marker. "Flow-through," "48% B-1, 2," and "100% B-1, 2" respectively indicate the "flow-through recovery solution," "48% B recovery solution," and "100% B recovery solution" obtained in (2) of Comparative Example 1. For SDS-PAGE analysis, a commercially available acrylamide gel (ATTO, e-PAGEL P-R16.5S) was used, and a sample solution for SDS-PAGE analysis prepared from the recovery solution by the method described in (Example 1-7) was used.

[0126] In Figure 4, a band was observed in the "extract" and "100% B recovery solution" near the molecular weight of the rBC2LCN(A) monomer (approximately 14 kDa), confirming the presence of rBC2LCN(A). These "100% B recovery solution-1" and "100% B recovery solution-2" were combined to form the purified rBC2LCN(A) solution, and the absorbance of the purified rBC2LCN(A) solution at 280 nm was measured using a microspectrophotometer. The protein concentration in the purified rBC2LCN(A) solution was calculated assuming a molar extinction coefficient of 1.0 for rBC2LCN(A). Based on the obtained protein concentration, the productivity per 1 L of purified rBC2LCN(A) culture solution was calculated, resulting in a productivity of 2.2 g / L of culture solution.

[0127] Example 3 Evaluation of rBC2LCN(A) binding to sugar chains In Example 3, the binding affinity of rBC2LCN(A) produced in Example 1 to H-type 3 glycans was evaluated by surface plasmon resonance. Specifically, a Biacore T100 (T200 Sensitivity Enhanced) instrument (GE Healthcare) was used to perform kinetic analysis using recombinant protein as the analyte and H-type 3 glycans as the solid phase. A dextran-coated Sensor Chip CM5 (GE Healthcare) was used as the sensor chip. Streptavidin (Fujifilm Wako Pure Chemical Industries) was immobilized to the dextran by the amine coupling method, and then biotin-labeled H-type 3 glycans (Glycotech) were added. Each glycan was immobilized on the sensor chip via a biotin-streptavidin reaction, resulting in the production of a sensor chip with immobilized H-type 3 glycans.

[0128] Glycan-binding affinity measurements were performed using HBS-EP+ (GE Healthcare) as the buffer solution, with a flow rate of 30 μL / min, a binding time of 3 minutes, and a dissociation time of 90 seconds. The sensor chip was regenerated using 25 mM sodium hydroxide at a flow rate of 30 μL / min and a regeneration time of 15 seconds. Analysis was performed using the analysis software (Biacore T100 Evaluation Software, version 1 or Biacore T200 Evaluation Software, version 1) included with the Biacore T100 (T200 Sensitivity Enhanced) instrument, and the dissociation constant (KD) was calculated using 1:1 binding fitting. The dissociation constant of rBC2LCN(A) for H-type 3 glycans was calculated to be 8.1 nM.

[0129] Example 4 Production of rBC2LCN(B)-1 Example 4 relates to the production of a fucose-binding protein consisting of 174 amino acids and represented by SEQ ID NO: 6, i.e., a fucose-binding protein in which an oligopeptide sequence containing histidine has been added to the N-terminus and an oligopeptide sequence containing cysteine ​​has been added to the C-terminus of the amino acid sequence of the fucose-binding protein consisting of 155 amino acids and represented by SEQ ID NO: 1 (hereinafter referred to as rBC2LCN(B)). (1) Construction of expression vector pTrc-rBC2LCN(B) and recombinant E. coli W3110 / pTrc-rBC2LCN(B) The expression vector pTrc-rBC2LCN(B) is an expression vector for expressing rBC2LCN(B). In the amino acid sequence of rBC2LCN(B) shown in SEQ ID NO: 6, positions 5 to 10 correspond to an oligopeptide sequence containing histidine, positions 15 to 141 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 168 to 174 correspond to an oligopeptide sequence containing cysteine.

[0130] The expression vector pTrc-rBC2LCN(B) and recombinant E. coli W3110 / pTrc-rBC2LCN(B) were prepared in the same manner as in Example 1, and recombinant E. coli W3110 / pTrc-BC2LCN(B)cys was obtained. Recombinant E. coli W3110 / pTrc-BC2LCN(B)cys was cultured and extracted from the cells using the method disclosed in JP 2018-000038 A to obtain the expression vector pTrc-BC2LCN(B)cys. Sequence analysis confirmed the base sequence, confirming that the expression vector pTrc-BC2LCN(B)cys contains the base sequence of SEQ ID NO: 11, which encodes the amino acid sequence of SEQ ID NO: 1. (2) Production of rBC2LCN(B) using recombinant E. coli The recombinant E. coli W3110 / pTrc-rBC2LCN(B) prepared in (1) above was inoculated into three 500 mL baffled Erlenmeyer flasks containing the preculture medium shown in Table 1 (autoclaved at 121°C for 20 minutes, cooled, and then kanamycin sulfate was added), and preculture was carried out at 30°C for 20 hours at a shaking rate of 130 rpm (amplitude 25 mm, circular rotation). The main culture medium and the fed-batch medium were prepared by the method described in Example 1.

[0131] After confirming that the turbidity (OD600) of the preculture solution was 3.0 or higher, 300 mL of this preculture solution was added to a 10 L fermenter containing the main culture medium to initiate the main culture. The main culture was carried out in the same manner as in Example 1 (2).

[0132] Eleven hours after the start of main cultivation, the agitation speed cascade control was terminated and the agitation speed was fixed at 800 rpm. When the DO concentration exceeded 3.0 mg / L, the feed medium (glucose, yeast extract, magnesium sulfate) shown in Table 3 was added dropwise in 2.0 g increments. A Watson-Marlowe 101U high-speed metering pump was used for the feed, with a flow rate of 6.0 mL / min. After 27 hours of main cultivation, the OD600 was confirmed to have reached 130, and the culture temperature was changed to 25°C. The amount of feed medium added at 27 hours was 1140 g (950 mL). After confirming that the culture temperature had reached 25°C, the agitation speed was changed to 600 rpm, and induction was initiated by adding 8.1 mL of a 0.5 M IPTG solution prepared by dissolving IPTG in purified water and sterilizing it through a 0.22 μm filter. 50 hours after the start of main cultivation, the OD600 reached 150, and the rotation speed of the agitator was reduced to 400 rpm. 70 hours after the start of main cultivation, the OD600 reached 170, and the cultivation was terminated. At the end of cultivation, the total amount of added feed medium was 2400 g (2000 mL), the total amount of added 14 wt% ammonia water was 215 mL, and the total amount of added 42 wt% phosphoric acid was 23 mL.

[0133] The fed-batch medium was added dropwise using a method in which a carbon source was added to the culture medium in conjunction with an increase in DO concentration (DO-stat method). As a result, the glucose concentration in the culture medium was maintained at 0.6 g / L or less from the start of feeding to the end of cultivation, and the dissolved oxygen concentration during the same period fluctuated between 0.0 mg / L and 3.3 mg / L. After cultivation was completed, the resulting 5,400 mL of culture medium was dispensed into 900 mL centrifuge vessels and centrifuged (4°C, 9,000 rpm, 120 minutes) to recover 1,100 g of bacterial cells. (3) Detergent extraction of rBC2LCN(B) Surfactant extraction of rBC2LCN(B) was performed using a method similar to that described in Example 1(3). Specifically, the extraction solution shown in Table 5 was added to approximately 30 g of the cells collected in Example 4(2) and stirred at room temperature for 30 minutes. The additives shown in Table 6 were then added and stirred overnight. The treated solution was then dispensed into 50 mL centrifuge vessels and centrifuged (4°C, 15,000 rpm, 30 minutes) using a centrifuge to recover 70 mL of supernatant as a soluble protein extract containing rBC2LCN(B). The recovered soluble protein extract containing rBC2LCN(B) was filtered through a 0.2 μm filter and then used for purification by cation exchange chromatography, as described below. The resulting soluble protein extract was used as the "extract" for analysis by SDS-PAGE, as described below. (4) Purification of rBC2LCN(B) by cation exchange chromatography Purification of rBC2LCN(B) by cation exchange chromatography was performed in the same manner as in Example 1(4). Purification of rBC2LCN(B) from the soluble protein extract recovered in Example 4(3) was performed by cation exchange chromatography using a GigaCap CM-650M. Specifically, rBC2LCN(B) was purified by the methods described in the following (Example 4-1) to (Example 4-6).

[0134] (Example 4-1): ToyoScreen GigaCap CM-650M (manufactured by Tosoh Corporation, volume 5 mL) was equilibrated by passing 20 mL of buffer C (20 mM sodium citrate buffer, pH 5.0) through it at a rate of 2.0 mL / min.

[0135] (Example 4-2): The pH of the soluble protein extract containing the rBC2LCN (B) was adjusted to pH 5.0 by adding 1.0 M citric acid. Furthermore, pure water was added to adjust the electrical conductivity to 7.5 mS / cm.

[0136] (Example 4-3): The extract whose pH and electrical conductivity had been adjusted in (Example 4-1) was added to the column equilibrated with buffer A in (Example 1-1) at a rate of 2.0 mL / min.

[0137] (Example 4-4): 50 mL of buffer C was added to the column to which the soluble protein extract had been added in (Example 4-3) at a rate of 2.0 mL / min to wash away substances that did not bind to the GigaCap CM-650M, and the wash solution was collected as "flow-through recovery solution." The first 15 mL from the start of collection was designated "flow-through recovery solution-1," the 15-30 mL portion was designated "flow-through recovery solution-2," and the solution collected from 30 mL onward was designated "flow-through recovery solution-3," and these were used for analysis by the SDS-PAGE method described below.

[0138] (Example 4-5): After washing with buffer C in (Example 4-4), 25 mL of a mixed solution of buffer C and buffer D (20 mM sodium citrate buffer containing 1 M sodium chloride, pH 5.0) (buffer C:buffer D = 45:55, v / v, sodium chloride concentration 550 mM) was added to the column at a rate of 2.0 mL / min to wash the GigaCap CM-650M, and the washing solution was recovered as "45% B recovery solution" and used for analysis by the SDS-PAGE method described below.

[0139] (Example 4-6): After washing the column with a mixed solution of buffer C and buffer D in (Example 4-5), 29 mL of buffer D was added at a rate of 2.0 mL / min to wash the GigaCap CM-650M, and the washing solution was collected as "100% B recovery solution." The first 5 mL from the start of collection was called "100% B recovery solution-1," the 5 mL to 10 mL was called "100% B recovery solution-2," and the recovery solution from 10 mL onward was called "100% B recovery solution-3," and these were used for analysis by the SDS-PAGE method described below. (5)Analysis The "extract," "flow-through recovery solution," "45% B recovery solution," and "100% B recovery solution" obtained in Examples 4(3) and (Example 4-4) to (Example 4-6) were analyzed by SDS-PAGE using a method similar to that described in Example 1(5). The results are shown in Figure 5. In Figure 5, "M" indicates a molecular weight marker. "Flow-through," "45% B," and "100% B-1, 2, and 3" respectively indicate the "flow-through recovery solution," "45% B recovery solution," and "100% B recovery solution" obtained in Example 4(3). For SDS-PAGE analysis, a commercially available acrylamide gel (ATTO, e-PAGEL P-R16.5S) was used, and the sample solution for SDS-PAGE analysis was prepared from the recovery solution by the method described in (Example 1-7).

[0140] In Figure 5, a band was observed in the "extract" and "100% B recovery solution" near the molecular weight of the rBC2LCN(B) monomer (approximately 17 kDa), confirming the presence of highly pure rBC2LCN(B) with almost no impurities in all "100% B recovery solutions." On the other hand, this band was barely observed in the "flow-through recovery solution" and "45% B recovery solution." The "100% B recovery solution" was used as the purified rBC2LCN(B) solution, and the absorbance of the purified rBC2LCN(B) solution at 280 nm was measured using a microspectrophotometer. The molar extinction coefficient of rBC2LCN(B) was set to 1.0 to calculate the protein concentration in the purified rBC2LCN(B) solution. Based on the obtained protein concentration, the productivity per 1 L of purified rBC2LCN(B) culture was calculated. The productivity was high, at 3.0 g / L culture. From the above results, it was found that rBC2LCN(B), which has a different amino acid sequence length from rBC2LCN(A) described in Examples 1 and 2, can also be obtained with high purity and high yield using the manufacturing method of the present invention without using animal-derived raw materials.

[0141] Example 5 Evaluation of rBC2LCN(B) binding to sugar chains The binding affinity of rBC2LCN(B) produced in Example 4 to H-type 3 glycans was evaluated using a method similar to that used in Example 3, and the dissociation constant of rBC2LCN(B) to H-type 3 glycans was calculated, resulting in a dissociation constant of 11 nM.

[0142] Comparative Example 3: Production of rBC2LCN (B)-2 (1) Detergent extraction of rBC2LCN (B) Using a method similar to that used in Comparative Example 2(1), approximately 200 g of the cells collected in Example 4(2) was added to the extract solution shown in Table 10 and stirred at room temperature for 30 minutes. The additives shown in Table 11 were then added, and the mixture was stirred overnight. The resulting solution was then centrifuged to recover 450 mL of a soluble protein extract containing rBC2LCN(B) as the supernatant. The recovered soluble protein extract containing rBC2LCN(B) was filtered through a 0.2 μm filter and used for purification by nickel chelate affinity chromatography, as described below. The resulting soluble protein extract was used as the "extract" for analysis by SDS-PAGE, as described below. (2) Purification of rBC2LCN(B) by nickel chelate affinity chromatography As in Comparative Example 2(2), rBC2LCN(B) was purified from the soluble protein extract recovered in Comparative Example 3(1) by nickel chelate affinity chromatography using Ni-NTA His·Bind Resin as the packing material. Specifically, rBC2LCN(B) was purified by the methods described below in (Comparison 3-1) to (Comparison 3-7).

[0143] (Ratio 3:1): A column (Vantage L Laboratory Column VL22 × 500) was packed with 70 mL of His·Bind Resin and equilibrated with 150 mL of Buffer A (20 mM Tris buffer, pH 8.3, containing 500 mM sodium chloride and 20 mM imidazole, Table 12) at a flow rate of 7.0 mL / min.

[0144] (Ratio 3-2): The soluble protein extract containing rBC2LCN (B) was loaded onto the column equilibrated with buffer A in (Ratio 3-1) above at a rate of 7.0 mL / min.

[0145] (Ratio 3-3): 500 mL of Buffer A was added to the column containing the soluble protein extract (Ratio 3-2) at a rate of 7.0 mL / min to wash away any substances that did not bind to the His·Bind Resin. The wash solution was collected as "Flow-through Recovery Solution." The first 50 mL of the recovery was designated "Flow-through Recovery Solution-1," the next 50 mL to 100 mL was designated "Flow-through Recovery Solution-2," and the remaining 100 mL and beyond was designated "Flow-through Recovery Solution-3." These were then used for SDS-PAGE analysis, as described below.

[0146] (Ratio 3-4): After washing with Buffer A in (Ratio 3-3), 200 mL of a mixture of Buffer A and Buffer B (20 mM Tris buffer containing 500 mM sodium chloride and 250 mM imidazole, pH 9.0; Table 12) (Buffer A:Buffer B = 9:1, v / v) was added to the column at a rate of 7.0 mL / min to wash the His·Bind Resin, and the wash solution was collected. The collected wash solution was used as the "10% B recovery solution" for SDS-PAGE analysis, as described below.

[0147] (Ratio 3-5): After washing the column with a mixture of Buffer A and Buffer B in the above (Ratio 3-4), a linear gradient elution was performed by gradually increasing the mixture ratio of Buffer A to Buffer B. The flow rate was 7.0 mL / min, and the mixture ratio of Buffer A to Buffer B was continuously varied from 9:1 (v / v) to 0:10 (v / v) over 40 minutes. The linear gradient wash solution was collected in 70 mL increments. The first 70 mL was designated "Gradient Recovery Solution-1," the 70-140 mL elution volume designated "Gradient Recovery Solution-2," the 140-210 mL elution volume designated "Gradient Recovery Solution-3," and the elution volume from 200 mL onward designated "Gradient Recovery Solution-4." These were used for SDS-PAGE analysis, as described below.

[0148] (Ratio 3-6): After washing the column with a mixture of Buffer A and Buffer B in (Ratio 3-5), 240 mL of Buffer B was added at a rate of 7.0 mL / min to wash the His·Bind Resin, and the wash solution was collected. The collected wash solution was designated "100% B recovery solution" and used for SDS-PAGE analysis, as described below.

[0149] (Ratio 3-7): After washing with Buffer B in (Ratio 3-6), 50 mL of Buffer C (20 mM Tris buffer containing 500 mM sodium chloride and 500 mM imidazole, pH 9.0; Table 12) was added to the column at a rate of 7.0 mL / min to wash the His·Bind Resin, and the wash solution was collected. This collected wash solution was designated "200% B recovery solution" and used for SDS-PAGE analysis, as described below. (3)Analysis The "extract," "flow-through recovery solution," "10% B recovery solution," "gradient elution recovery solution," "100% B recovery solution," and "200% B recovery solution" obtained in Comparative Example 3 (1) and (Ratio 3-3) to (Ratio 3-7) were analyzed by SDS-PAGE using a method similar to that used in Comparative Example 2 (3). The results are shown in Figure 6. In Figure 6, "M" indicates a molecular weight marker. Furthermore, "flow-through," "10% B," "gradient," "100% B," and "200% B" refer to the "flow-through recovery solution," "10% B recovery solution," "gradient recovery solution," "100% B recovery solution," and "200% B recovery solution" obtained in Comparative Example 3 (3), respectively. For SDS-PAGE analysis, a commercially available acrylamide gel (ATTO, e-PAGEL P-R16.5S) was used, and the sample solution for SDS-PAGE analysis was prepared from the recovery solution using the method described in (Example 1-7).

[0150] In Figure 6, bands were observed near the molecular weight of the rBC2LCN(B) monomer (approximately 17 kDa) containing almost no impurities in "Gradient Elution Recovery Solution-4," "100% B Recovery Solution," and "200% B Recovery Solution." These recovery solutions containing highly purified rBC2LCN(B) were combined to form a purified rBC2LCN(B) solution, and the absorbance of the purified rBC2LCN(B) solution at 280 nm was measured using a microspectrophotometer. The protein concentration in the purified rBC2LCN(B) solution was calculated assuming a molar extinction coefficient of 1.0 for rBC2LCN(B). Based on the obtained protein concentration, the productivity of the purified rBC2LCN(B) solution per liter of culture medium was calculated. The productivity was 0.85 g / L of culture medium, which was lower than the productivity in Example 4 (3.0 g / L of culture medium).

Claims

1. A method for producing a fucose-binding protein using Escherichia coli obtained by transformation with an expression vector containing DNA encoding the fucose-binding protein, comprising the following steps (1) to (3): Step (1) adding a medium containing a carbon source and a nitrogen source to a culture vessel, and culturing Escherichia coli obtained by transformation with an expression vector containing DNA encoding a fucose-binding protein with stirring to obtain a culture; step (2) adding a solution containing at least one surfactant to the culture obtained in step (1) and disrupting the E. coli without ultrasonic irradiation to obtain an aqueous solution containing a fucose-binding protein; step (3) purifying the fucose-binding protein from the solution containing the fucose-binding protein obtained in step (2) by cation exchange chromatography; The step (3) includes the following steps (4) to (7): Step (4) washing the cation exchange chromatography support with an equilibration buffer; step (5) contacting a solution containing a fucose-binding protein with a cation exchange chromatography carrier washed with an equilibration buffer to adsorb the fucose-binding protein onto the carrier; Step (6) washing the cation exchange chromatography carrier onto which the fucose-binding protein has been adsorbed with a first washing solution; Step (7) using a second washing solution to elute the fucose-binding protein adsorbed on the cation exchange chromatography carrier; the equilibration buffer solution, the solution containing a fucose-binding protein, the first washing solution, and the second washing solution are all buffer solutions containing citric acid; A method in which the fucose-binding protein is any one of the following (a) to (d): (a) a fucose-binding protein comprising an amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or more; (b) a fucose-binding protein comprising an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, and which has binding affinity to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, where X is an integer of 120 or more; (c) a fucose-binding protein comprising an amino acid sequence containing one or more of the amino acid substitutions listed in (I) to (III) below in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or more; (I) Substitution of the glutamine residue at position 39 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (II) Substitution of the cysteine ​​residue at position 72 of the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from a glycine residue and an alanine residue. (III) Substitution of the glutamine residue at position 65 of the amino acid sequence represented by SEQ ID NO: 1 with a leucine residue (d) A fucose-binding protein comprising an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted or added in a region other than positions 39, 65 and 72 of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of (c), and having binding affinity to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

2. The method according to claim 1, wherein the equilibration buffer and the first washing solution are buffers containing less than 750 mmol / L of an alkali metal salt.

3. 3. The method according to claim 1, wherein the second washing solution is a buffer solution containing an alkali metal salt at 750 mmol / L or more and 3000 mmol / L or less.

4. 4. The method according to claim 2 or 3, wherein the alkali metal salt is sodium chloride.

5. 5. The method according to claim 1, wherein the step (3) is carried out using a column packed with a carrier for cation exchange chromatography.

6. The method according to claim 1 , wherein one of the surfactants used in step (2) is a cationic surfactant.

7. 7. The method according to claim 6, wherein the cationic surfactant is a hexadecyltrimethylammonium halide.

8. The method according to any one of claims 1 to 7, wherein the surfactant used in step (2) is not an animal-derived surfactant.

9. The method according to any one of claims 1 to 8, wherein the carbon source and nitrogen source used in step (1) are not derived from animals.

10. The production method according to any one of claims 1 to 9, wherein the fucose-binding protein is any one of the following (e) to (h): (e) a fucose-binding protein consisting of an amino acid sequence comprising the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, to which an oligopeptide containing a polyhistidine sequence is further added at the N-terminus and an oligopeptide containing cysteine ​​is further added at the C-terminus, wherein X is an integer of 120 or more; (f) a fucose-binding protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, and further comprising a polyhistidine sequence added to the N-terminus and an oligopeptide containing cysteine ​​added to the C-terminus, the fucose-binding protein having a binding affinity to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more; (g) A fucose-binding protein comprising an amino acid sequence comprising the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, to which an oligopeptide containing a polyhistidine sequence is added at the N-terminus and an oligopeptide containing cysteine ​​is added at the C-terminus, and which comprises an amino acid sequence containing one or more of the amino acid substitutions listed in (IX) to (XI) below, wherein X is an integer of 120 or greater; (IX) Substitution of the glutamine residue at position 39 of the amino acid sequence represented by SEQ ID NO: 1 with a leucine residue (X) Substitution of the cysteine ​​residue at position 72 of the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from a glycine residue and an alanine residue. (XI) Substitution of the glutamine residue at position 65 of the amino acid sequence represented by SEQ ID NO: 1 with a leucine residue (h) A fucose-binding protein having an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted or added in a region other than positions 39, 65 and 72 of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of (g) above, and further comprising an oligopeptide containing a polyhistidine sequence added to the N-terminus and an oligopeptide containing cysteine ​​added to the C-terminus, and having binding affinity for sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

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