JP2018000038A - Fucose-binding lectins with cysteine tags and adsorbents using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide fucose-binding lectin which can be immobilized to a support with high efficiency and site-specificity, methods for producing the lectin, and adsorbents for fucose-containing sugar chains and/or glycoconjugates obtainable by immobilizing the lectin to a water-insoluble support.SOLUTION: To solve the problem, the invention provides a protein comprising a fucose-binding lectin whose N- or C-terminus is added with an oligopeptide containing one or more cysteines, a method for producing the protein, and an adsorbent obtainable by immobilizing the protein to a water-insoluble support.SELECTED DRAWING: None

Description

  The present invention relates to a lectin having a binding property to a fucose-containing sugar chain and / or complex carbohydrate that can be immobilized on a carrier with high efficiency and position-selection, a polynucleotide encoding the lectin, and production of the lectin The present invention relates to a method and an adsorbent obtained by immobilizing the lectin on a carrier insoluble in water.

  Lectin is a general term for proteins that bind to saccharides, and is known to bind not only to monosaccharides and sugar chains, but also to complex carbohydrates in which monosaccharides and sugar chains are bound to proteins and lipids. Since it specifically recognizes sugar chains, it has been widely used for detection and separation / purification of sugar chains and complex carbohydrates. For example, Patent Document 1 discloses a sugar chain profiling technique using a substrate on which a lectin is immobilized, and Non-Patent Document 1 discloses a method for separating and purifying a glycoprotein using agarose on which a lectin is immobilized. ing.

  In recent years, it has been clarified that sugar chains play an important role in the function of biomolecules such as glycoproteins, cell differentiation and recognition, as research related to functional analysis of sugar chains progresses. Among the sugar chains, fucose-containing sugar chains are often associated with physiological functions. For example, Lewis blood group antigens disclosed in Non-Patent Document 2 and sialyl Lewis sugar chain antigens as tumor markers are known. In Patent Document 2, antibody-dependent cytotoxicity (ADCC) of an antibody composition is removed by removing fucose α1-6 linked to N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain bound to the antibody composition. ) A method for promoting activity is disclosed.

  As a method for separating and purifying fucose-containing sugar chains and complex carbohydrates having various physiological functions with high efficiency, the fucose-containing sugar chains and complex carbohydrates are adsorbed on a carrier on which a fucose-binding lectin is immobilized. Thereafter, a method of desorption is common. As a lectin that binds to a fucose-containing sugar chain, for example, a lectin derived from a bamboo shoot that binds to an α1-2-linked fucose-containing sugar chain disclosed in Non-Patent Document 3 or α1 disclosed in Patent Document 3 A lectin derived from Sugitake that binds to a -6-linked fucose-containing sugar chain is known.

  As a method for immobilizing a lectin on a carrier, for example, reductive amination disclosed in a product catalog of ConA-Agarose (manufactured by J-Oil Mills) in which concanavalin A, which is a mannose-binding lectin, is immobilized on an agarose gel. In general, the lysine ε-amino group in the lectin molecule is reacted with a functional group such as a formyl group or an epoxy group introduced into the carrier and immobilized by covalent bonding. However, when the lysine in the lectin is embedded in the lectin molecule, the reactivity between the lectin and the functional group introduced into the carrier is reduced, so that the rate of immobilization on the carrier is reduced or the carrier is immobilized on the carrier. It was a problem that there was a case where it was not converted. In addition, when a large number of lysines are present in the lectin, since the lectin is immobilized on the carrier at random, a part of the sugar chain binding region is immobilized in a form facing the carrier side. The problem is that the binding properties of chains and complex carbohydrates may be impaired.

Japanese Patent No. 4824119 Japanese Patent No. 5547754 Japanese Patent No. 4514163

Biochem. Biophys. Res. Commun. 165 (2), 703-707, 1989. Protein Nucleic Acid Enzyme, 43 (16), 2394-2403, 1998 Biochemistry, 19, 2841-2846, 1980

  An object of the present invention is to provide a protein that can be immobilized on a carrier with high efficiency and position-selectivity when immobilizing a lectin having binding ability to a fucose-containing sugar chain and / or complex carbohydrate on the carrier, It is to provide a protein production method and an adsorbent obtained by immobilizing the protein on a carrier insoluble in water.

  As a result of intensive studies to solve the above problems, the present inventors have found that oligopeptides containing one or more cysteines on the N-terminal side or C-terminal side of lectins having binding properties to fucose-containing sugar chains and / or complex carbohydrates It was found that the protein added with can be immobilized on a carrier with high efficiency and position selection. Furthermore, the present inventors have found that the adsorbent obtained by immobilizing the lectin of the present invention on a water-insoluble carrier has excellent performance as an adsorbent for fucose-containing sugar chains and / or complex carbohydrates, thereby completing the present invention. .

  That is, the present invention provides the inventions described in the following (1) to (13).

(1) A protein having an oligopeptide containing one or more cysteines at the N-terminus or C-terminus of a fucose-binding lectin.
(2) The fucose-binding lectin is
(A) a protein comprising at least the second proline to 156th glycine of the amino acid sequence set forth in SEQ ID NO: 1, or (B) one or more in the amino acid sequence of the protein of (A) above A protein in which one amino acid is deleted, substituted or added,
(1) The protein according to the description.
(3) The protein according to (1) or (2), wherein the oligopeptide comprising one or more cysteines is an oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.
(4) A polynucleotide encoding the protein according to any one of (1) to (3).
(5) An expression vector comprising the polynucleotide according to (4).
(6) A transformant obtained by transforming a host with the expression vector according to (5).
(7) The transformant according to (6), wherein the host is Escherichia coli.
(8) A transformant obtained by introducing an expression vector containing a polynucleotide encoding a protein having an oligopeptide containing one or more cysteines at the N-terminus or C-terminus of a fucose-binding lectin into a host is cultured. A method for producing a protein, comprising: a step; and a step of recovering the protein from the obtained culture.
(9) The fucose-binding lectin is
(A) a protein comprising at least the second proline to 156th glycine of the amino acid sequence set forth in SEQ ID NO: 1, or (B) one or more in the amino acid sequence of the protein of (A) above (8) The method for producing a protein according to (8), wherein the amino acid is a protein in which one amino acid is deleted, substituted or added.
(10) An adsorbent for fucose-containing sugar chains and / or complex carbohydrates, wherein the protein according to (8) or (9) is immobilized on a carrier insoluble in water.
(11) The adsorbent according to (8) or (9), wherein the water-insoluble carrier has a functional group that reacts with a mercapto group of cysteine to form a covalent bond.
(12) A method for purifying fucose-containing sugar chains and / or complex carbohydrates, wherein the adsorbent according to (10) or (11) is used.

  The present invention is described in further detail below.

  In the present invention, the fucose-binding lectin is a lectin having binding properties to a fucose-containing sugar chain and / or complex carbohydrate consisting of the amino acid sequence shown in SEQ ID NO: 1. The protein of the present invention is a protein in which an oligopeptide containing one or more cysteines (hereinafter referred to as a cysteine tag) is added to the N-terminal or C-terminal of a fucose-binding lectin consisting of the amino acid sequence shown in SEQ ID NO: 1. It is characterized by. More specifically, for example, at the C-terminal of the region containing at least the second proline to the 156th glycine of the fucose-binding lectin consisting of the amino acid sequence shown in SEQ ID NO: 1 shown in SEQ ID NO: 1, It is a sequence to which a cysteine tag shown in No. 2 is added.

  The protein of the present invention may contain a function capable of binding to a fucose-containing sugar chain and / or complex carbohydrate and a cysteine capable of reacting with a functional group that reacts with a thiol group introduced into a water-insoluble carrier. For example, in the amino acid sequence shown in SEQ ID NO: 1, a protein or sequence in which one or more amino acids in the region from the second proline to the 156th glycine are deleted, substituted or added No. 31 (introducing a linker consisting of the amino acid sequence shown in SEQ ID NO: 32 into the C-terminal of the 156th glycine from the second proline of the amino acid sequence shown in SEQ ID NO: 1, The amino acid encoding the linker or domain is added before the cysteine tag. Proteins also, to include the function of binding fucose-containing sugar chains and / or glycoconjugates, capable of reacting cysteine with a functional group that reacts with the introduced mercapto group to the carrier, are included in the proteins of the present invention.

  In the present invention, the length of the cysteine tag is in the range of 1 to 50 amino acids as long as the fucose-containing sugar chain and / or the complex carbohydrate possessed by the fucose-binding lectin to which the tag is added is not impaired. It can be set arbitrarily from the inside, and is particularly preferably set in the range of 2 to 20 amino acids. One or more cysteines may be included in the cysteine tag. However, when two or more cysteines are included, it is necessary to design the amino acid sequence of the cysteine tag so that an SS bond is not formed between the cysteines. As the types of amino acids other than cysteine constituting the cysteine tag, considering the physical properties of the water-insoluble carrier for immobilizing the protein of the present invention, an amino acid having physical properties that allow the protein of the present invention to easily approach the carrier is appropriately selected. do it. An example of a cysteine tag in the present invention is an oligopeptide having the amino acid sequence set forth in SEQ ID NO: 2.

The polynucleotide used for producing the fucose binding protein of the present invention by genetic engineering may be a polynucleotide comprising a sequence encoding the fucose binding protein of the present invention. A polynucleotide comprising a sequence encoding the fucose binding protein of the present invention can be prepared by a known method. As an example of the polynucleotide preparation method,
(A) a method of artificially synthesizing a polynucleotide containing the nucleotide sequence by converting the amino acid sequence of the protein of the present invention into a nucleotide sequence,
(B) A polynucleotide encoding a fucose-binding protein among the proteins of the present invention is prepared directly or artificially or from a genomic DNA of Burkholderia cenocepacia using a DNA amplification method such as a PCR method, and then the prepared poly Examples thereof include a method of adding an oligonucleotide encoding the cysteine tag described in SEQ ID NO: 2 to the 5 ′ end side or 3 ′ end side of the nucleotide by a genetic engineering technique.

  In the preparation method, when designing the polynucleotide sequence, it is preferable to design the polynucleotide sequence in consideration of the frequency of codon usage in the host to be transformed, from the viewpoint of efficient protein expression using the host. Further, amplification is performed by PCR using a primer set designed to substitute, delete and / or add amino acids using the polynucleotide prepared in (B) as a template in the preparation method described above. Alternatively, a polynucleotide encoding a fucose-binding protein having one or more amino acid substitutions and / or deletions and / or additions can be prepared by performing amplification using the error-prone PCR method. .

  The polynucleotide comprising the sequence encoding the protein of the present invention obtained by the above method is introduced into a corresponding vector system used in the field of normal genetic engineering to express the protein of the present invention in various hosts. can do. Examples of the host include bacteria, yeasts, molds, insect cells, animal cells, and plant cells. However, the host / vector system for gene manipulation has been improved because of ease of handling, ease of culture, high density culture, and the like. In particular, bacteria, particularly bacteria belonging to the genus Escherichia represented by Escherichia coli are preferable. Examples of Escherichia coli used for transformation include, but are not limited to, strains such as JM109, JM110, HB101, BL21, BL21 (DE3), and BLR (DE3).

  A polynucleotide comprising a sequence encoding the protein of the present invention is transformed into a host (for example, E. coli JM109 or HB101) after preparing a plasmid having the polynucleotide by inserting it into an appropriate plasmid using a restriction enzyme. Thus, a host capable of expressing the protein of the present invention can be prepared. Examples of plasmids for expressing the protein of the present invention include pET-28a (+) (Merck), pTrc99A (GenBank ACCESSION No. U13872), pSTV28 (Takara Bio), pSTV29 (Takara Bio), Examples include pCDF-1b (Merck) and pRSF-1b (Merck).

  When the polynucleotide encoding the protein of the present invention is inserted into the plasmid, it may be inserted into a multicloning site in the plasmid so as not to cause frame shift of the polynucleotide. In addition, the fucose-binding protein of the present invention is encoded by using a plasmid designed so that an oligonucleotide encoding an appropriate amino acid is added to the 5 ′ end side or 3 ′ end side of the inserted polynucleotide. A polynucleotide comprising a sequence having an additional sequence in addition to the sequence to be prepared can also be prepared. As an example, by using the restriction enzyme NcoI site on the multicloning site of plasmid pET-26b (+) (manufactured by Merck), a PelB signal sequence (in addition, an arbitrary amino acid is added to the N-terminal side of the protein of the present invention). A plasmid can be prepared in which a polynucleotide comprising a sequence encoding a protein linked to (which may be added) is inserted. As another example, by using the restriction enzyme EcoRI site of plasmid pTrc99A (GenBank ACCESSION No. U13872), methionine and glutamic acid (an arbitrary amino acid may be further added) to the N-terminal side of the protein of the present invention. A plasmid into which a polynucleotide comprising a sequence encoding a protein to which is added can be prepared.

The method for producing the protein of the present invention comprises:
(A) producing the protein of the present invention by culturing the transformant of the present invention,
(A) recovering the protein of the present invention from the culture obtained in step (A),
These two steps are included. In the present specification, the culture includes not only cells of the cultured transformant itself and cell secretions but also a medium used for culturing the transformant. Details of each step (a) and step (a) will be described below.

(A) Step of producing the protein of the present invention by culturing the transformant of the present invention The transformant used in the protein production method of the present invention can be cultured in a medium suitable for culturing the target host. Well, for example, when the host is Escherichia coli, a terrific broth (TB) medium and a Luria-Bertani (LB) medium supplemented with necessary nutrients are examples of preferable media. In addition to carbon, nitrogen and inorganic salts, nutrient media common in the technical field may be added to the medium.

  In order to selectively grow transformants depending on the presence or absence of introduction of a vector containing the polynucleotide of the present invention, it is preferable to add a drug corresponding to the drug resistance gene contained in the vector to the medium and culture, For example, when the vector contains a kanamycin resistance gene, kanamycin may be added to the medium. Furthermore, a reagent such as glycine may be added to the medium in order to promote the secretion of the protein from the transformant to the culture medium. Specifically, when the host is Escherichia coli, 2 glycines are added to the medium. % (W / v) or less is preferably added.

  The culture temperature is not particularly limited as long as it is a general temperature in the art. For example, when the host is Escherichia coli, it is 10 ° C. to 40 ° C., preferably 25 ° C. to 35 ° C., more preferably around 30 ° C., What is necessary is just to select suitably according to the characteristic of the protein to express. In addition, the pH of the medium may be appropriately selected from a general range in the technical field according to various conditions such as the type of the host. For example, when the host is Escherichia coli, the pH is preferably 6.8 to 7.4. The range is preferred, more preferably around pH 7.0.

  In addition, since the protein of the present invention is expressed without adding an inducing agent depending on the expression vector, an inducing agent is not necessarily required. However, when the inducible promoter is included in the expression vector of the present invention, It is preferable to induce protein expression by adding an inducer under conditions that allow the protein of the present invention to be expressed well. As a preferable inducer, IPTG (isopropyl-β-D-thiogalactopyranoside) can be exemplified. The IPTG addition concentration may be appropriately selected from the range of 0.005 mM to 1.0 mM, and preferably 0.01 mM to 0.5 mM. Various conditions relating to IPTG induction may be performed under conditions well known in the art. For example, when the host is Escherichia coli, the turbidity (absorbance at 600 nm) of the culture solution is about 0.1 to 1.0. The protein expression of the present invention can be induced by culturing after adding an appropriate amount of IPTG.

(A) Step of recovering the protein of the present invention from the culture obtained in step (a) To recover the protein of the present invention from the culture obtained by culturing the transformant of the present invention, However, the method of the present invention may be appropriately selected from the methods usually used, and the extraction of the protein of the present invention from the aforementioned culture may be carried out according to the form of expression. When the protein of the present invention is expressed in cells (including periplasm in prokaryotes), the cells are collected by centrifugation, and then added with an enzyme treatment agent or a surfactant. What is necessary is just to crush and extract the protein of this invention. On the other hand, when secreted into the culture solution, the cells may be separated by centrifugation, and the protein of the present invention may be extracted from the resulting culture supernatant.

  In order to separate and purify the protein of the present invention from the extract obtained by the above-described method, a method known in the art may be used, and examples thereof include separation and purification using liquid chromatography. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, and the like. By performing separation and purification by combining these chromatography, the protein of the present invention can be enhanced. It can be recovered to purity.

  By immobilizing the protein of the present invention on a carrier insoluble in water, an adsorbent for separating and purifying the fucose-containing sugar chain and / or complex carbohydrate of the present invention (hereinafter referred to as the adsorbent of the present invention) is produced. can do. There are no particular limitations on the water-insoluble carrier used in the preparation of the adsorbent of the present invention, and inorganic carriers such as silica gel or glass deposited with a gold thin film, agarose, cellulose, chitin, chitosan, dextran, pullulan, starch, alginic acid. Polysaccharide carriers made from polysaccharides such as salt and carrageenan, crosslinked polysaccharide carriers obtained by crosslinking them with a crosslinking agent, synthetic polymer carriers such as polymethacrylate, polyvinyl alcohol, polyurethane, polystyrene and the like and crosslinked with a crosslinking agent Examples of such crosslinked synthetic polymer-based carriers can be given. Among these carriers, polysaccharide carriers and cross-linked polysaccharide carriers having no charge such as agarose, cellulose, dextran, pullulan, and the like in terms of high hydrophilicity and low non-specific adsorption of substances to be separated and purified. A hydrophilic synthetic polymer carrier such as methacrylate and polyvinyl alcohol and a crosslinked hydrophilic synthetic polymer carrier are preferred. The shape of the carrier insoluble in water and the presence or absence of pores are not particularly limited, and the shape may be any of particulate, sponge, flat membrane, flat plate, hollow, fibrous or non-particulate. The presence or absence of pores may be either porous or nonporous. As the water-insoluble carrier for immobilizing the protein of the present invention, an active functional group for immobilizing the protein of the present invention on the carrier can be easily introduced, and the fucose-containing sugar chain to be separated and purified and / or A particulate carrier having a hydroxyl group is preferred in terms of increasing the amount of complex carbohydrate adsorbed, and a porous particulate carrier is more preferred. Furthermore, the water-insoluble carrier used for preparing the adsorbent of the present invention may be a commercially available product. For example, Toyopearl (manufactured by Tosoh Corporation) using polymethacrylate as a raw material, or Sepharose (GE) using agarose as a raw material. Cellufine (manufactured by JNC) using cellulose as a raw material can be used.

  The above-described method for immobilizing the protein of the present invention on a water-insoluble carrier is not particularly limited as long as it is a general protein immobilization method. For example, it can be shared by using a coordination bond or an affinity bond. A method of immobilizing the protein of the present invention on a carrier without forming a bond, a method of reacting an active functional group for immobilization introduced into the protein of the present invention with a carrier to form a covalent bond and immobilizing it on the carrier, Examples thereof include a method in which an active functional group introduced into a carrier is reacted with the protein of the present invention to form a covalent bond and immobilized on the carrier.

  Since the protein of the present invention has a cysteine tag containing one or more cysteines, the protein of the present invention can be immobilized on a carrier in a position-selective manner by utilizing a selective reaction with the mercapto group of cysteine. it can.

  As a method of immobilizing the protein of the present invention on a carrier without forming a covalent bond, a method of immobilizing the protein of the present invention on a glass on which a gold thin film is deposited using a coordinate bond between a mercapto group and gold And a method of immobilizing a protein of the present invention into which biotin has been introduced on an avidin-immobilized carrier such as streptavidin sepharose high performance (manufactured by GE Healthcare) using affinity binding of avidin-biotin. Can do. Examples of the method for introducing biotin into the protein of the present invention include biotinylation reagents having a maleimide group such as N-biotinyl-N ′-[2- (N-maleimido) ethyl] piperazine hydrochloride and the protein of the present invention. A method of reacting a mercapto group can be exemplified.

  In addition, as a method of reacting an active functional group for immobilization introduced into the protein of the present invention with a carrier to form a covalent bond and immobilizing, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid 3- The maleimide group of a compound having both a maleimide group and an active ester group, such as sulfo-N-hydroxysuccinimide ester sodium salt, and the mercapto group of the protein of the present invention are reacted under neutral conditions around pH 7 to give the protein of the present invention. Examples thereof include a method of reacting with a carrier having an amino group introduced, such as Toyopearl AF-amino-650M (manufactured by Tosoh Corporation) after introducing an active ester group.

  Furthermore, as a method of reacting the active functional group introduced into the carrier with the protein of the present invention to form a covalent bond and immobilize it on the carrier, there are epoxy group, maleimide group, haloacetyl group, haloalkyl group, vinyl introduced into the carrier. Examples include a method of reacting a mercapto group of the protein of the present invention with a functional group that reacts with a mercapto group such as an isocyanate group or an isothiocyanate group to form a covalent bond. Among the functional groups that react with these mercapto groups to form a covalent bond, the protein of the present invention is immobilized on the carrier in the pH range of 6.5 to 7.5, that is, in an aqueous solution having a pH near neutral. An epoxy group, a maleimide group, a haloacetyl group, a haloalkyl group, and a vinyl group are preferable in that the reaction can be performed with high efficiency, and an epoxy group, a maleimide group, and a haloacetyl group are more preferable.

  As a method for introducing an epoxy group into a water-insoluble carrier, for example, a carrier having a hydroxyl group and an epoxy group-containing compound such as epichlorohydrin, ethylene glycol diglycidyl ether, or 1,4-butanediol diglycidyl ether are used. The method of making it react on basic conditions can be mention | raise | lifted.

  Examples of a method for introducing a maleimide group into a water-insoluble carrier include a carrier having a hydroxyl group and / or an amino group, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, 4- (N- And a method of reacting a carboxylic acid having a maleimide group such as maleimidomethyl) cyclohexanecarboxylic acid in the presence of a condensing agent such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC). . Furthermore, the method of making the N-hydroxysuccinimide ester and N-hydroxysulfosuccinimide ester of the carboxylic acid which has the above-mentioned maleimide group react can be illustrated. The carrier having an amino group is prepared, for example, by reacting a carrier having an epoxy group introduced thereinto with a compound having an amino group such as aqueous ammonia, ethylenediamine, diethylenetriamine, or tris (2-aminoethyl) amine. be able to.

  Examples of a method for introducing a haloacetyl group into a water-insoluble carrier include, for example, a carrier having a hydroxyl group, a carrier into which an amino group has been introduced by the above-described method, and an acid halogen such as chloroacetic acid chloride, bromoacetic acid chloride, and bromoacetic acid bromide. And a method of reacting a halogenated acetic acid such as chloroacetic acid, bromoacetic acid and iodoacetic acid in the presence of a condensing agent such as EDC. Furthermore, a method of reacting the aforementioned N-hydroxysuccinimide ester or N-hydroxysulfosuccinimide ester of halogenated acetic acid can be mentioned.

  The protein of the present invention can be immobilized on a water-insoluble carrier by reacting the above-described carrier into which the active functional group has been introduced with the protein of the present invention dissolved in a buffer solution. There is no restriction | limiting in particular in the buffer solution which melt | dissolves the protein of this invention, Acetate buffer solution, phosphate buffer solution, 2-morpholino ethanesulfonic acid (MES) buffer solution, 4- (2-hydroxyethyl) -1-piperazine ethanesulfone Examples thereof include an acid (HEPES) buffer solution and a tris (hydroxymethyl) aminomethane (Tris) buffer solution. In addition, for the purpose of increasing the efficiency of the immobilization reaction, an inorganic salt such as sodium chloride or a surfactant such as polyoxyethylene sorbitan monolaurate (Tween 20) may be added to the buffer solution. The reaction temperature and pH when immobilizing the protein of the present invention on a carrier are 0 ° C. or more and 50 ° C. or less for the reaction temperature in consideration of the reactivity of the active functional group and the stability of the protein of the present invention. May be appropriately set within the range of pH 5 or more and pH 9 or less, and the reaction temperature is preferably 15 ° C. or more and 40 ° C. or less, and the pH is preferably within the range of pH 6 or more and pH 8 or less from the viewpoint of suppressing protein deactivation.

  The amount of the protein of the present invention immobilized on a water-insoluble carrier may be appropriately set in consideration of the concentration and amount of fucose-containing sugar chains and / or complex carbohydrates in the solution for separation and purification, For example, when the carrier is a porous particulate carrier, 0.01 mg to 50 mg per 1 mL carrier is preferable, and 0.05 mg to 30 mg is more preferable. The amount of the protein of the present invention immobilized on the carrier can be adjusted by adjusting the amount of the protein of the present invention used during the immobilization reaction or the amount of active functional group introduced into the carrier. The amount of the protein of the present invention immobilized on the carrier is determined by collecting the immobilized reaction solution and the washing solution after the reaction to determine the amount of the unreacted protein of the present invention, and then using the amount of the protein of the present invention used for the immobilization reaction. It can be calculated by subtracting the amount of unreacted protein of the present invention from

  The fucose-containing sugar chain and / or complex carbohydrate that can be purified by the adsorbent of the present invention is not particularly limited as long as it is a complex carbohydrate such as protein or lipid to which the fucose-containing sugar chain and the fucose-containing sugar chain are bound. A Lewis Y-type sugar chain containing an α1-2-linked fucose and an α1-3-linked fucose, a Lewis b-type sugar chain containing an α1-2-linked fucose and an α1-4-linked fucose, and an α1-2-linked fucose H type 1 sugar chain containing, H type 2 type sugar chain, H type 3 type sugar chain, Lewis X type sugar chain containing α1-3 linked fucose, Lewis a type sugar chain containing α1-4 linked fucose, N-glycoside-linked sugar chains containing fucose α1-6 linked to N-acetylglucosamine at the reducing end, and proteins and lipids to which these fucose-containing sugar chains are bound And if carbohydrate, these fucose-containing sugar chains and / or glycoconjugates can be mentioned cells having the cell surface.

  The method for purifying fucose-containing sugar chains and / or complex carbohydrates using the adsorbent of the present invention is not particularly limited, but is capable of efficiently separating and purifying fucose-containing sugar chains and / or complex carbohydrates. After mixing the adsorbent of the present invention with a solution containing fucose-containing sugar chains and / or complex carbohydrates before separation and purification, the adsorbent of the present invention to which the fucose-containing sugar chains and / or complex carbohydrates are adsorbed is not adsorbed. A solution containing fucose-containing sugar chains and / or complex carbohydrates before separation and purification is passed through a chromatography column packed with the adsorbent of the present invention to separate components, and fucose-containing sugar chains and / or complexes A method of adsorbing a carbohydrate to the adsorbent of the present invention is preferred. Moreover, what is necessary is just to set the usage-amount of the adsorption agent of this invention used for isolation | separation refinement | purification of a fucose containing sugar chain and / or complex carbohydrate according to a processing amount.

  The adsorption of fucose-containing sugar chains and / or complex carbohydrates to the adsorbent of the present invention is carried out by, for example, adsorbing agent equilibrated with a buffer solution having a pH of 5 to 9, preferably 6 to 8, and fucose before separation and purification. What is necessary is just to make the solution containing a containing sugar chain and / or complex carbohydrate contact. There is no particular limitation on the buffer used for the adsorption of the fucose-containing sugar chain and / or complex carbohydrate to the adsorbent of the present invention, and an acetate buffer, a phosphate buffer, a 2-morpholinoethanesulfonic acid (MES) buffer, Examples thereof include 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer and tris (hydroxymethyl) aminomethane (Tris) buffer. Further, an inorganic salt such as sodium chloride or a surfactant such as polyoxyethylene sorbitan monolaurate (Tween 20) may be added to the buffer solution.

  The fucose-containing sugar chain and / or complex carbohydrate adsorbed on the adsorbent of the present invention can be desorbed by contacting with a buffer containing L-fucose. As the buffer solution containing L-fucose, for example, the buffer solution used for adsorption of the sugar chain and / or complex carbohydrate containing fucose is 0.1 mM to 1 M, preferably 1 mM to 100 mM L-. A buffer solution to which fucose has been added can be mentioned.

  When it is desired to obtain fucose-containing sugar chains and / or complex carbohydrates from a solution containing fucose-containing sugar chains and / or complex sugars before separation and purification, the above-described adsorption and desorption operations are performed. Fucose-containing sugar chains and / or complex carbohydrates can be obtained. On the other hand, when it is desired to remove the fucose-containing sugar chains and / or complex carbohydrates from the solution containing the fucose-containing sugar chains and / or complex sugars before separation and purification, only the above-described adsorption operation is performed. A solution from which the fucose-containing sugar chains and / or complex carbohydrates are removed can be obtained.

  The protein of the present invention is a protein having a cysteine tag containing one or more cysteines at the N-terminus or C-terminus of the fucose-binding lectin, and the above-mentioned fucose-binding lectin can be immobilized on a carrier in a position-selective manner. . Further, by using an adsorbent for fucose-containing sugar chains and / or complex carbohydrates in which the protein of the present invention is immobilized on a water-insoluble carrier, fucose-containing sugar chains and / or complex carbohydrates, fucose on the cell surface Purification and removal of cells having a sugar chain can be performed.

  In addition, the adsorbent of the present invention has the above-described fucose-binding lectin immobilized on a carrier in a position-selective manner, and is capable of binding fucose-containing glycans and / or complex carbohydrates. Since the ratio is improved, fucose-containing sugar chains and / or complex carbohydrates and fucose on the cell surface are compared to the adsorbent obtained by immobilizing fucose-binding lectin without cysteine tag attached to the carrier. The amount of adsorption of cells having a sugar chain can be improved.

The result of the sugar chain affinity evaluation of the protein of the present invention is shown.

EXAMPLES Hereinafter, although an Example, a comparative example, and a reference example are given and this invention is demonstrated further in detail, this invention is not limited to these.
Example 1 Preparation of Plasmid pET-BC2LCNcys A polynucleotide of SEQ ID NO: 4 encoding the amino acid sequence of fucose-binding lectin shown in SEQ ID NO: 3 was prepared by the methods described in (1) to (5) below.

(1) A first-stage PCR reaction was performed with the following reagent composition and reaction conditions.
(Reagent composition, total reaction volume: 50 μL)
・ 0.025 unit / μL PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.)
・ 30 nM each of the primers shown in SEQ ID NOs: 5 to 28 and buffer attached to the enzyme (reaction conditions)
Using a thermal cycler, PCR was carried out for 5 cycles of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 60 seconds.

(2) Next, using the reaction solution of (1), a second-stage PCR reaction was performed with the following reagent composition and reaction conditions.
(Reagent composition, total reaction volume: 50 μL)
・ 0.025unit / μL PrimeSTAR HS DNA Polymerase
(Takara Bio)
・ 500 nM each of the primers shown in SEQ ID NOs: 29 and 30 ・ 1 μL First stage PCR reaction solution ・ Buffer attached to the enzyme (reaction conditions)
Using a thermal cycler, PCR was carried out for 30 cycles of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, 72 ° C. for 60 seconds.

  (3) After electrophoresis of the reaction solution of (2) after the second-stage PCR reaction by agarose gel electrophoresis, a 0.5 kbp band corresponding to the target product was cut out to purify the PCR product.

  (4) The PCR product obtained in the above (3) is double-digested with restriction enzymes NcoI and XhoI, and the digested DNA is transferred to the restriction enzyme NcoI-XhoI site of plasmid pET-28 (+). Kit ver. The plasmid was prepared by ligation using 2.1 (manufactured by Takara Bio Inc.) and then transformed into E. coli BL21 (DE3) to obtain recombinant E. coli BL21 (DE3) / pET-BC2LCNcys.

  (5) The recombinant E. coli BL21 (DE3) / pET-BC2LCNcys obtained in (4) above is cultured and collected, and then the plasmid pET-BC2LCNcys (5) using QIAprep Spin Miniprep Kit (manufactured by Qiagen). 0.6 kbp) was obtained. As a result of analyzing the base sequence encoding the fucose-binding lectin inserted in the plasmid pET-BC2LCNcys obtained by this method, it was confirmed that the base sequence was as designed.

Example 2 Production of a fucose-binding lectin to which a cysteine tag was added From the recombinant Escherichia coli BL21 (DE3) / pET-BC2LCNcys obtained in Example 1, the cysteine tag was prepared by the method described in (1) to (5) below. An added fucose-binding lectin was prepared.
(1) Recombinant E. coli BL21 (DE3) / pET-BC2LCNcys was inoculated into an LB / Km liquid medium supplemented with 30 μg / mL kanamycin, and precultured by shaking overnight at 37 ° C. After inoculation so that the turbidity (OD600) of the culture solution was 0.6, it was cultured at 37 ° C.
(2) The preculture was inoculated into 1 L of LB / Km liquid medium supplemented with 30 μg / mL kanamycin and cultured at 37 ° C. with shaking. When the turbidity (OD600) of the culture solution reached approximately 0.6, the culture temperature was switched to 20 ° C. and the culture was performed overnight.
(3) After completion of the culture, the culture solution was ice-cooled and then collected by centrifugation. The collected bacteria were treated with BugBuster Protein Extraction Reagent (Merck) supplemented with 20 mM Tris-HCl buffer (pH 8.0) and 1 mM phenylmethylsulfonyl fluoride (PMSF) to obtain 150 mL of a soluble fraction. .

(4) A carrier (His Bind Resin) obtained by equilibrating the solution of the soluble fraction obtained in the above (3) with a 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM sodium chloride and 20 mM imidazole. The solution was passed through a column packed with 15 mL of a carrier volume manufactured by Merck, and a fucose-binding lectin added with a cysteine tag contained in the soluble fraction was adsorbed. The fucose-binding lectin added with a cysteine tag adsorbed on a carrier is eluted with a 20 mM Tris-HCl buffer solution (pH 8.0) containing 150 mM sodium chloride and 250 mM imidazole, so that the fucose-binding property added with the desired cysteine tag is added. 50 mL of eluate containing lectin was obtained.
(5) The target cysteine is obtained by dialyzing the eluate containing the fucose-binding lectin added with the cysteine tag obtained in the above (4) against D-PBS (-) (manufactured by Wako Pure Chemical Industries). 75 mL of a D-PBS (-) solution of fucose-binding lectin with a tag added thereto was obtained.

The concentration of fucose-binding lectin added with a cysteine tag in the obtained D-PBS (−) solution was measured by an ultraviolet absorption method, and the concentration of fucose-binding lectin added with a cysteine tag when the absorbance at 280 nm was 1.0. As a result of calculating the concentration with 1.0 mg / mL, the concentration was 1.2 mg / mL.
Example 3 Affinity evaluation of fucose-binding lectin added with a cysteine tag to a sugar chain In order to evaluate the affinity of a fucose-binding lectin added with a cysteine tag obtained in Example 2 to a fucose-containing sugar chain, Surface plasmon resonance (SPR) measurement using BIACORE T100 (manufactured by GE Healthcare) was performed. CM5 sensor chip in which streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) is immobilized using Lewis Y-type sugar chains containing α1-2 linked fucose and α1-3 linked fucose as sugar chains for evaluating affinity A biotinylated Lewis Y sugar chain (Ley-PAA-biotin, manufactured by Glycotech) was immobilized on (manufactured by GE Healthcare) using a biotin-avidin bond. The structure of Lewis Y-type sugar chain is shown below.

Lewis Y-type sugar chain Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc
The fucose-binding lectin to which a cysteine tag was added was diluted with HBS-EP + buffer (manufactured by GE Healthcare), and kinetic analysis was performed at 12 concentrations ranging from 0 to 1013 nM. SPR measurement was performed by injecting a fucose-binding lectin solution added with a cysteine tag at each concentration for 6 minutes at a flow rate of 30 μL / min, and a fucose-binding lectin with a cysteine tag added to a sensor chip on which Lewis Y sugar chains were immobilized. Were combined. Dissociation of the fucose-binding lectin bound to the sensor chip from the sensor chip was performed by injecting a 10 mM aqueous sodium hydroxide solution at a flow rate of 30 μL / min for 30 seconds. As a result of analyzing the obtained results with the software (version 1.1.1) attached to BIACORE T100, the dissociation constant (Kd value) of the fucose-binding lectin added with a cysteine tag with respect to the Lewis Y sugar chain is 46 nM. there were.

FIG. 1 shows a sensorgram of SPR measurement using BIACORE T100 performed in Example 3.
Example 4 Immobilization of fucose-binding lectin having a cysteine tag added to a carrier into which an epoxy group was introduced and evaluation as an adsorbent (part 1)
In Examples 4 to 9 below, preparation of an adsorbent for fucose-containing sugar chains and / or complex carbohydrates by immobilizing a fucose-binding lectin having a cysteine tag attached to a carrier, and fucose-containing sugar chains and The present invention relates to evaluation as an adsorbent using a sugar chain not containing fucose.

  In the following examples, comparative examples, and reference examples, fucose-containing sugar chains (PA-Sugar chain 046, Leb-hexacaccharide, manufactured by Takara Bio Inc.), which are fluorescently labeled with 2-aminopyridine as a fucose-containing sugar chain, are described below. , Referred to as fucose-containing sugar chain). Further, as a sugar chain not containing fucose, an oligomannose type sugar chain (PA-Sugar chain 016, M3B, manufactured by Takara Bio Inc., hereinafter referred to as an oligomannose type sugar chain) whose reducing end is fluorescently labeled with 2-aminopyridine is used. Using. The structures of fucose-containing sugar chains and oligomannose type sugar chains are shown below. The reducing terminal 2-aminopyridine was omitted.

Fucose-containing sugar chain Fucα1-2Galβ1-3 (Fucα1-4) GlcNAcβ1-3Galβ1-4Glc
Oligomannose type sugar chain Manα1-3 (Manα1-6) Manβ1-4GlcNAcβ1-4GlcNAc
Example 4 relates to immobilization of a fucose-binding lectin having a cysteine tag added to a synthetic polymer carrier into which an epoxy group has been introduced, and evaluation as an adsorbent.
(1) Production of Synthetic Polymer Carrier Introduced With Epoxy Group As a synthetic polymer carrier, Toyopearl HW-65F (manufactured by Tosoh Corporation) suspended in water was used after suction filtration with a glass filter. In a reaction vessel (PFA bottle, 100 mL volume, manufactured by ASONE), water-containing Toyopearl HW-65F (10.0 g), 1,4-butanediol diglycidyl ether (Aldrich, 2.0 g), water ( 15.0 g) was added, and the mixture was stirred at 50 ° C. for 30 minutes, followed by addition of 48% aqueous sodium hydroxide (manufactured by Kanto Chemical Co., Inc., 280 mg) and further stirring at 50 ° C. for 8 hours to carry out the epoxidation reaction It was. After the reaction, a glass filter was used to wash with a large amount of water until the filtrate became neutral, thereby obtaining Toyopearl HW-65F (hereinafter, epoxidized Toyopearl HW-65F) into which an epoxy group was introduced.

(2) Immobilization of fucose-binding lectin with a cysteine tag attached to a synthetic polymer carrier into which an epoxy group has been introduced (Preparation of adsorbent 1)
50% by volume suspension (200 μL) prepared by adding water to the epoxidized Toyopearl HW-65F prepared in (1) was added to a reaction vessel (mini bio spin chromatography column, manufactured by BIO-RAD). , Sodium dihydrogen phosphate dihydrate (manufactured by Wako Pure Chemical Industries, Ltd.) and phosphoric acid, including 500 mM sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) and 20 mM ethylenediaminetetraacetic acid (EDTA, manufactured by Dojin Chemical Laboratory Co., Ltd.) The plate was washed 5 times with 200 mM phosphate buffer (pH 7.4, 200 μL) prepared from disodium hydrogen (manufactured by Wako Pure Chemical Industries, Ltd.). The 50% by volume suspension of epoxidized Toyopearl HW-65F is prepared by allowing epoxidized Toyopearl HW-65F suspended in water to settle in a graduated cylinder and leaving it until the volume is constant by tapping occasionally. It was prepared by adding water so that the volume of epoxidized Toyopearl HW-65F was 50%.

  Next, the amount of fucose-binding lectin charged with a cysteine tag per mL of carrier in a reaction vessel containing epoxidized Toyopearl HW-65F (100 μL) was adjusted to 2.24 mg, and the cysteine prepared in Example 2 described above. By adding a D-PBS (−) solution (concentration 2.80 mg / mL, 80 μL) of a fucose-binding lectin to which a tag has been added, and stirring at 40 ° C. for 15 hours, A fucose-binding lectin with a cysteine tag was immobilized on epoxidized Toyopearl HW-65F. After the immobilization reaction, the carrier is washed with D-PBS (-) (manufactured by Wako Pure Chemical Industries, Ltd.) containing 0.05% by weight of Tween 20 (manufactured by Sigma) to thereby remove the fucose-binding lectin added with a cysteine tag. An adsorbent 1 for fucose-containing sugar chains and / or complex carbohydrates immobilized on epoxidized Toyopearl HW-65F was obtained.

  The amount of fucose-binding lectin added with a cysteine tag in the immobilized reaction solution and the collected washing solution was measured by SPR measurement using CM5 sensor chip on which Lewis Y-type sugar chain was immobilized and BIACORE T100 described in Example 3. By calculating and subtracting the amount of fucose-binding lectin added with unreacted cysteine tag contained in the washing solution after the immobilized reaction from the amount of fucose-binding lectin added with the cysteine tag used for the immobilization reaction, As a result of calculating the immobilization amount and the immobilization rate of the fucose-binding lectin to the adsorbent 1, the immobilization amount was 0.92 mg per mL of the carrier, and the immobilization rate was 41.1%.

(3) Measurement of adsorption rate of fucose-containing sugar chain to adsorbent 1 25 vol% suspension (100 μL) prepared by adding D-PBS (−) to adsorbent 1 prepared in (2) It added to the reaction container (BIO-RAD company make, a mini bio spin chromatography column), and it wash | cleaned 4 times by D-PBS (-) (100 microliters). In addition, the 25 volume% suspension of adsorbent 1E was prepared by adding D-PBS (-) (300 microliters) to the reaction container containing the adsorbent 1 (100 microliters) produced by (2).

  Next, a D-PBS (−) solution of fucose-containing sugar chains (concentration 500 pmol / mL, 50 μL, addition amount 25 pmol) is added to the reaction vessel containing the adsorbent 1 (25 μL), and stirred at 25 ° C. for 2 hours. By doing so, the fucose-containing sugar chain was adsorbed to the adsorbent 1. The adsorbent on which the fucose-containing sugar chain was adsorbed was washed with D-PBS (−), and the entire amount of the washing solution was recovered.

Included in the amount of fucose-containing glycans used in the adsorption treatment and the washing solution by measuring the fluorescence intensity derived from the fluorescently labeled fucose-containing glycans using a microplate reader (infinite M200, TECAN, Exit 320 nm, Emission 400 nm) After the amount of fucose-containing sugar chains was determined, the adsorption rate of the fucose-containing sugar chains to the adsorbent 1 was calculated from the formula shown below. As a result, the adsorption rate was 88%.
Adsorption rate = ((fucose-containing sugar chain amount used in the adsorption treatment−fucose-containing sugar chain amount contained in the washing solution) / fucose-containing sugar chain amount used in the adsorption treatment) × 100
(4) Measurement of recovery rate of fucose-containing sugar chain from adsorbent 1 adsorbed with fucose-containing sugar chain (3) In a reaction vessel containing adsorbent 1 (25 μL) to which fucose-containing sugar chain was adsorbed, -Adsorbed to the adsorbent 1 by adding D-PBS (-) solution (concentration 15 mM, 50 μL, L-fucose addition amount 750 nmol) of fucose (manufactured by Wako Pure Chemical Industries, Ltd.) and stirring for 1 hour at 25 ° C. The fucose-containing sugar chain was desorbed. The adsorbent from which the fucose-containing sugar chain was desorbed was washed with D-PBS (−), and the entire amount of the washing solution was recovered.

  In the same manner as in the operation (3) described above, the amount of fucose-containing sugar chains desorbed from the adsorbent 1 was obtained using a microplate reader, and then the fucose-containing sugar chains from the adsorbent 1 were calculated from the formula shown below. As a result of calculating the recovery rate, the recovery rate was 82%.

Recovery rate = (amount of desorbed fucose-containing sugar chain / amount of fucose-containing sugar chain used for adsorption treatment) × 100
(5) Measurement of adsorption rate of fucose-containing sugar chain to synthetic polymer carrier into which epoxy group is introduced 50% by volume of epoxidized Toyopearl HW-65F prepared in (1) by the method described in (2) The suspension (200 μL) was added to the reaction vessel, and washed 5 times with 200 mM phosphate buffer (pH 7.4, 200 μL) containing 500 mM sodium chloride and 20 mM ethylenediaminetetraacetic acid (EDTA).

  Next, in a reaction vessel containing epoxidized Toyopearl HW-65F (100 μL), α-thioglycerin / D-PBS (−) prepared from α-thioglycerin (manufactured by Tokyo Chemical Industry Co., Ltd.) and D-PBS (−). The solution (concentration 10 mg / mL, 100 μL) was added, and the mixture was stirred at 40 ° C. for 15 hours to react epoxidized Toyopearl HW-65F with α-thioglycerin. After completion of the reaction, the carrier was washed with D-PBS (-) containing 0.05% by weight of Tween 20 to obtain an adsorbent 1B in which the epoxy group was blocked with α-thioglycerin.

  As a result of calculating the adsorption rate of the fucose-containing sugar chain to the adsorbent 1B by the method described in (3), the adsorption rate was 5%. Therefore, it was revealed that the fucose-containing sugar chain is not adsorbed to the adsorbent 1B in which the epoxy group introduced into the carrier is blocked with α-thioglycerin.

(6) Measurement of adsorption rate of oligomannose-type sugar chain to adsorbent 1 According to the method described in (3), 25 vol% suspension (100 μL) of adsorbent 1 was added to the reaction vessel, and D-PBS Washed 4 times with (-) (100 μL). Next, a D-PBS (−) solution of oligomannose type sugar chain (concentration 500 pmol / mL, 50 μL, addition amount 25 pmol) is added to the reaction vessel containing the adsorbent 1 (25 μL), and the mixture is added at 25 ° C. for 60 minutes. By stirring, the oligomannose type sugar chain was adsorbed to the adsorbent 1. The adsorbent adsorbing the oligomannose-type sugar chain was washed with D-PBS (−), and the entire amount of the washing solution was recovered.

  By measuring the fluorescence intensity derived from the oligomannose sugar chain by the method described in (3), the amount of oligomannose sugar chain used in the adsorption treatment and the amount of oligomannose sugar chain contained in the washing solution were determined. Then, as a result of calculating the adsorption rate of the oligomannose type sugar chain to the adsorbent 1, the adsorption rate was 6%. Therefore, it was revealed that the adsorbent 1 does not adsorb oligomannose type sugar chains not containing fucose.

Example 5 Immobilization of fucose-binding lectin having a cysteine tag added to a carrier into which an epoxy group was introduced and evaluation as an adsorbent (part 2)
Example 5 relates to immobilization of a fucose-binding lectin having a cysteine tag added to a polysaccharide-based carrier into which an epoxy group has been introduced, and evaluation as an adsorbent.

(1) Preparation of a polysaccharide carrier into which an epoxy group has been introduced As a polysaccharide carrier, a product obtained by subjecting Sepharose 4 Fast Flow (manufactured by GE Healthcare, hereinafter referred to as Sepharose 4FF) suspended in water to suction filtration with a glass filter is used. Then, Sepharose 4FF into which an epoxy group was introduced (hereinafter referred to as epoxidized Sepharose 4FF) was obtained by the method described in Example 4 (1).
(2) Immobilization of fucose-binding lectin with a cysteine tag attached to a polysaccharide carrier into which an epoxy group has been introduced (Preparation of adsorbent 2)
Adsorbent 2 for fucose-containing sugar chains and / or complex carbohydrates, in which a fucose-binding lectin having a cysteine tag added to the epoxidized Sepharose 4FF produced in (1) was immobilized by the method described in (2) of Example 4. Got. Similarly, the amount of fucose-binding lectin added with a cysteine tag to adsorbent 2 was calculated by the method described in Example 4 (2). As a result, the amount of immobilization was 1.13 mg per mL of carrier. The rate was 50.4%.

(3) Measurement of the adsorption rate of fucose-containing sugar chains on the adsorbent 2 The adsorption rate of fucose-containing sugar chains on the adsorbent 2 prepared in (2) was calculated by the method described in (4) of Example 4. As a result, the adsorption rate was 90%.
(4) Measurement of recovery rate of fucose-containing sugar chain from adsorbent 2 adsorbed with fucose-containing sugar chain Adsorption by which fucose-containing sugar chain was adsorbed in (3) by the method described in (4) of Example 4 As a result of calculating the recovery rate of fucose-containing sugar chains from Agent 2, the recovery rate was 84%.
(5) Measurement of adsorption rate of fucose-containing sugar chain to polysaccharide-based carrier into which epoxy group has been introduced The epoxy group was converted from the epoxidized Sepharose 4FF prepared in (1) by the method described in (4) of Example 4. -Adsorbent 2B blocked with thioglycerin was obtained. As a result of calculating the adsorption rate of the fucose-containing sugar chain to the adsorbent 2B by the method described in Example 4 (3), the adsorption rate was 3%. Therefore, it was revealed that the fucose-containing sugar chain is not adsorbed on the adsorbent 2B.

(6) Measurement of adsorption rate of oligomannose type sugar chain to adsorbent 2 Adsorption rate of oligomannose type sugar chain to adsorbent 2 prepared in (2) by the method described in (4) of Example 4 As a result, the adsorption rate was 5%. Therefore, it was revealed that the adsorbent 2 does not adsorb oligomannose type sugar chains.

Comparative Example 1 Immobilization of fucose-binding lectin having no cysteine tag added to a carrier into which an epoxy group was introduced and evaluation as an adsorbent (Part 1)
Comparative Example 1 relates to immobilization of a fucose-binding lectin not added with a cysteine tag to a synthetic polymer carrier into which an epoxy group has been introduced, and evaluation as an adsorbent.
A commercially available fucose-binding lectin (AiLecS1, Sum, which has binding properties to H-type 1 type sugar chains and H-type 3 type sugar chains containing fucose linked with α1-2, as a fucose-binding lectin not added with a cysteine tag. A product made by Kojun Pharmaceutical Co., Ltd., hereinafter referred to as commercially available fucose-binding lectin) was used. The structures of H type 1 type sugar chain and H type 3 type sugar chain are shown below.

H type 1 sugar chain Fucα1-2Galβ1-3GlcNAc
H type 3 sugar chain Fucα1-2Galβ1-3GalNAc
In Comparative Example 1, the commercially available fucose-binding lectin was immobilized on a synthetic polymer carrier into which an epoxy group was introduced at a pH of 7.4 in the same manner as in Example 1.

(1) Immobilization of a commercially available fucose-binding lectin on a synthetic polymer carrier into which an epoxy group has been introduced (Preparation of adsorbent 3)
A 50% by volume suspension (200 μL) of epoxidized Toyopearl HW-65F prepared in (1) of Example 4 was added to the reaction vessel, and 200 mM containing 500 mM sodium chloride and 20 mM ethylenediaminetetraacetic acid (EDTA) was added. Washed 5 times with phosphate buffer (pH 7.4, 200 μL).

  Next, in a reaction vessel containing epoxidized Toyopearl HW-65F (100 μL), the amount of commercial fucose-binding lectin charged per mL of carrier was 2.24 mg, and the above-mentioned commercial fucose-binding lectin D-PBS (− ) The solution (concentration 1.60 mg / mL, 140 μL) was added and stirred at 40 ° C. for 15 hours to immobilize the commercially available fucose-binding lectin on epoxidized Toyopearl HW-65F. After the immobilization reaction, the carrier is washed with D-PBS (-) containing 0.05% by weight of Tween 20, whereby a commercially available fucose-binding lectin is immobilized on epoxidized Toyopearl HW-65F, and / or Alternatively, complex carbohydrate adsorbent 3 was obtained.

  The amount of commercial fucose-binding lectin immobilized on the adsorbent 3 was calculated by the method described in Example 4 (2). As a result, the amount of commercial fucose-binding lectin used in the reaction and the unreacted commercial fucose binding Since the amount of the sex lectin was the same, it was revealed that the commercially available fucose-binding lectin was not immobilized on the epoxidized Toyopearl HW-65F. Therefore, the adsorption rate of fucose-containing sugar chains and oligomannose type sugar chains to the adsorbent 3 was not measured.

Comparative Example 2 Immobilization of fucose-binding lectin having no cysteine tag added to a carrier into which an epoxy group was introduced and evaluation as an adsorbent (part 2)
In Comparative Example 2, the commercially available fucose-binding lectin was immobilized on a synthetic polymer-based carrier into which an epoxy group was introduced at pH 8.4 and then evaluated as an adsorbent.
(1) Immobilization of a commercially available fucose-binding lectin on a synthetic polymer carrier into which an epoxy group has been introduced (Preparation of adsorbent 4)
A 50% by volume suspension (200 μL) of epoxidized Toyopearl HW-65F prepared in (1) of Example 4 was added to the reaction vessel, and 200 mM containing 500 mM sodium chloride and 20 mM ethylenediaminetetraacetic acid (EDTA) was added. Washed 5 times with Tris-HCl buffer (pH 8.4, 200 μL).
Next, in a reaction vessel containing epoxidized Toyopearl HW-65F (100 μL), the amount of commercial fucose-binding lectin charged per mL of carrier was 2.24 mg, and the above-mentioned commercial fucose-binding lectin D-PBS (− ) The solution (concentration 1.60 mg / mL, 140 μL) was added and stirred at 40 ° C. for 15 hours to immobilize the commercially available fucose-binding lectin on epoxidized Toyopearl HW-65F. After the immobilization reaction, the carrier is washed with D-PBS (-) containing 0.05% by weight of Tween 20, whereby a commercially available fucose-binding lectin is immobilized on epoxidized Toyopearl HW-65F, and / or Alternatively, complex carbohydrate adsorbent 4 was obtained. As a result of calculating the immobilization amount and immobilization rate of the commercially available fucose-binding lectin to the adsorbent 4 by the method described in Example 4 (2), the immobilization amount was 0.53 mg per mL of carrier, and the immobilization rate. Was 23.8%.

(2) Measurement of the adsorption rate of fucose-containing sugar chains on the adsorbent 4 The adsorption rate of fucose-containing sugar chains on the adsorbent 4 prepared in (1) was calculated by the method described in (4) of Example 4. As a result, the adsorption rate was 15%. Therefore, it became clear that the adsorption rate of the fucose-containing sugar chain to the adsorbent 4 is low. The reason why the adsorption rate of the adsorbent 4 was low was considered to be the inactivation of the commercially available fucose-binding lectin during the immobilization reaction on the carrier.

(3) Measurement of recovery rate of fucose-containing sugar chain from adsorbent 4 adsorbed with fucose-containing sugar chain Adsorption by which fucose-containing sugar chain was adsorbed in (2) by the method described in Example 4 (4) As a result of calculating the recovery rate of the fucose-containing sugar chain from the agent 4, the recovery rate was 5%.
(4) Measurement of adsorption rate of oligomannose type sugar chain to adsorbent 4 Adsorption rate of oligomannose type sugar chain to adsorbent 4 prepared in (1) by the method described in (4) of Example 4 As a result, the adsorption rate was 8%. Therefore, it was revealed that the adsorbent 4 does not adsorb oligomannose type sugar chains.

Example 6 Immobilization of fucose-binding lectin having a cysteine tag added to a carrier into which a maleimide group was introduced and evaluation as an adsorbent (part 1)
Example 6 relates to immobilization of a fucose-binding lectin having a cysteine tag added to a synthetic polymer carrier into which a maleimide group has been introduced, and evaluation as an adsorbent.

(1) Production of Synthetic Polymer Carrier Introduced with Maleimide Group As a synthetic polymer carrier, Toyopearl HW-65F (manufactured by Tosoh Corp.) suspended in water and suction filtered with a glass filter was used. Epoxidized Toyopearl HW-65F was obtained by the method described in 4 (1).

Next, the obtained epoxidized Toyopearl HW-65F (2.0 g), ethylenediamine (manufactured by Tokyo Chemical Industry Co., Ltd.) and 0.5 M ethylenediamine aqueous solution (4.0 mL) prepared from water were mixed and mixed at 40 ° C. for 15 The amination reaction was carried out by stirring for a period of time. After the reaction, aminated Toyopearl HW-65F was obtained by washing with a large amount of water until the filtrate became neutral on the glass filter.
Next, the obtained aminated Toyopearl HW-65F (2.0 g), dimethyl sulfoxide (manufactured by Kanto Chemical Co., 2.0 mL, hereinafter referred to as DMSO), 3-maleimidopropionic acid N-succinimidyl (manufactured by Tokyo Chemical Industry Co., Ltd.) and A maleimidation reaction was performed by mixing a DMSO solution of N-succinimidyl 3-maleimidopropionate prepared from DMSO (concentration 10 mg / mL, 2.0 mL) and stirring at 35 ° C. for 4 hours. After the reaction, a glass filter was used to sequentially wash with DMSO and water to obtain Toyopearl HW-65F into which a maleimide group was introduced (hereinafter referred to as maleimidated Toyopearl HW-65F).

(2) Immobilization of a fucose-binding lectin with a cysteine tag attached to a synthetic polymer carrier into which a maleimide group has been introduced (Preparation of adsorbent 5)
A 50% by volume suspension (200 μL) of maleimidized Toyopearl HW-65F prepared in (1) is added to a reaction vessel, and a 200 mM phosphate buffer containing 500 mM sodium chloride and 20 mM ethylenediaminetetraacetic acid (EDTA). Washed 5 times (pH 7.4, 200 μL).
Next, the amount of fucose-binding lectin added with a cysteine tag per mL of carrier in a reaction vessel containing maleimidized Toyopearl HW-65F (100 μL) was 2.24 mg, and the cysteine prepared in Example 2 described above. By adding a D-PBS (−) solution (concentration 2.80 mg / mL, 80 μL) of a fucose-binding lectin to which a tag has been added, and stirring at 35 ° C. for 4 hours, A fucose-binding lectin to which a cysteine tag was added was immobilized on maleimidized Toyopearl HW-65F. After the immobilization reaction, the carrier is washed with D-PBS (-) containing 0.05% by weight of Tween 20, so that fucose-binding lectin with a cysteine tag is immobilized on maleimidized Toyopearl HW-65F. An adsorbent 5 for sugar chains and / or complex carbohydrates was obtained.

As a result of calculating the immobilization amount and the immobilization rate of the fucose-binding lectin to which the cysteine tag was added to the adsorbent 5 by the method described in Example 4 (2), the immobilization amount was 1.07 mg per mL of the carrier. The immobilization rate was 47.6%.
(3) Measurement of adsorption rate of fucose-containing sugar chain to adsorbent 5 By the method described in Example 3, (3), the adsorption rate of fucose-containing sugar chain to adsorbent 5 prepared in (2) was calculated. As a result, the adsorption rate was 90%.
(4) Measurement of recovery rate of fucose-containing sugar chain from adsorbent 5 adsorbed with fucose-containing sugar chain Adsorption by which fucose-containing sugar chain was adsorbed in (3) by the method described in (4) of Example 4 As a result of calculating the recovery rate of the fucose-containing sugar chain from Agent 5, the recovery rate was 88%.
(5) Measurement of adsorption rate of fucose-containing sugar chain on polysaccharide carrier into which maleimide group has been introduced 50% by volume suspension of maleimidized Toyopearl HW-65F prepared in (1) by the method described in (2) (200 μL) was added to the reaction vessel and washed 5 times with 200 mM phosphate buffer (pH 7.4, 200 μL) containing 500 mM sodium chloride and 20 mM ethylenediaminetetraacetic acid (EDTA).

Next, α-thioglycerin / D-PBS (−) prepared from α-thioglycerin (manufactured by Tokyo Chemical Industry Co., Ltd.) and D-PBS (−) in a reaction vessel containing maleimidized Toyopearl HW-65F (100 μL). A solution (concentration 10 mg / mL, 100 μL) was added, and the mixture was stirred at 35 ° C. for 4 hours to react maleimidated Toyopearl HW-65F with α-thioglycerin. After completion of the reaction, the carrier was washed with D-PBS (-) containing 0.05% by weight of Tween 20 to obtain an adsorbent 5B in which the maleimide group was blocked with α-thioglycerin.
As a result of calculating the adsorption rate of the fucose-containing sugar chain to the adsorbent 5B by the method described in (3), the adsorption rate was 5%. Therefore, it was revealed that the fucose-containing sugar chain is not adsorbed on the adsorbent 5B.

(6) Measurement of adsorption rate of oligomannose type sugar chain to adsorbent 5 Adsorption rate of oligomannose type sugar chain to adsorbent 5 prepared in (2) by the method described in (6) of Example 4 As a result, the adsorption rate was 6%. Therefore, it became clear that the adsorbent 5 does not adsorb oligomannose type sugar chains.

Example 7 Immobilization of a fucose-binding lectin having a cysteine tag added to a carrier into which a maleimide group was introduced and evaluation as an adsorbent (part 2)
Example 7 relates to immobilization of a fucose-binding lectin having a cysteine tag added to a polysaccharide carrier into which a maleimide group has been introduced, and evaluation as an adsorbent.

(1) Production of a polysaccharide carrier having a maleimide group introduced As a polysaccharide carrier, a product obtained by subjecting Sepharose 4FF suspended in water to suction filtration with a glass filter was used, and maleimide was obtained by the method described in (1) of Example 6. Sepharose 4FF into which a group was introduced (hereinafter referred to as maleimidated Sepharose 4FF) was obtained.
(2) Immobilization of a fucose-binding lectin with a cysteine tag attached to a polysaccharide carrier into which a maleimide group has been introduced (preparation of adsorbent 6)
By the method described in Example 6 (1), the fucose-containing sugar chain and / or complex carbohydrate adsorbent 6 in which the fucose-binding lectin having a cysteine tag added to the maleimidated Sepharose 4FF prepared in (1) is immobilized. Got. The amount of fucose-binding lectin added with a cysteine tag to the adsorbent 6 was calculated by the method described in Example 4 (2). As a result, the amount of immobilization was 1.18 mg per mL of carrier, the immobilization rate. Was 52.5%.

(3) Measurement of the adsorption rate of fucose-containing sugar chains on the adsorbent 6 The adsorption rate of fucose-containing sugar chains on the adsorbent 6 prepared in (2) was calculated by the method described in (4) of Example 4. As a result, the adsorption rate was 95%.
(4) Measurement of recovery rate of fucose-containing sugar chain from adsorbent 6 adsorbed with fucose-containing sugar chain Adsorption with fucose-containing sugar chain adsorbed in (3) by the method described in Example 4 (4) As a result of calculating the recovery rate of the fucose-containing sugar chain from the agent 6, the recovery rate was 90%.

(5) Measurement of adsorption rate of fucose-containing sugar chain to polysaccharide-based carrier into which maleimide group has been introduced According to the method described in Example 6 (5), maleimide group is converted to α from maleimidated Sepharose 4FF prepared in (1). -Adsorbent 6B blocked with thioglycerin was obtained. As a result of calculating the adsorption rate of the fucose-containing sugar chain to the adsorbent 6B by the method described in Example 4 (3), the adsorption rate was 3%. Therefore, it was revealed that the fucose-containing sugar chain is not adsorbed on the adsorbent 6B.
(6) Measurement of adsorption rate of oligomannose type sugar chain to adsorbent 6 Adsorption rate of oligomannose type sugar chain to adsorbent 6 prepared in (2) by the method described in (4) of Example 4 As a result, the adsorption rate was 3%. Therefore, it was revealed that the adsorbent 6 does not adsorb oligomannose type sugar chains not containing fucose.
Comparative Example 3 Immobilization of fucose-binding lectin with no cysteine tag added to a carrier into which a maleimide group was introduced and evaluation as an adsorbent Comparative Example 3 was a commercial fucose on a synthetic polymer carrier into which a maleimide group was introduced. The present invention relates to immobilization of binding lectin and evaluation as an adsorbent.

(1) Immobilization of a commercially available fucose-binding lectin on a synthetic polymer carrier into which maleimide groups have been introduced (preparation of adsorbent 7)
A 50% by volume suspension (200 μL) of maleimidated Toyopearl HW-65F prepared in (1) of Example 6 was added to the reaction vessel, and 200 mM containing 500 mM sodium chloride and 20 mM ethylenediaminetetraacetic acid (EDTA) was added. Washed 5 times with phosphate buffer (pH 7.4, 200 μL).

  Next, in a reaction vessel containing maleimidized Toyopearl HW-65F (100 μL), the amount of commercially available fucose-binding lectin per mL of carrier was 2.24 mg, and a D-PBS (−) solution of a commercially available fucose-binding lectin was used. (Concentration 1.60 mg / mL, 140 μL) was added and stirred at 35 ° C. for 4 hours to immobilize the commercially available fucose-binding lectin on maleimidated Toyopearl HW-65F. After the immobilization reaction, the carrier is washed with D-PBS (-) containing 0.05% by weight of Tween 20, whereby a commercially available fucose-binding lectin is immobilized on maleimidized Toyopearl HW-65F, and / or Alternatively, complex carbohydrate adsorbent 7 was obtained.

  The amount of the commercially available fucose-binding lectin immobilized on the adsorbent 7 was calculated by the method described in Example 4 (2). As a result, the amount of the commercially available fucose-binding lectin used in the reaction and the unreacted commercial fucose-binding property were calculated. Since the amount of lectin was the same, it was revealed that the commercially available fucose-binding lectin was not immobilized on maleimidated Toyopearl HW-65F. Therefore, the adsorption rate of fucose-containing sugar chains and oligomannose type sugar chains to the adsorbent 7 was not measured.

Example 8 Immobilization of a fucose-binding lectin having a cysteine tag attached to a carrier into which a haloacetyl group was introduced and evaluation as an adsorbent (part 1)
Example 8 relates to immobilization of a fucose-binding lectin having a cysteine tag added to a synthetic polymer carrier into which a haloacetyl group has been introduced, and evaluation as an adsorbent.

(1) Production of Synthetic Polymer Carrier Introduced with Haloacetyl Group As a synthetic polymer carrier, Toyopearl HW-65F (manufactured by Tosoh Corp.) suspended in water was suction filtered with a glass filter. Aminated Toyopearl HW-65F was obtained by the method described in 4 (1).

  Next, 3-maleimidopropion prepared from the aminated Toyopearl HW-65F (2.0 g), DMSO (manufactured by Kanto Chemical Co., Ltd., 2.0 mL), N-succinimidyl bromoacetate (manufactured by Tokyo Chemical Industry Co., Ltd.) and DMSO A bromoacetylation reaction was performed by mixing a solution of acid N-succinimidyl in DMSO (concentration 10 mg / mL, 2.0 mL) and stirring at 35 ° C. for 4 hours. After the reaction, a glass filter was used to sequentially wash with DMSO and water to obtain Toyopearl HW-65F into which a bromoacetyl group was introduced (hereinafter referred to as bromoacetylated Toyopearl HW-65F).

(2) Immobilization of fucose-binding lectin with a cysteine tag attached to a synthetic polymer carrier into which a haloacetyl group has been introduced (production of adsorbent 8)
A fucose-containing sugar chain and / or a complex sugar in which a fucose-binding lectin having a cysteine tag added thereto is immobilized on the bromoacetylated Toyopearl HW-65F produced in (1) by the method described in Example 6 (1). A quality adsorbent 8 was obtained. The amount of fucose-binding lectin added with a cysteine tag to the adsorbent 8 was calculated by the method described in Example 4 (2). As a result, the amount of immobilization was 1.10 mg per mL of carrier, the immobilization rate. Was 48.9%.

(3) Measurement of adsorption rate of fucose-containing sugar chain to adsorbent 8 By the method described in (4) of Example 4, the adsorption rate of fucose-containing sugar chain to adsorbent 8 prepared in (2) was calculated. As a result, the adsorption rate was 89%.
(4) Measurement of recovery rate of fucose-containing sugar chain from adsorbent 2 adsorbed with fucose-containing sugar chain Adsorption with fucose-containing sugar chain adsorbed in (3) by the method described in Example 4 (4) As a result of calculating the recovery rate of the fucose-containing sugar chain from the agent 8, the recovery rate was 86%.
(5) Measurement of adsorption rate of fucose-containing sugar chain to polysaccharide carrier into which maleimide group has been introduced. Bromo of bromoacetylated Toyopearl HW-65F prepared in (1) by the method described in (5) of Example 6. Adsorbent 8B in which the acetyl group was blocked with α-thioglycerin was obtained. As a result of calculating the adsorption rate of the fucose-containing sugar chain to the adsorbent 8B by the method described in Example 4 (3), the adsorption rate was 4%. Therefore, it was revealed that the fucose-containing sugar chain is not adsorbed on the adsorbent 8B.

(6) Measurement of adsorption rate of oligomannose type sugar chain to adsorbent 8 Adsorption rate of oligomannose type sugar chain to adsorbent 8 produced in (2) by the method described in (6) of Example 4 As a result, the adsorption rate was 5%. Therefore, it was clarified that the adsorbent 8 does not adsorb oligomannose type sugar chains not containing fucose.

Example 9 Immobilization of fucose-binding lectin having a cysteine tag added to a carrier into which a haloacetyl group was introduced and evaluation as an adsorbent (part 2)
Example 9 relates to immobilization of a fucose-binding lectin having a cysteine tag added to a polysaccharide carrier into which a haloacetyl group has been introduced, and evaluation as an adsorbent.

(1) Production of polysaccharide carrier into which haloacetyl group was introduced As a polysaccharide carrier, Sepharose 4FF suspended in water was filtered by suction with a glass filter, and bromide was obtained by the method described in (1) of Example 8. Sepharose 4FF into which an acetyl group was introduced (hereinafter referred to as bromoacetylated Sepharose 4FF) was obtained.
(2) Immobilization of a fucose-binding lectin with a cysteine tag added to a polysaccharide carrier into which a haloacetyl group has been introduced (preparation of adsorbent 9)
An adsorbent for fucose-containing sugar chains and / or complex carbohydrates in which a fucose-binding lectin having a cysteine tag added thereto is immobilized on the bromoacetylated Sepharose 4FF produced in (1) by the method described in Example 8 (1) 9 was obtained. As a result of calculating the immobilization amount of the fucose-binding lectin to which the cysteine tag was added to the adsorbent 9 by the method described in Example 4 (2), the immobilization amount was 1.21 mg per mL of the carrier, the immobilization rate Was 53.8%.
(3) Measurement of the adsorption rate of fucose-containing sugar chains on the adsorbent 9 The adsorption rate of fucose-containing sugar chains on the adsorbent 9 produced in (2) was calculated by the method described in (4) of Example 4. As a result, the adsorption rate was 94%.
(4) Measurement of recovery rate of fucose-containing sugar chain from adsorbent 6 adsorbed with fucose-containing sugar chain Adsorption with fucose-containing sugar chain adsorbed in (3) by the method described in Example 4 (4) As a result of calculating the recovery rate of the fucose-containing sugar chain from the agent 9, the recovery rate was 91%.

(5) Measurement of adsorption rate of fucose-containing sugar chain to polysaccharide-based carrier into which maleimide group has been introduced According to the method described in Example 6 (5), maleimide group is converted to α from maleimidated Sepharose 4FF prepared in (1). -Adsorbent 9B blocked with thioglycerin was obtained. As a result of calculating the adsorption rate of the fucose-containing sugar chain to the adsorbent 9B by the method described in Example 4 (3), the adsorption rate was 4%. Therefore, it was revealed that the fucose-containing sugar chain is not adsorbed on the adsorbent 9B.
(6) Measurement of adsorption rate of oligomannose type sugar chain to adsorbent 9 Adsorption rate of oligomannose type sugar chain to adsorbent 9 prepared in (2) by the method described in (6) of Example 4 As a result, the adsorption rate was 4%. Therefore, it was revealed that the adsorbent 9 does not adsorb oligomannose type sugar chains not containing fucose.

Comparative Example 4 Immobilization of Fucose-Binding Lectin without Addition of Cysteine Tag to Carrier Introduced with Haloacetyl Group and Evaluation as Adsorbent Comparative Example 4 is a commercially available fucose to a synthetic polymer carrier into which a haloacetyl group was introduced The present invention relates to immobilization of binding lectin and evaluation as an adsorbent.

(1) Immobilization of a commercially available fucose-binding lectin on a synthetic polymer carrier into which a haloacetyl group is introduced (preparation of adsorbent 10)
A fucose-containing sugar chain and / or a complex sugar in which a commercially available fucose-binding lectin is immobilized on the bromoacetylated Toyopearl HW-65F produced in (1) of Example 8 by the method described in (1) of Comparative Example 3 A quality adsorbent 10 was obtained. The amount of the commercially available fucose-binding lectin immobilized on the adsorbent 10 was calculated by the method described in Example 4 (2). As a result, the amount of commercially available fucose-binding lectin used in the reaction and the unreacted commercial fucose-binding property were calculated. Since the amount of lectin was the same, it was revealed that commercially available fucose-binding lectin was not immobilized on bromoacetylated Toyopearl HW-65F. Therefore, the adsorption rate of fucose-containing sugar chains and oligomannose type sugar chains to the adsorbent 10 was not measured.

Reference Example 1 Immobilization of fucose-binding lectin with a cysteine tag added to a carrier into which a formyl group was introduced and evaluation as an adsorbent Reference Example 1 was a synthesis in which a formyl group was introduced using the ε-amino group of lysine. The present invention relates to immobilization of fucose-binding lectin with a cysteine tag attached to a polymer carrier and evaluation as an adsorbent.

(1) Production of Synthetic Polymer Carrier Introducing Formyl Group As a synthetic polymer carrier, Toyopearl HW-65F (manufactured by Tosoh Corp.) suspended in water was suction filtered with a glass filter. Epoxidized Toyopearl HW-65F was obtained by the method described in 4 (1).

The obtained epoxidized Toyopearl HW-65F (2.0 g), D-glucamine (manufactured by Tokyo Chemical Industry Co., Ltd.) and 0.5 M D-glucamine aqueous solution (4.0 mL) prepared from water were mixed at 40 ° C. D-glucamine was introduced into the carrier by stirring for 15 hours. After the reaction, D-glucamine-introduced Toyopearl HW-65F was obtained by washing with a large amount of water until the filtrate became neutral on the glass filter.
Next, sodium periodate aqueous solution (10 mg / mL, 1.5 mL) prepared from the obtained D-glucamine-introduced Toyopearl HW-65F (2.0 g), sodium periodate (manufactured by Kanto Chemical Co., Inc.) and water Water (1.5 mL) was mixed, and the formylation reaction was performed by stirring at 25 ° C. for 60 minutes. After the reaction, it was washed with a large amount of water using a glass filter to obtain Toyopearl HW-65F into which formyl group was introduced (hereinafter referred to as formylated Toyopearl HW-65F).

(2) Immobilization of fucose-binding lectin with a cysteine tag attached to a synthetic polymer carrier into which formyl group has been introduced (production of adsorbent 11)
A 50% by volume suspension (200 μL) of formylated Toyopearl HW-65F prepared in (1) was added to the reaction vessel, and a 200 mM Tris-HCl buffer containing 500 mM sodium chloride and 20 mM ethylenediaminetetraacetic acid (EDTA). Washed 5 times with the solution (pH 8.0, 200 μL).

  Add the cysteine tag prepared in Example 2 above to a reaction vessel containing formylated Toyopearl HW-65F (100 μL) with a charge of 2.24 mg of fucose-binding lectin added with a cysteine tag per mL of carrier. A D-PBS (−) solution (concentration 2.80 mg / mL, 80 μL) of the fucose-binding lectin and D-PBS (−) (20 μL) were added and stirred at 30 ° C. for 1 hour. Next, a cysteine tag was added by adding sodium borohydride aqueous solution (5 mg / mL, 10 μL) prepared from sodium borohydride (manufactured by Kanto Chemical Co., Inc.) and water, and further stirring at 30 ° C. for 1 hour. The fucose-binding lectin was immobilized on formylated Toyopearl HW-65F. After the immobilization reaction, the carrier is washed with D-PBS (-) containing 0.05% by weight of Tween 20, so that fucose-binding lectin added with a cysteine tag is immobilized on formylated Toyopearl HW-65F. The sugar chain and / or complex carbohydrate adsorbent 11 was obtained.

  The amount of fucose-binding lectin added with a cysteine tag used in the reaction was calculated as a result of calculating the amount of fucose-binding lectin added with a cysteine tag to the adsorbent 11 by the method described in Example 4 (2). The amount of fucose-binding lectin to which unreacted cysteine tag was added was the same, so that it was revealed that the fucose-binding lectin to which cysteine tag was added was not immobilized on formylated Toyopearl HW-65F. . Therefore, the adsorption rate of fucose-containing sugar chains and oligomannose type sugar chains to the adsorbent 11 was not measured.

Table 1 shows the results of immobilizing the fucose-binding lectin on the carrier in Examples 4 to 9, Comparative Examples 1 to 4 and Reference Example 1.
Table 2 shows the evaluation results of the adsorbents prepared in Examples 4 to 9 and Comparative Example 2 as adsorbents for fucose-containing sugar chains and / or complex carbohydrates.

Claims (12)

A protein having an oligopeptide containing one or more cysteines at the N-terminus or C-terminus of a fucose-binding lectin. Fucose-binding lectin
(A) a protein comprising at least the second proline to 156th glycine of the amino acid sequence set forth in SEQ ID NO: 1, or (B) one or more in the amino acid sequence of the protein of (A) above A protein in which one amino acid is deleted, substituted or added,
The protein according to claim 1. The protein according to claim 1 or 2, wherein the oligopeptide comprising one or more cysteines is an oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2. A polynucleotide encoding the protein according to any one of claims 1 to 3. An expression vector comprising the polynucleotide according to claim 4. A transformant obtained by transforming a host with the expression vector according to claim 5. The transformant according to claim 6, wherein the host is Escherichia coli. Culturing a transformant obtained by introducing an expression vector containing a polynucleotide encoding a protein having an oligopeptide containing one or more cysteines at the N-terminus or C-terminus of a fucose-binding lectin, And a step of recovering the protein from the obtained culture. Fucose-binding lectin
(A) a protein comprising at least the second proline to 156th glycine of the amino acid sequence set forth in SEQ ID NO: 1, or (B) one or more in the amino acid sequence of the protein of (A) above The method for producing a protein according to claim 8, wherein the amino acid is a protein in which one amino acid is deleted, substituted or added. An adsorbent for fucose-containing sugar chains and / or complex carbohydrates, wherein the protein according to claim 8 or 9 is immobilized on a carrier insoluble in water. The adsorbent according to claim 8 or 9, wherein the water-insoluble carrier has a functional group that reacts with a mercapto group of cysteine to form a covalent bond. A method for purifying fucose-containing sugar chains and / or complex carbohydrates, wherein the adsorbent according to claim 10 or 11 is used.

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