JP2023042742A - Method for separating and collecting cells using adsorbent with large particle size - Google Patents

Abstract

To provide technologies that can separate and purify both highly undifferentiated cells and low undifferentiated cells from a large number of cells in a short time and obtain each cell with high recovery rate and high purity, in the preparation of cells for regenerative medicine.SOLUTION: The above problem can be solved by using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier having a wet particle size of 300 μm or more and less than 600 μm, contacting a cell suspension containing cell populations with different degrees of undifferentiation with the adsorbent to bind the cells that bind to the fucose-binding protein to the adsorbent, recovering the cells that were not adsorbed to the adsorbent, and subsequently contacting the fucose-containing aqueous solution with the adsorbent to which the cells are bound and collecting the desorbed cells.SELECTED DRAWING: None

Description

The present invention is a method for separating and recovering cells using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier having a wet particle size of 300 μm or more, and a mixture of cells with different degrees of undifferentiation is brought into contact with the adsorbent. After recovering the cells that were not adsorbed to the adsorbent, the fucose-containing aqueous solution was applied to desorb and recover the cells bound to the adsorbent, thereby obtaining heterogeneous cells with different degrees of undifferentiation at a high recovery rate. on how to.

BC2LCN, derived from the N-terminal domain of BC2L-C lectin produced by Gram-negative bacteria (Burkholderia cenocepacia), is a protein having binding affinity to sugar chains containing fucose residues. In addition to H-type 1 sugar chain (Fucα1-2Galβ1-3GlcNAc) and H-type 3 sugar chain (Fucα1-2Galβ1-3GalNAc) known as undifferentiated sugar chain markers described in Document 1 and Patent Document 2, Lewis High binding affinity to multiple types of sugar chains containing fucose residues such as Y-type sugar chains (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and Lewis X-type sugar chains (Galβ1-4(Fucα1-3)GlcNAc) (Non-Patent Document 2). In addition, BC2LCN binds to undifferentiated human iPS cells and human ES cells in which H type 1 and H type 3 sugar chains are highly expressed, but does not bind to human somatic cells. known (Non-Patent Document 3). Furthermore, since BC2LCN has binding properties to the undifferentiated glycan markers, it can be used, for example, to detect complex carbohydrates containing undifferentiated glycan markers and to detect undifferentiated cells such as human iPS cells and human ES cells. (Patent Document 1 and Patent Document 2). In addition, it is known that the H-type 1 sugar chain is highly expressed in specific cancer cells as SSEA-5 (Non-Patent Document 4).

Although BC2LCN has the same ability to detect undifferentiated stem cells as anti-Nanog antibody, which is a known antibody for detecting undifferentiated cells (Patent Document 2), the binding of BC2LCN and sugar chains of undifferentiated cells is due to electrostatic interaction. and the bond strength is affected by external environment such as solvent and salt concentration. Therefore, depending on the experimental conditions, in the detection of the undifferentiated cells and/or the glycoconjugate containing the undifferentiated sugar chain marker, the binding affinity between the sugar chain and BC2LCN may be low. There is a need for BC2LCN with improved binding affinity to undifferentiated glycan markers. As a cell separation method using an adsorbent in which BC2LCN is immobilized on a water-insoluble carrier, a cell separation method that removes undifferentiated cells such as human iPS cells is known (Patent Document 3). A method of peeling and collecting bound undifferentiated cells is known (Patent Document 4).

However, in the methods disclosed in Patent Documents 3 and 4, from a heterogeneous cell mixture of cells with high expression of H type 1 sugar chains and H type 3 sugar chains and cells with low expression, Cells with different expression of H-type 1 and H-type 3 sugar chains could be separated from each other, but Toyopearl has a distribution of particle sizes ranging from 150 to 212 μm when wet with pure water. Cells with low expression of H-type 1 glycans and H-type 3 glycans that are not adsorbed to the cell adsorbent are recovered because the particles with fucose-binding protein immobilized thereon are used as the cell adsorbent. In this process, cell types with particularly large cell diameters are trapped between the Toyopearl particles, resulting in a decrease in the recovery rate. In particular, in Patent Document 4, after cells are brought into contact with an adsorbent, a fucose-containing aqueous solution is acted to desorb and recover cells with high expression of H-type 1 sugar chains and H-type 3 sugar chains bound to the adsorbent. In this process, cells with a relatively large cell diameter, such as human iPS cells, are similarly trapped between Toyopearl particles, resulting in a decrease in the recovery rate. rice field. Furthermore, in these cases, when a fucose-containing aqueous solution is acted to desorb cells with high expression of H-type 1 sugar chains and H-type 3 sugar chains bound to the adsorbent, when the cells are in contact with the adsorbent, Cells with low expression of H-type 1 sugar chains and H-type 3 sugar chains sandwiched between Toyopearl particles also flow out and are mixed into the desorbed cells, resulting in a decrease in the purity of the desired desorbed cells. was a problem.

However, at present, cells with high expression of H type 1 and H type 3 sugar chains and cells with low expression of H type 1 type sugar chain and H type 3 type sugar chain are separated and recovered from a large amount of cells at a high recovery rate for use in regenerative medicine. There was a strong demand for the development of technology that could

WO2013/065302 WO2013/128914 JP 2018-134073 JP 2019-000063

Tateno, H. et al., Stem Cells Transl Med. 2013, 2(4):265-273. Sulak, O. et al., Structure. 2010, 18(1):59-72. Tateno, H. et al., J Biol Chem. 2011, 286(23): 20345-20353. Tang, C et al., Nat Biotechnol. 2011, 29(9):829-835.

An object of the present invention is to develop a technique for separating and purifying target cells with different degrees of undifferentiation from a large amount of cells in a short period of time in the preparation of cells for use in regenerative medicine, and for obtaining the target cells at a high recovery rate. to provide. Specifically, undifferentiated markers present on the cell surface, such as sugar chains containing structures consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, are extracted from heterologous cell populations with different expression levels of these undifferentiated markers. It is an object of the present invention to provide a technique for separating and recovering both cells with a high expression level and cells with a low expression level of , at a high recovery rate. More specifically, a cell population is attached to an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier having a particle size when wet with water (hereinafter abbreviated as wet particle size) of 300 μm or more and less than 600 μm. After contacting and recovering the cells that were not adsorbed to the adsorbent, the cells bound to the adsorbent were desorbed and recovered using a fucose-containing aqueous solution, whereby cells with high and low expression levels of the undifferentiated marker were identified. It is to provide a technique for collecting both easily and efficiently.

As a result of intensive studies to solve the above-mentioned problems, the present inventors found that a water-insoluble carrier having a wet particle size of 300 μm or more and less than 600 μm was added with a fucose-binding protein in order to suppress clogging of cells between adsorbent particles. is immobilized as an adsorbent, expression of a sugar chain or the like containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, which are undifferentiated markers present on the cell surface, to the adsorbent Heterogeneous cell populations with different amounts are brought into contact, first the cell group with low undifferentiated marker expression that was not adsorbed to the adsorbent is collected, and then fucose is added to the adsorbent bound with the cell group with high undifferentiated marker expression. It was found that by desorbing and recovering cells bound to the adsorbent by allowing the containing aqueous solution to act, it is possible to obtain cells with high and low expression of undifferentiated markers on the cell surface at high recovery rates. , have completed the present invention.

That is, the present invention includes the inventions described in [1] to [6] below.
[1]
A method for separating and recovering cells using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier having a wet particle size of 300 μm or more and less than 600 μm, wherein the cells contain cell populations with different degrees of undifferentiation. A step of contacting the suspension with the adsorbent to bind the cells that bind to the fucose-binding protein to the adsorbent, and then recovering the cells that did not bind to the fucose-binding protein; A method for separating and recovering cells with different degrees of undifferentiation, comprising the step of contacting with an aqueous fucose-containing solution and recovering detached cells.
[2]
The method according to [1], wherein the cell population with different degrees of undifferentiation is a mixture of heterologous cells with different expression of sugar chains containing structures consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc.
[3]
The method according to any one of [1] to [2], wherein the cells highly expressing sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc are human iPS cells.
[4]
The method according to any one of [1] to [3], wherein the fucose-binding protein is any one of (a) to (d) below.
(a) A fucose-binding protein comprising an amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, wherein X is an integer of 120 or more. sex protein.
(b) 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 in the amino acid sequence shown by SEQ ID NO: 1 A fucose-binding protein comprising fucose and having binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more binding protein.
(c) any one of the amino acid substitutions described in (1) to (3) below in the 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 Fucose-binding protein (1) substitution of leucine residue for glutamine residue at position 39 in the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or more, comprising an amino acid sequence containing the above (2) Substitution of the 72nd cysteine residue in the amino acid sequence shown by SEQ ID NO: 1 with one type of amino acid residue selected from glycine residues and alanine residues (3) 65th amino acid sequence shown in SEQ ID NO: 1 (d) in the amino acid sequence of the fucose-binding protein of (c), one or more in regions other than 39th, 65th and 72nd of SEQ ID NO: 1 and has binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, fucose-binding protein.

[5]
The method according to any one of [1] to [3], wherein the fucose-binding protein is any one of (e) to (h) below.
(e) to the amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is A fucose-binding protein consisting of an amino acid sequence to which a cysteine-containing oligopeptide is added, wherein X is an integer of 120 or more.
(f) 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 in the amino acid sequence shown by SEQ ID NO: 1 On the other hand, a sugar comprising an amino acid sequence to which a polyhistidine sequence is added to the N-terminus and an oligopeptide containing cysteine is added to the C-terminus, and which has a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. A fucose-binding protein having binding affinity to a chain, wherein X is an integer of 120 or greater.

(g) to the amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is In the amino acid sequence to which an oligopeptide containing cysteine is added, an amino acid sequence containing any one or more of the amino acid substitutions described in (4) to (6) below, wherein X is an integer of 120 or more, fucose-binding protein (4) replacement of the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (5) substitution of the 72nd cysteine residue in the amino acid sequence shown in SEQ ID NO: 1, Substitution of one amino acid residue selected from glycine residues and alanine residues (6) Substitution of the 65th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (h) The above ( An amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added to regions other than the 39th, 65th and 72nd regions of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of g) to which an oligopeptide containing a polyhistidine sequence is added at the N-terminus and an oligopeptide containing a cysteine sequence is added at the C-terminus, comprising an amino acid sequence, Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc A fucose-binding protein having binding affinity to sugar chains comprising a structure of
[6]
The method according to any one of [1] to [5], wherein an adsorbent packed in a column is used.
The present invention will be described in detail below.

The cell separation and recovery method using the large particle size adsorbent of the present invention (hereinafter sometimes abbreviated as the cell separation and recovery method of the present invention) is a water-insoluble carrier having a wet particle size of 300 μm or more and less than 600 μm. Using an adsorbent on which a fucose-binding protein was immobilized, cell populations of heterogeneous cells with different degrees of undifferentiation were brought into contact, then the cell group that did not bind to the adsorbent was first separated and then bound to the adsorbent. A cell separation and recovery method characterized by high cell recovery, including a step of desorbing cells using a fucose-containing aqueous solution.

In the present invention, an undifferentiated marker is a molecule present on the cell surface that serves as an indicator of the degree of undifferentiation, specifically known as a fucose-containing sugar chain to which the fucose-binding protein can bind. A structure consisting of Fucα1-2Galβ1-3GlcNAc having a sugar chain comprising an H-type 1-type sugar chain structure, an H-type 3-type sugar chain structure, a Lewis Y-type sugar chain structure, and/or a Lewis b-type sugar chain structure A sugar chain and/or a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc can be exemplified.

In the present invention, cells with different degrees of undifferentiation refer to cells with different expression levels of the undifferentiated markers on the cell surface. Taking sugar chains to which the fucose-binding protein can bind as an undifferentiated marker, the expression of the undifferentiated marker molecule in each cell type can be evaluated, for example, in K562 cells (human chronic myeloid leukemia) and NHDF cells (human chronic myelogenous leukemia). Dermal fibroblasts), human mesenchymal stem cells, Ramos cells (human Burkitt's lymphoma), RD cells (rhabdomyosarcoma), etc. contain cells having a sugar chain that binds to the fucose-binding protein. known not to. In 2102Ep cells (human embryonic tumor cells), about 50% to 80% of the cell population express sugar chains that bind to the fucose-binding protein, and human iPS cell line 201B7 (hereinafter referred to as 201B7 cells), nearly 100% of the cells are known to express sugar chains that bind to the fucose-binding protein. Thus, different cell types (hereafter abbreviated as heterologous cells) are considered to have different levels of undifferentiated marker molecule expression. In comparison of the undifferentiated degree of heterologous cells, even if each cell type expresses the sugar chain that binds to the fucose-binding protein in the same number of cells, if the heterologous cell is a cell surface It is thought that the numbers and densities of undifferentiated marker molecules present in the cells are different. In the cell separation and recovery method of the present invention, both cells with high expression and low expression of sugar chains that bind to fucose-binding proteins are separated and recovered at a high recovery rate from the mixture of these heterologous cells. be able to.

In addition, as another form of heterologous cells, even if it is a single cell type, it includes the case where the numbers and densities of undifferentiated marker molecules present on the cell surface are mixed for each individual cell. be For example, when the undifferentiated marker is a sugar chain that can bind to the fucose-binding protein, for example, in the case of 201B7 cells, which are human iPS cells, nearly 100% of the cells bind to the fucose-binding protein as described above. Although it is known to express sugar chains, it is known that there are undifferentiated deviant cells with a low degree of undifferentiation depending on the culture conditions. Again, in the context of the present invention, 201B7 cells and undifferentiated deviated 201B7 cells can be considered heterologous cells even if they are of a single cell type. In the cell separation and collection method of the present invention, even heterogeneous cell mixtures in these forms can be subjected to separation and collection. Since both undifferentiated deviant cells with a low concentration and highly undifferentiated 201B7 cells adsorbed to the adsorbent can be obtained at a high recovery rate, it is possible to prepare large amounts of highly undifferentiated iPS cells and undifferentiated deviant cells This is an extremely effective means for analyzing the properties of

In the cell separation and recovery method of the present invention, after contacting a cell population with an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier, the cell group that does not bind to the adsorbent is first separated and then bound to the adsorbent. It includes a step of contacting the cells with a fucose-containing aqueous solution to obtain a detached cell group. In this cell desorption process, the fucose molecules in the solution bind to the fucose-binding sugar chains on the cell surface, thereby dissociating the bonds between the fucose-binding protein of the adsorbent and the cells, and the cells bound to the adsorbent are released. It is considered to be removable. The solution for preparing the fucose-containing aqueous solution is not particularly limited as long as it does not interfere with the viability of the cells. Water, MACS buffer (0.5% (w /v) Various solvents such as bovine serum albumin (hereinafter abbreviated as BSA) and 2 mM ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA)) or an aqueous solution of mannitol can be used. In addition, the composition of the fucose-containing aqueous solution can be appropriately adjusted as necessary. For example, when cells are difficult to desorb even when using an adsorbent having a wet particle size of 300 μm or more and less than 600 μm in the present invention, mannitol may be added. By adjusting the concentration to 0.5 to 0.7 M, cell contraction due to osmotic pressure is caused, and by adding about 10 mM EDTA to suppress non-specific adsorption of cells to the adsorbent, cell desorption is suppressed. can be promoted.

In addition, it is also possible to add a reagent that enhances cell viability, or to use a buffer solution as the base solution to suppress pH fluctuations. The fucose concentration of the fucose-containing aqueous solution in the present invention is preferably 20 mM to 1.0 M, more preferably 40 mM to 800 mM, most preferably 60 mM to 700 mM. As for the structure of fucose used in the aqueous solution, either the D-form or the L-form may be used, but it is preferable from an economic point of view to use the L-form, which generally exists in nature. In the present invention, the cell density and the volume of the cell liquid in which the heterogeneous cell mixture is brought into contact with the adsorbent are not particularly limited. Approximately 1 mL to 100 mL of the cell fluid can be passed through a column filled with an adsorbent for treatment.

In the cell separation and recovery method of the present invention, a large amount of cell fluid can be continuously treated by using an adsorbent packed in a column. This is an extremely effective means for directly separating and recovering target cells at a high recovery rate from a cell solution with a low cell density and a large liquid volume, such as a suspension of cultured cells recovered using the method. If contaminants contained in the medium or cell suspension are thought to interfere with the binding of highly undifferentiated cells to the adsorbent, dilute the collected cell sap with MACS buffer as appropriate before adding the adsorbent. You should make contact with

Next, the fucose-binding protein in the cell separation and recovery method of the present invention will be explained. The fucose-binding proteins in the cell separation and recovery method of the present invention include H type 1 sugar chain (Fucα1-2Galβ1-3GlcNAc), H type 3 sugar chain (Fucα1-2Galβ1-3GalNAc), Lewis Y sugar chain (Fucα1 -2Galβ1-4(Fucα1-3)GlcNAc), Lewis b-type sugar chain (Fucα1-2Galβ1-3(Fucα1-4)GlcNAc), etc. BC2LCN lectin is also included in the fucose-binding protein in the cell separation and collection method of the present invention. Specifically, the fucose-binding protein in the cell separation and recovery method of the present invention is (a): the amino acid sequence of the recombinant BC2LCN lectin shown in SEQ ID NO: 1 (of the amino acid sequence registered with GenPept as accession number WP_006490828). matches the amino acid sequence from 2nd to 156th), wherein X is an integer of 120 or more, or (b) : 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 to the Xth amino acid in the amino acid sequence shown by SEQ ID NO: 1, and H type A protein having a binding property to type 1 sugar chains and/or H type 3 sugar chains and X being an integer of 120 or more is expressed as a recombinant protein in a transformant of E. coli. The fucose-binding protein in the cell separation and recovery method of the present invention has binding properties to the fucose-containing sugar chain, particularly to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. As long as 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 less, Preferably no more than 10 amino acids may be deleted, substituted or added. Moreover, X may be 120 or more and 155 or less, and may be 125 or more and 155 or less. As disclosed in JP-A-2020-25535, the fucose-binding protein in the cell separation and recovery method of the present invention deletes a plurality of amino acid residues on the C-terminal side of the amino acid sequence shown in SEQ ID NO: 1. As a result, the productivity (expression level) can be improved when produced in a transformant of E. coli compared to when the amino acid residue is not deleted.

In addition, the fucose-binding protein in the cell separation and recovery method of the present invention has the following advantages in terms of improving heat stability: Substitution, (ii) replacement of the 72nd cysteine residue of the amino acid sequence shown by SEQ ID NO: 1 with one type of amino acid residue selected from glycine residue and / or alanine residue, (iii) SEQ ID NO: 1 substitution of the 65th glutamine residue in the amino acid sequences shown with a leucine residue. As disclosed in Japanese Unexamined Patent Application Publication No. 2020-25535, by performing the amino acid substitutions described in (i) to (iii) above, the heat stability of the fucose-binding protein in the cell separation and recovery method of the present invention is improved. can be made The amino acid substitutions described in (i) to (iii) above are effective in improving the stability against heat whether they are used alone or in combination, but they can further improve the stability against heat. , It is more preferable to combine multiple amino acid substitutions described in (i) to (iii) above. Furthermore, the fucose-binding protein in the cell separation and recovery method of the present invention has the binding property to sugar chains containing structures consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, as long as it has SEQ ID NO: 1 In the amino acid sequence from the 1st proline to the Xth amino acid in the amino acid sequence represented by, one or more amino acid residues in the region other than the positions substituted by the substitutions (i) to (iii) Deletions, substitutions or insertions may be made, for example no more than 15, preferably no more than 10 amino acid residues may be deleted, substituted or inserted.

Specific examples of the fucose-binding protein in the cell separation and recovery method of the present invention include SEQ ID NO: 1, SEQ ID NO: 2 (amino acid sequences from 1st to 127th in the amino acid sequence shown in SEQ ID NO: 1), and SEQ ID NO: 3. (Amino acid sequence in which the 72nd cysteine residue of SEQ ID NO: 2 is replaced with a glycine residue), SEQ ID NO: 4 (the 39th glutamine residue of SEQ ID NO: 2 is a leucine residue, and the 72nd cysteine residue is glycine amino acid sequence substituted with a residue), SEQ ID NO: 5 (the 39th glutamine residue of SEQ ID NO: 2 is a leucine residue, the 65th glutamine residue is a leucine residue, and the 72nd cysteine residue is a glycine residue SEQ ID NO: 6 (amino acid sequence obtained by adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the amino acid sequence shown by SEQ ID NO: 1 and an oligopeptide containing a cysteine residue to the C-terminus), SEQ ID NO: 7 (amino acid sequence obtained by adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the amino acid sequence shown by SEQ ID NO: 2 and an oligopeptide containing a cysteine residue to the C-terminus), SEQ ID NO: 8 (SEQ ID NO: 3 An amino acid sequence obtained by adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the indicated amino acid sequence and an oligopeptide containing a cysteine residue to the C-terminus), SEQ ID NO: 9 (at the N-terminus of the amino acid sequence shown by An oligopeptide containing a polyhistidine sequence, an amino acid sequence obtained by adding an oligopeptide containing a cysteine residue to the C-terminus) and SEQ ID NO: 10 (an oligopeptide containing a polyhistidine sequence at the N-terminus of the amino acid sequence shown in SEQ ID NO: 5) , an amino acid sequence in which an oligopeptide containing a cysteine residue is added to the C-terminus).

As long as the fucose-binding protein in the cell separation and recovery method of the present invention has a binding property to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, its N-terminal side and / Or at the C-terminal side, it may have an additional amino acid sequence useful for detecting fucose-binding proteins.

The additional amino acid sequences include oligopeptides containing a polyhistidine sequence, glutathione S-transferase (hereinafter referred to as GST), maltose binding protein, cellulose binding domain, myc tag, FLAG tag and the like. Among these additional amino acid sequences, the productivity is high when produced using E. coli, and the fucose-binding protein can be easily detected by using a fluorescently labeled anti-polyhistidine antibody or anti-GST antibody. , preferably an oligopeptide or GST containing a polyhistidine sequence, more preferably an oligopeptide containing a polyhistidine sequence. The number of histidine repeat sequences in an oligopeptide containing a polyhistidine sequence is not particularly limited. connectivity may be compromised.

Therefore, the length of the repeating sequence of histidines in an oligopeptide containing a polyhistidine sequence is preferably a repeating sequence consisting of 5 to 15 histidines, more preferably a repeating sequence consisting of 5 to 10 histidines. preferable. The position at which the oligopeptide containing the polyhistidine sequence is attached to the fucose-binding protein is not particularly limited, and may be both the N-terminal side and the C-terminal side, or either the N-terminal side or the C-terminal side. However, it is preferable that the oligopeptide containing the polyhistidine sequence is added to the N-terminal side of the fucose-binding protein in terms of efficient detection with an anti-polyhistidine antibody.

Furthermore, the fucose-binding protein in the cell separation and recovery method of the present invention has, on its N-terminal side and/or C-terminal side, a cysteine residue or It may have an additional amino acid sequence consisting of an oligopeptide containing a lysine residue (hereinafter referred to as carrier immobilization tag). By immobilizing a fucose-binding protein on a carrier, for example, an undifferentiated cell adsorbent for removing undifferentiated cells such as human iPS cells described in Patent Document 3 can be produced. The length of the carrier-immobilizing tag is not particularly limited as long as the fucose-binding protein has the ability to bind to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. do not have. As the tag for carrier immobilization, an oligopeptide consisting of 2 to 10 amino acid residues containing one or more cysteine residues is preferable, since immobilization to a water-insoluble carrier can be performed with high selectivity and high efficiency. includes an oligopeptide consisting of 3 amino acid residues of "Gly-Gly-Cys", an oligopeptide consisting of 5 amino acid residues of "Ala-Ser-Gly-Gly-Cys" and an oligopeptide consisting of 5 amino acid residues of "Gly-Gly-Gly-Ser- An oligopeptide consisting of 7 amino acid residues "Gly-Gly-Cys" can be exemplified. The position at which the oligopeptide containing one or more cysteines is attached to the fucose-binding protein is not particularly limited, and may be both the N-terminal side and the C-terminal side, or either the N-terminal side or the C-terminal side. , the fucose-binding protein can be efficiently immobilized on a carrier, and the binding activity is less likely to be inhibited because the fucose-binding protein is away from the active center of the fucose-binding protein. The peptide is preferably added to the C-terminal side of the fucose-binding protein.

A signal peptide for promoting efficient expression in the host may be added to the N-terminal side of the fucose-binding protein in the cell separation and recovery method of the present invention. Examples of the signal peptide when the host is Escherichia coli include signal peptides such as PelB, DsbA, MalE, and TorT that secrete proteins into the periplasm. A DNA encoding a fucose-binding protein in the cell separation and recovery method of the present invention can be prepared by a known method. As a method for preparing the DNA, a method of converting the amino acid sequence of the fucose-binding protein into a base sequence in the cell separation and collection method of the present invention and artificially synthesizing DNA containing the base sequence, or a method of cell separation and collection of the present invention. A method of directly and artificially preparing the DNA encoding the fucose-binding protein in the method, or a method of preparing it from Burkholderia cenocepacia genomic DNA or the like using a DNA amplification method such as PCR can be exemplified.

In the preparation method, when designing the base sequence, it is preferable to consider the frequency of codon usage in E. coli to be transformed. Ile), ATA, leucine (Leu), CTA, glycine (Gly), GGA, and proline (Pro), CCC, are rare codons (rare codons). Selective conversion is preferred. Analysis of codon usage frequency is performed using a public database (for example, Codon Usage Database on the homepage of Kazusa DNA Research Institute, http://www.kazusa.or.jp/codon/, access date: May 7, 2020). It is also possible by using

In order to transform Escherichia coli with the DNA encoding the fucose-binding protein prepared by the above method, the DNA itself may be used for transformation. It is preferable to insert the DNA into an appropriate position in a vector based on a commonly used bacteriophage, cosmid, plasmid, or the like to form an expression vector, and to transform using the expression vector, since stable transformation can be performed. . Here, the appropriate position means a position that does not destroy the replication function of the expression vector, the desired antibiotic marker, and the region involved in transmissibility. When inserting the DNA into a vector, it is preferable to insert into the vector in a state of being linked to a functional DNA such as a promoter required for expression. The vector used as the expression vector is not particularly limited as long as it can stably exist and replicate in the host, and pET vector, pUC vector, pTrc vector, pCDF vector, pBBR vector and the like can be exemplified.
Examples of the promoter include trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and λ phage λPL promoter and λPR promoter. Transformation of Escherichia coli as a host with the expression vector may be carried out by a method commonly used by those skilled in the art. When Escherichia coli W3110 strain or the like is selected, methods described in known literature (for example, Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) can be used.

Next, a method for producing a fucose-binding protein in the present invention will be described. The fucose-binding protein in the present invention is produced by culturing the transformant to produce the fucose-binding protein (hereinafter referred to as the first step) and recovering the fucose-binding protein from the resulting culture. It can be manufactured by a process including two processes (hereinafter referred to as a second process). In the present specification, the culture includes the cultured transformant cells themselves and cell secretions, as well as the medium used for culture. In the first step, the transformant may be cultured in a medium suitable for the culture. For example, when Escherichia coli is used as a host, it is preferable to use Terrific Broth (TB) medium, Luria-Bertani (LB) medium, etc. supplemented with necessary nutrients. When the expression vector contains a drug resistance gene, selective growth of the transformant becomes possible by adding a drug corresponding to the gene to the medium and carrying out the first step. For example, the expression vector contains a kanamycin resistance gene kanamycin is preferably added to the medium. The culture temperature may be any temperature that is generally known for the host to be used. It can be determined appropriately while taking into account the above.

In addition, the pH of the medium may be within the generally known pH range for the host to be used. For example, when the host is Escherichia coli, the pH is in the range of pH 6.8 to pH 7.4, preferably around pH 7.0. , may be appropriately determined in consideration of the production amount of the fucose-binding protein and the like. When an inducible promoter is introduced into the expression vector, the expression can be induced by adding an inducer to the medium under conditions that allow the fucose-binding protein to be produced well. Preferred inducers include, for example, isopropyl-β-D-thiogalactopyranoside (IPTG) when using the tac promoter or lac promoter, and the concentration to be added is in the range of 0.005 mM to 1.0 mM, preferably It ranges from 0.01 mM to 0.5 mM. Induction of expression by adding IPTG may be performed under conditions generally known for the host to be used.

In the second step, the fucose-binding protein is recovered from the culture obtained in the first step by a generally known recovery method. For example, when the fucose-binding protein is secreted and produced in the culture medium, the cells may be separated by centrifugation, and the fucose-binding protein may be recovered from the resulting culture supernatant. ), the cells are collected by centrifugation, the cells are disrupted by adding an enzyme treatment agent or surfactant, etc., and the fucose-binding protein can be recovered from the cell lysate. .

Moreover, when it is desired to improve the purity of the fucose-binding protein, a method known in the art may be used, and an example thereof is a separation and purification method using liquid chromatography. As liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like are preferably used, and a combination of these chromatographies is more preferred. In addition, the purity and molecular weight of the fucose-binding protein purified by the chromatography may be examined using methods known in the art. Examples include SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. Graphical methods may be mentioned.

The binding affinity of the fucose-binding protein to sugar chains in the present invention can be evaluated by Enzyme-linked immunosorbent assay method, surface plasmon resonance method, or the like. As an example, the surface plasmon resonance method will be described. Binding affinity evaluation by the surface plasmon resonance method is performed, for example, using a Biacore T200 instrument (manufactured by Cytiva), using a fucose-binding protein as the analyte and a sugar chain (Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc as the solid phase). It can be measured as a sugar chain containing a structure consisting of Sugar chain-immobilized sensor chips are produced by using biotin-labeled sugar chains to prepare streptavidin-coated sensor chips (Sensor Chip SA, manufactured by Cytiva) and dextran-coated sensor chips (Sensor Chip CM5, Cytiva (manufactured) to which streptavidin has been immobilized in advance. In addition, binding affinity evaluation can be performed using a kinetics analysis program attached to the instrument.

Next, the adsorbent used in the cell separation and recovery method of the present invention will be described. There are no particular restrictions on the raw materials for the water-insoluble carrier used as the adsorbent in the cell separation and recovery method of the present invention. Inorganic carriers such as silica gel and glass deposited with a thin gold film, and polysaccharides such as agarose, cellulose, chitin and chitosan. and water-insoluble polysaccharide carriers made from the raw material and crosslinked polysaccharide carriers obtained by crosslinking them with a crosslinking agent, and crosslinked polysaccharide carriers obtained by crosslinking water-soluble polysaccharides such as dextran, pullulan, starch, alginate, and carrageenan with a crosslinking agent. , poly(meth)acrylate, polyvinyl alcohol, polyurethane, polystyrene and the like, and crosslinked synthetic polymer carriers obtained by crosslinking these with a crosslinking agent. Among these carriers, uncharged polysaccharide carriers such as agarose, cellulose, dextran, and pullulan, which have hydroxyl groups and can be easily modified with a hydrophilic polymer to be described later, and their cross-linking agents. Crosslinked crosslinked polysaccharide carriers, hydrophilic synthetic polymer carriers such as poly(meth)acrylate and polyurethane, and crosslinked hydrophilic synthetic polymer carriers obtained by crosslinking these with a crosslinking agent are preferred.
In addition, the water-insoluble carrier used for the adsorbent is preferably modified with a hydrophilic polymer on the surface of the water-insoluble carrier from the viewpoint of suppressing non-specific adsorption of cells, and the hydrophilic polymer is water-insoluble. More preferably, it is fixed to the carrier by covalent bonding. Hydrophilic polymers that modify the surface of water-insoluble carriers include neutral polysaccharides such as agarose, cellulose, dextran, pullulan and starch, and hydroxyl groups such as poly(2-hydroxyethyl (meth)acrylate) and polyvinyl alcohol. can be exemplified by synthetic polymers having Among these hydrophilic polymers, neutral polysaccharides such as dextran, pullulan and starch are preferred, and dextran and pullulan are more preferred, because they are highly hydrophilic and can be easily fixed to the surface of an insoluble carrier by covalent bonding. . Although the molecular weights of dextran and pullulan are not particularly limited, those having a number average molecular weight of 10,000 to 1,000,000 are preferred in terms of sufficient hydrophilic modification of the surface of the insoluble carrier. The shape of the water-insoluble carrier used for the adsorbent is not particularly limited, and may be particulate, sponge-like, flat membrane-like, flat plate-like, hollow, or fibrous. A particulate carrier is preferable, and a spherical particulate carrier is more preferable, because it can be carried out efficiently.

The average particle diameter (median diameter) of the water-insoluble carrier used as the adsorbent in the cell separation and recovery method of the present invention when swollen in pure water is In order for the cells to be separated and collected to be in sufficient contact with the surface of the adsorbent, and for both the cells not bound to the adsorbent and the cells adsorbed and desorbed from the adsorbent by the fucose solution to pass through the gap between the adsorbents without stagnation, the gap should be 300 μm or more. It is characterized by using a particulate carrier having a particle size of less than 600 μm. If the particle size is less than 300 μm, cells that are not bound to the adsorbent and cells that are desorbed and collected by the fucose solution are less likely to pass through the gaps between the adsorbents, resulting in a lower cell recovery rate. If the particle size exceeds 600 μm, the contact between the adsorbent-bound cells and the surface of the adsorbent is insufficient, and the efficiency of separating cells that bind to the adsorbent and cells that do not bind to the adsorbent decreases. The cell separation and recovery method of the present invention is particularly effective when cells to be separated include cells with a relatively large cell diameter (cells with a cell diameter of 20 μm or more) such as megakaryocytes and human mesenchymal stem cells, and are 300 μm or more and less than 600 μm. By using a water-insoluble carrier with a wet particle size of 1, it suppresses clogging between adsorbent particles of cells with a large cell diameter, and both cells that are not adsorbed to the adsorbent and desorbed cells that are adsorbed to the adsorbent. By smooth passage, it is possible to obtain both cells with high recovery and high purity. The particle size of the insoluble carrier can be measured, for example, using a precision particle size distribution analyzer (product name: "Multisizer 3") manufactured by Beckman Coulter, Inc., or the like.
Alternatively, after photographing an image of a slide glass with a scale using an optical microscope, images of a plurality of particles to be measured are photographed at the same magnification, and the particle size of the photographed plurality of carriers is measured using a ruler. , can be obtained by calculating the average value thereof. The particulate carrier having a wet particle size of 300 μm or more and 600 μm or less used in the present invention can be prepared by a classification operation such as wet classification using a stainless steel standard sieve. In this classifying operation, it is preferable to apply ultrasonic waves to the sieve in order to classify efficiently. Specifically, first, the target suspension is classified by a stainless steel sieve with an opening of 600 μm equipped with an ultrasonic oscillator of 35 kHz, and the suspension that has passed through the sieve is collected.

Next, this suspension is similarly classified with a stainless steel sieve having an opening of 300 μm equipped with a similar ultrasonic oscillator, and the particles remaining on the sieve are collected as particulate carriers. Especially in the latter stage (300 μm sieve), it is important to use a particle size distribution analyzer to confirm that unintended particles (less than 300 μm) are properly removed, as this will affect the subsequent performance of the adsorbent. I can say. The presence or absence of pores in the water-insoluble carrier used for the adsorbent is not particularly limited, and may be either porous or non-porous. In addition, the water-insoluble carrier used in the adsorbent of the present invention is a particle having a hydroxyl group in that active functional groups can be easily introduced for immobilizing the fucose-binding protein used in the adsorbent of the present invention on the carrier. A shape carrier is preferred. Furthermore, the water-insoluble carrier used in the adsorbent of the present invention may be a commercially available product. (manufactured by Asahi Kasei Corp.), Cellophia (manufactured by Asahi Kasei Corp.) using cellulose as a raw material, and the like can be used.

The adsorbent in the cell separation and recovery method of the present invention is a step of preparing a water-insoluble carrier having a wet particle size of 300 μm or more and less than 600 μm by classification, and then producing a reactive water-insoluble carrier from the water-insoluble carrier (hereinafter referred to as step X ), and a step of immobilizing the reactive water-insoluble carrier by reacting the fucose-binding protein (hereinafter referred to as step Y). The details of process X and process Y are described below.

Step X is a step of introducing a reactive functional group for immobilizing a fucose-binding protein into a water-insoluble carrier to produce a reactive water-insoluble carrier. The reactive functional group for immobilizing the fucose-binding protein of the present invention on a water-insoluble carrier is not particularly limited as long as it is a general functional group for protein immobilization, and includes an epoxy group, a formyl group, a carboxyl group, an active Examples include an ester group, an amino group, a maleimide group, a haloacetyl group and the like. The method for introducing the functional group into the water-insoluble carrier is not particularly limited as long as it is a general method for introducing the functional group.

Step Y is a step of immobilizing the fucose-binding protein of the present invention on the reactive water-insoluble carrier produced in step X. The method for immobilizing the fucose-binding protein on the reactive water-insoluble carrier obtained in step X is not particularly limited as long as it is a general protein immobilization method. may be performed according to the method described in .

The amount of fucose-binding protein immobilized on the water-insoluble carrier may be appropriately set in consideration of the binding property between the cells to be separated and recovered and the fucose-binding protein in the cell separation and recovery method of the present invention. is preferably 0.01 mg or more and 50 mg or less, more preferably 0.05 mg or more and 30 mg or less per water-insoluble carrier. In addition, the amount of fucose-binding protein immobilized on the water-insoluble carrier can be adjusted by adjusting the amount of the protein used during the immobilization reaction and the amount of active functional group introduced into the water-insoluble carrier. The amount of fucose-binding protein immobilized on the water-insoluble carrier was determined by recovering the immobilization reaction solution and the post-reaction washing solution to determine the amount of unreacted fucose-binding protein. It can be calculated by subtracting the amount of unreacted fucose-binding protein of the present invention from the amount of protein.

In addition, as described above, the water-insoluble carrier used for the adsorbent in the present invention preferably has a hydrophilic polymer fixed by a covalent bond in order to suppress non-specific adsorption of cells. When producing an agent, before introducing the functional group for immobilizing the fucose-binding protein of the present invention in step X, the hydrophilic polymer can be immobilized on the water-insoluble carrier by covalent bonding. . The method for covalently immobilizing a hydrophilic polymer on a water-insoluble carrier is not particularly limited as long as it is a general covalent bond formation reaction. Epoxy groups are introduced into the water-insoluble carrier by reacting epoxy group-containing compounds such as ether and 1,4-butanediol diglycidyl ether under basic conditions. A method of reacting under conditions can be mentioned.

The cell separation and recovery method of the present invention uses an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier having a wet particle size of 300 μm or more and less than 600 μm, whereby sugars containing a structure consisting of Fucα1-2Galβ1-3GlcNAc are used. It is a particularly effective cell separation and recovery method for separating and recovering, at a high recovery rate, both cells with high sugar chain expression and cells with low sugar chain expression containing a structure consisting of a chain and/or Fucα1-2Galβ1-3GalNAc. . According to the cell separation and recovery method of the present invention, the expression of a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc is low, for example, by inducing differentiation from human iPS cells. It is possible to purify various differentiated cells for use in regenerative medicine with a high recovery rate. Characteristic analysis of cells can be easily performed.

Furthermore, the recovery rate of high-quality human iPS cells with a high degree of undifferentiated expression, that is, a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc. can get higher. In addition, the cell separation and recovery method of the present invention can easily process a large amount of cells in a short time compared to the existing methods of cell separation and recovery using magnetic beads, cell sorting using a cell sorter, and cell separation and recovery using a cell rolling column. It is an extremely effective means for large-scale purification of high-quality human iPS cells and differentiated cells produced from human iPS cells by differentiation induction, especially for regenerative medicine applications.

FIG. 2 shows microscopic images of the carriers after classification and the prepared cell adsorbent in the wet state in Preparation Examples 2, 3, and 4. FIG. FIG. 2 is a diagram showing particle size distributions in wet conditions of classified carriers and prepared cell adsorbents in Preparation Examples 2, 3, and 4. FIG. FIG. 10 is a diagram showing microscope images of the carrier after classification and the prepared cell adsorbent in a wet state in Preparation Example 5. FIG. FIG. 10 is a diagram showing particle size distributions in a wet state of the classified carrier and the prepared cell adsorbent in Preparation Example 5. FIG. FIG. 10 is a diagram showing microscopic images of the carrier after classification and the prepared cell adsorbent in a wet state in Preparation Example 6. FIG. FIG. 10 is a diagram showing particle size distributions in a wet state of the classified carrier and the prepared cell adsorbent in Preparation Example 6. FIG. FIG. 10 is a diagram showing microscopic images of the carrier after classification and the prepared cell adsorbent in a wet state in Preparation Example 7. FIG. FIG. 10 is a diagram showing particle size distributions in a wet state of the classified carrier and the prepared cell adsorbent in Preparation Example 7. FIG. 1 is a graph showing recovery rates of 201B7 cells and human mesenchymal stem cells in effluent cell sap in Example 1, Comparative Example 1, and Reference Example 1. FIG. 1 is a graph showing recovery rates of 201B7 cells and human mesenchymal stem cells in desorbed cell sap in Example 1, Comparative Example 1, and Reference Example 1. FIG.

EXAMPLES The present invention will be described in more detail below with reference to Preparation Examples, Examples, Comparative Examples, and Reference Examples, but the present invention is not limited to these.

Preparation Example 1 Preparation of rBC2LCN Lectin 127Q39L/C72G Preparation Example 1 relates to preparation of a fucose-binding protein (rBC2LCN lectin 127Q39L/C72G) represented by SEQ ID NO: A consisting of 148 amino acids. Namely, replacement of the 39th glutamine residue with a leucine residue counting from the first proline residue in the amino acid sequence of the fucose-binding protein represented by SEQ ID NO: 4 consisting of 127 amino acids, and the 72nd cysteine residue. A fucose-binding protein obtained by adding an oligopeptide sequence containing histidine to the N-terminus and an oligopeptide sequence containing cysteine to the C-terminus (hereinafter referred to as rBC2LCN ( A).) is related to the production. The manufacturing method will be described below in (1) to (7).
(1) Preparation of expression vector pTrc-rBC2LCN(A) and recombinant E. coli W3110/pTrc-rBC2LCN(A) Expression vector pTrc-rBC2LCN(A) is an expression vector for expressing rBC2LCN lectin 127Q39L/C72G. The 5th to 10th amino acid sequence of rBC2LCN (A) represented by SEQ ID NO: A is an oligopeptide sequence containing histidine, the 15th to 141st is the amino acid sequence of SEQ ID NO: 4, and the 142nd to 148th is cysteine. corresponds to an oligopeptide sequence containing

Expression vector pTrc-rBC2LCN(A) and recombinant E. coli W3110/pTrc-rBC2LCN(A) were prepared according to the method described in Example 6 of JP-A-2020-58343. That is, using the expression vector pET-BC2LCN (127Q39L/C72G) cys described in (1) of Example 3 of JP-A-2020-58343 as a template, PCR by the method disclosed in JP-A-2018-000038. carried out.

The resulting PCR product was digested with restriction enzymes NcoI and HindIII, and the expression vector pTrc-PelBV3Km similarly digested with restriction enzymes NcoI and HindIII (Carbenicillin in the expression vector pTrc-PelBV3 prepared by the method described in WO2015/199154 expression vector into which a kanamycin resistance gene was introduced instead of the resistance gene), and a ligation reaction was performed. This ligation product was used to transform E. coli W3110 to obtain recombinant E. coli W3110/pTrc-BC2LCN(A). The recombinant E. coli W3110/pTrc-BC2LCN (A) obtained is cultured by the method disclosed in JP-A-2018-000038, and the expression vector pTrc-BC2LCN (127Q39L/C72G) is extracted from the cells. got As a result of confirming the nucleotide sequence by sequence analysis, it was confirmed that the expression vector pTrc-BC2LCN(A) contained the nucleotide sequence of SEQ ID NO:C encoding the amino acid sequence of SEQ ID NO:A.
(2) Production of rBC2LCN lectin 127Q39L/C72G using recombinant E. coli. Sterilized and added kanamycin sulfate after cooling) was inoculated into a 500 mL baffled Erlenmeyer flask (three), and shaken at 30° C. for 20 hours at a shaking speed of 130 times per minute (amplitude 25 mm, circular motion). was pre-cultured.

For the preparation of 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.

Next, an aqueous solution of each component shown in Table 3 was separately prepared, and the aqueous solutions other than 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 Advantech). Add these sterilized aqueous solutions of each component to a 10 L fermenter containing the sterilized solution containing the components shown in Table 2 above (add the amount shown in Table 3) to prepare the main culture medium. bottom.

Next, feed media shown in Table 4 were prepared. Specifically, an aqueous glucose solution, an aqueous yeast extract solution, and an aqueous magnesium sulfate heptahydrate solution prepared at predetermined concentrations were separately autoclaved (121° C., 30 minutes), cooled to room temperature, and then autoclaved. , was prepared by mixing filter-sterilized aqueous kanamycin sulfate solutions in the proportions shown in Table 4 in a sterile container.

After confirming that the turbidity (OD600) of the pre-culture solution was 3.0 or more, 300 mL of this pre-culture solution was added to a 10-L fermentor filled with a main culture medium to initiate main culture. In the main culture, the culturing apparatus was BMS-10 manufactured by ABLE, the stirring blades were four φ95 mm impeller blades of ABLE manufactured by combining two stages with a basket-shaped net, and the DO sensor was SDOC-16 type DO electrode manufactured by ABLE. Inpro 3030 type pH electrode manufactured by Mettler Tredo was used as a pH sensor, the air flow rate after 0.22 μm filter sterilization was 6.0 L/min, the culture temperature was 30° C., and the pH of the culture solution was 6.9 to 7.1. was adjusted within the range of

A 14% by weight ammonia aqueous solution and a 42% by weight phosphoric acid aqueous solution were used to adjust the pH of the culture solution. In addition, the stirring rotation speed at the start of the main culture is set to 400 rpm, and when the dissolved oxygen concentration falls below 2.2 mg / L, the stirring rotation speed is increased in a stepwise manner. continued culturing. In the culture apparatus used for the main culture, the DO concentration in the culture solution, that is, the consumption state of the carbon source introduced at the beginning of the main culture, is detected by detecting the signal from the DO electrode using a computer installed with the ABLE culture control program. It was confirmed. In this DO electrode, the above-mentioned main culture medium was added to the fermenter and the temperature was set to 30 ° C., and then the dissolved oxygen concentration was stirred for 45 minutes at an aeration rate of 6.0 L / min and a stirring blade speed of 700 rpm. was used after calibration as 7.5 mg/L (saturated concentration). Sampling liquids taken periodically during the culture were measured with a spectrophotometer (Hitachi High-Tech Science, U-2900 model) for OD600 (turbidity at 600 nm), and further with a glucose analyzer (YSI model, 2700). to measure glucose concentration.

About 10 hours after the start of the main culture, the DO concentration did not decrease (the stirring rotation speed in the cascade control did not increase), and the analysis values of the sample solution at this time were OD600 = 24 and glucose concentration = 0.1 g/L. rice field. In other words, the glucose added to the culture solution at the start of the culture was metabolized by E. coli and depleted. As a result, the growth of E. coli was suppressed, resulting in a decrease in oxygen consumption and an increase in the DO concentration indicated by the DO sensor. ing.

After 11 hours from the start of the main culture, the stirring rotation speed cascade control was terminated and the stirring rotation speed was fixed at 800 rpm. When the DO concentration exceeded 3.0 mg / L, the feed medium (glucose, yeast Extract, magnesium sulfate) was set to be supplied dropwise at 2.0 g each. A Watson Marlowe metering pump 101U high speed type was used for the supply, and the flow rate was set to 6.0 mL/min.

After confirming that OD600 had reached 130 27 hours after the start of the main culture, the set temperature of the culture solution was changed to 25°C. Incidentally, the amount of the feed medium dropped after 27 hours was 1200 g (1000 mL). After confirming that the temperature of the culture solution reached 25° C., the stirring speed was changed to 600 rpm, IPTG (manufactured by Fujifilm Wako Pure Chemical Industries) was dissolved in pure water, and filtered through a 0.22 μm filter (manufactured by Advantech). Induction was initiated by addition of 8.1 mL of 0.5 M IPTG aqueous solution prepared by filter sterilization (IPTG final concentration = 0.83 mM). OD600 reached 160 50 hours after the start of the main culture, and the rotational speed of the stirring blade was lowered to 400 rpm. OD600 reached 180 68 hours after the start of the main culture, and the culture was terminated. At the end of the culture, the total amount of feed medium added was 2500 g (2083 mL), the total amount of 14% by weight ammonia water added was 220 mL, and the total amount of 42% by weight phosphoric acid added was 25 mL.

As a result of dropping the feed medium by a method of adding a carbon source to the culture solution in conjunction with the increase in DO concentration (DO stat method), the change from the start of the feed batch (11 hours after the start of the main culture) to the end of the culture (68 hours after the start of the main culture) ), the glucose concentration in the culture medium was maintained at 0.6 g/L or less, and the dissolved oxygen concentration during the same period varied in the range from 0.0 mg/L to 3.3 mg/L. This indicates that the carbon source and the nitrogen source were intermittently supplied using the DO concentration, which increased in response to the shortage of glucose during the culture, as an index. In addition, the total amount of carbon source (glucose) input through the main culture was 951 g (including initial input amount), and the total amount of nitrogen source (yeast extract) was 419 g (including initial input amount).

After the culture is completed, the obtained 5500 mL culture solution is dispensed into a 900 mL centrifugal container and centrifuged (4 °C, 9,000 rpm, 120 minutes), 1170 g of cells were recovered.

In addition, the culture solution was diluted 10-fold with BugBuster HT (manufactured by Merck), incubated at 25 ° C. for 1 hour, and then subjected to SDS-PAGE (manufactured by ATTO, staining solution: EzApply, gel: P-R16.5S, running buffer: EzRunT, running Apparatus: WSE-1150P) method was used to confirm the band concentration around 14 kDa. As a result, the productivity of rBC2LCN lectin 127Q39L/C72G per 1 L of culture medium was confirmed to be approximately 7.0 g/L of culture medium. bottom.
(3) Surfactant extraction of rBC2LCN lectin 127Q39L/C72G To about 30 g of the cells collected in Example 1 (2), an extraction solution whose composition is shown in Table 5 was added and stirred at room temperature for 30 minutes. 6 was added and stirred overnight. After that, the treated liquid is dispensed into a 50 mL centrifugal container (himac50TC tube manufactured by Koki Holdings) and centrifuged using a centrifuge (body: CR20GII manufactured by Koki Holdings, rotor: R15A manufactured by Koki Holdings) ( 4° C., 15,000 rpm, 30 min), 75 mL of supernatant was collected as a soluble protein extract containing the rBC2LCN lectin 127Q39L/C72G. The recovered soluble protein extract containing rBC2LCN lectin 127Q39L/C72G was filtered through a 0.2 μm filter and then used for purification by cation exchange chromatography as described below. The prepared soluble protein extract was used as an "extract" for analysis by the SDS-PAGE method described later.

(4) Purification of rBC2LCN lectin 127Q39L/C72G by cation exchange chromatography Purification of rBC2LCN lectin 127Q39L/C72G from the soluble protein extract recovered in (3) of Example 1 was performed using TOYOPEARL GigaCap CM-650M (manufactured by Tosoh , a packing material for cation exchange chromatography, hereinafter abbreviated as GigaCap CM-650M). Specifically, rBC2LCN lectin 127Q39L/C72G was purified by the methods described in (4-1) to (4-6) below.

(4-1): ToyoScreen GigaCap CM-650M (manufactured by Tosoh, the GigaCap CM-650M packed column, volume is 5 mL), 20 mL of buffer A (20 mM sodium citrate buffer containing 50 mM sodium chloride, pH 5.0 , listed in Table 7) was passed through at a rate of 2.0 mL/min for equilibration.
(4-2): The pH of the soluble protein extract containing the rBC2LCN lectin 127Q39L/C72G was adjusted to pH 5.0 by adding 1.0 M citric acid. Furthermore, pure water was added to adjust the electric conductivity to 7.5 mS/cm.
(4-3): To the column equilibrated with buffer A in (4-1), the extract solution adjusted for pH and electrical conductivity in (Ex. 1-1) was applied at a rate of 2.0 mL / min. was added with
(4-4): Add 50 mL of buffer A at a rate of 2.0 mL/min to the column to which the soluble protein extract was added in (4-3) above to wash substances that do not bind to GigaCap CM-650M. Removed.

(4-5): To the column washed with buffer A in (4-4), 25 mL 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 is 500 mM) was added at a rate of 2.0 mL/min to wash the GigaCap CM-650M. .

(4-6): To the column washed with the mixed solution of buffer A and buffer B in (4-5), 29 mL of buffer B was added at a rate of 2.0 mL / min, and GigaCap CM- While washing 650M, the washing liquid was recovered. The recovered liquid was used as the "100% B recovered liquid" for analysis by the SDS-PAGE method described later.

(5) Analysis The “100% B recovered solution” obtained in (4-6) above was analyzed by SDS-PAGE. For analysis by the SDS-PAGE method, a commercially available acrylamide gel (manufactured by ATTO, P-R16.5S) was used, and SDS-PAGE prepared from the recovered solution by the method described in (5-1) below. An analytical sample solution was used.

(5-1): The protein concentration of the "100% B recovery solution" was adjusted to 0.20 mg/mL with BugBuster HT (manufactured by Merck). The extract was diluted 10-fold with BugBuster HT. After mixing 15 μL of the undiluted flow-through recovery solution and each of the prepared samples with 15 μL of commercially available SDS sample buffer (manufactured by ATTO, EzApply), the mixture was heated at 94° C. for 5 minutes. 10 μL of the resulting solution was added to an SDS-PAGE lane and analyzed by the SDS-PAGE method (electrophoresis buffer: EzRunT manufactured by ATTO, electrophoresis tank: WSE-1150 manufactured by ATTO, staining solution: AE-1340 manufactured by ATTO).

A single band was confirmed near the molecular weight of the rBC2LCN lectin 127Q39L/C72G monomer (approximately 14 kDa). It was confirmed that there existed, that is, high-purity purification could be achieved.

The absorbance at 280 nm of these "100% B recovery solutions" was measured using a microvolume spectrophotometer as a purified rBC2LCN lectin 127Q39L/C72G solution. After calculating the protein concentration in the purified rBC2LCN lectin 127Q39L/C72G solution with the molar extinction coefficient of rBC2LCN lectin 127Q39L/C72G as 1.0, based on the obtained protein concentration, As a result of calculating the productivity, the productivity was as high as 4.9 g/L-culture solution.
(6) Concentration of rBC2LCN lectin 127Q39L/C72G The "100% B recovery liquid" obtained in (4-6) was filtered through an ultrafiltration membrane (Merck, Amicon Ultra-15, molecular weight cut off = 10,000). and buffer-exchanged with D-PBS(-) (manufactured by Cell Science Laboratory). The resulting purified lectin solution was sterile-filtered through a 0.2 μm filter, sealed, and stored in a refrigerator at 4°C. The concentration of this purified lectin solution was 10.2 mg/mL (280 nm absorbance).
(7) Preparation of solution for immobilization reaction Subsequently, a buffer solution for immobilization reaction was prepared. The properties of the buffer are 0.20 M phosphate, 0.50 M NaCl, 0.02 M EDTA, pH 7.4. 20.0 mL of the purified lectin solution, 122.4 mL of the immobilization reaction buffer, 9.6 mL of D-PBS(-), and 1.3 mL of 0.1 M TCEP are uniformly mixed to prepare a solution for the immobilization reaction, It was allowed to stand at 23°C for 4 hours. The lectin concentration of this immobilization reaction solution is 1.33 mg/mL. TCEP (Nacalai Tesque, Tris(2-carboxyethyl)phosphine hydrochloride) is a reducing agent for cleaving the double bond (disulfide bond) of the cysteine tag at the end of the lectin molecule. Reduction of the disulfide bond and exposure of the —SH group of the cysteine tag greatly improve the reactivity with the maleimide group immobilized on the water-insoluble carrier.

Preparation Example 2 Production of Adsorbent 127Q39L/C72G-A Toyopearl HW-40EC was classified with a stainless steel standard sieve having an opening of 600 μm equipped with an ultrasonic oscillator of 35 kHz, and the fraction that passed through the sieve was collected. This recovered suspension was carefully classified again with a stainless steel standard sieve with an opening of 300 μm equipped with an ultrasonic oscillator, and the particle size distribution of the fraction remaining on the sieve was measured using a laser diffraction particle size distribution meter (Microtrac Bell). MT-3200 II type, using pure water as a dispersion medium), and confirmed that the mixing ratio of particles of less than 300 μm was less than 5.0%, and was collected as a carrier.

Next, in a 500 mL Teflon (registered trademark) container, 60.0 g of the previous classified Toyopearl HW-40EC suction gel (hydrous gel), 30.0 g of pure water, 33.0 g of DMSO (dimethyl sulfoxide) , manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 30.0 g of epichlorohydrin (manufactured by Tokyo Kasei) are sequentially charged and suspended, and 65.0 mL of a 5.0 M NaOH aqueous solution (manufactured by Kanto Kagaku) is added thereto, Toyopearl HW-40EC was epoxidized by shaking in a shaker at 30°C for 3 hours. After completion of the reaction, the suspension was filtered through a glass filter and washed with pure water until the filtrate became neutral. As a result of quantifying the epoxy groups, it was confirmed that 317 mmol/L of epoxy groups were introduced into the carrier wetted with pure water.

Subsequently, 55.0 g of the Toyopearl HW-40EC suction gel epoxidized by introducing epichlorohydrin in the previous stage was placed in a 500 mL Teflon (registered trademark) container, and 82.5 g of 30% by weight prepared in advance was charged. A dextran aqueous solution (weight average molecular weight: 500,000, manufactured by Sigma-Aldrich) was added to mix the two together, and then shaken in a shaker at 30°C for 30 minutes. Next, 5.8 mL of 48% NaOH aqueous solution (manufactured by Kanto Kagaku) was added to the reaction vessel, and the mixture was shaken in a shaker at 30°C for an additional 18 hours to immobilize the dextran on the epoxidized Toyopearl HW-40EC. After completion of the reaction, the suspension was filtered through a glass filter and washed with pure water until the filtrate became neutral to prepare dextran-modified Toyopearl HW-40EC. As a result of quantifying dextran, it was confirmed that 570 mg/L-pure water-wet carrier dextran had been introduced.

Subsequently, 50.0 g of the dextran-modified Toyopearl HW-40EC suction gel in the previous stage and 100.0 mL of a previously prepared tetraethylene glycol diglycidyl ether aqueous solution (Denacol manufactured by Nagase ChemteX Corporation) were added to a 500 mL Teflon (registered trademark) container. Prepared from EX-821, concentration 100 mg / mL), after shaking for 30 minutes in a shaker at 30 ° C., 1.05 mL of 48% NaOH aqueous solution (Kanto Kagaku) was added to the reaction vessel, The mixture was shaken in a shaker for 8 hours, and tetraethylene glycol diglycidyl ether was added. After completion of the reaction, the suspension was filtered through a glass filter and washed with pure water until the filtrate became neutral. As a result of quantitative determination of epoxy groups, it was confirmed that epoxy groups were introduced into the carrier wetted with 74 mmol/L-pure water.

Subsequently, 45.0 g of Toyopearl HW-40EC suction gel to which tetraethylene glycol diglycidyl ether was added in the previous stage was added to a 500 mL Teflon (registered trademark) container, and 93.0 mL of 0.5 M ethylenediamine aqueous solution ( (prepared from anhydrous ethylenediamine manufactured by Tokyo Chemical Industry Co., Ltd.) was added, and the epoxy groups were aminated by shaking in a shaker at 50°C for 3 hours. After completion of the reaction, the suspension was filtered through a glass filter and washed with pure water until the filtrate became neutral.

Subsequently, 45.0 g of the amino group-introduced Toyopearl HW-40EC suction gel collected in the previous step was added to a 500 mL Teflon (registered trademark) container, and the previously prepared N-succinimidyl 3-maleimidopropionate/DMSO After adding 90.0 mL of a solution (prepared from N-succinimidyl 3-maleimidopropionate manufactured by Tokyo Kasei, concentration 10 mg/mL), maleimide groups were introduced into the carrier by shaking in a shaker at 35° C. for 4 hours. After completion of the reaction, the suspension was filtered through a glass filter, and the filter was washed with 90 mL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) three times to remove unreacted N-succinimidyl 3-maleimidopropionate. Subsequently, it was thoroughly washed with pure water until no residual DMSO was left. As a result of quantifying maleimide groups, it was confirmed that 14 mmol/L of maleimide groups were introduced into the support wetted with pure water.

Subsequently, using the rBC2LCN lectin immobilization reaction solution prepared in Preparation Example 1(7), a reaction was carried out with Toyopearl HW-40EC introduced with a maleimide group in the previous step. Into a 500 mL Teflon (registered trademark) container, 40.0 g of the maleimide group-introduced HW-40EC suction gel in the previous stage was collected, and the BC2LCN lectin prepared in advance at a concentration of 1.33 mg / mL in Preparation Example 1 (7). 61 mL of the immobilization reaction solution was added, and the reaction was slowly shaken at 35° C. for 15 hours. After the reaction, the suspension was filtered through a glass filter, 100 mL of D-PBS(−) was poured into the wet gel on the filter, gently stirred and washed with a spatula, and filtered again. This washing operation was repeated 5 times to collect 40 g of suction gel, which was dispersed in D-PBS(-) to obtain a cell adsorbent 127Q39L/C72G-A (the volume of the cell adsorbent in the suspension was 52 mL). The amount of rBC2LCN lectin contained in the supernatant before and after the reaction was analyzed by Pierce Protein Assay (manufactured by Thermo). The immobilized amount was assumed to be 302 mg/L-wet carrier. In addition, it was confirmed that the particle size distribution of the cell adsorbent 127Q39L/C72G-A was the same as that of Toyopearl HW-40EC, which was first wet-classified in the previous step, and had not changed (particle size range in wet state = 300 to 600 μm, less than 300 μm <5.0%). FIG. 1 shows microscopic images of the carrier after classification and the cell adsorbent 127Q39L/C72G-A prepared by immobilizing the rBC2LCN lectin in a wet state, and FIG. 2 shows the particle size distribution.

Preparation Example 3 Production of Adsorbent 127Q39L/C72G-B In Preparation Example 3, compared to Preparation Example 2, the amount of immobilized rBC2LCN127Q39L/C72G, which is a fucose-binding protein, was doubled to prepare adsorbent 127Q39L/C72G. -B is a preparation example. The concentration of the immobilization reaction solution prepared in Preparation Example 1(7) was changed from 1.33 mg/mL to twice the concentration (2.66 mg/mL). Subsequently, in the same procedure as in Preparation Example 2, a reaction from wet classification of Toyopearl HW-40EC (manufactured by Tosoh) to introduction of a maleimide group was carried out. 40.0 g of maleimide group-introduced Toyopearl HW-40EC suction gel was placed in a 500 mL Teflon (registered trademark) container, and 61 mL of a BC2LCN lectin immobilization reaction solution prepared in advance to a concentration of 2.66 mg/mL was added. , 35° C. for 15 hours with slow shaking. A washing operation was performed with D-PBS(-) in the same manner as in Preparation Example 2, 40 g of suction gel was collected, and this was dispersed in D-PBS(-) to obtain cell adsorbent 127Q39L/C72G-B (suspension Volume of cell adsorbent in suspension is 52 mL). The amount of rBC2LCN lectin immobilized on the cell adsorbent 127Q39L/C72G-B was 632 mg/L-wet carrier. As for the particle size distribution, the wet state particle size range was 300 to 600 μm, and the proportion of particles less than 300 μm was <5.0%. Fig. 1 shows microscopic images of the carrier after classification and the cell adsorbent 127Q39L/C72G-B prepared by immobilizing the rBC2LCN lectin in a wet state, and Fig. 2 shows the particle size distribution.

Preparation Example 4 Preparation of Adsorbent 127Q39L/C72G-C Preparation Example 4 is an adsorbent 127Q39L/C72G in which the immobilized amount of rBC2LCN127Q39L/C72G, which is a fucose-binding protein, is increased threefold compared to Preparation Example 2. -C preparation example. The concentration of the immobilization reaction solution prepared in Preparation Example 1(7) was changed from 1.33 mg/mL to 3 times the concentration (3.99 mg/mL). Subsequently, in the same procedure as in Preparation Example 2, a reaction from wet classification of Toyopearl HW-40EC (manufactured by Tosoh) to introduction of a maleimide group was carried out. 40.0 g of maleimide group-introduced Toyopearl HW-40EC suction gel was placed in a 500 mL Teflon (registered trademark) container, and 61 mL of a BC2LCN lectin immobilization reaction solution prepared in advance to a concentration of 3.99 mg/mL was added. , 35° C. for 15 hours with slow shaking. A washing operation was performed with D-PBS(-) in the same manner as in Preparation Example 2, 40 g of suction gel was collected, and this was dispersed in D-PBS(-) to obtain cell adsorbent 127Q39L/C72G-C (suspension Volume of cell adsorbent in suspension is 52 mL). The amount of rBC2LCN lectin immobilized on the cell adsorbent 127Q39L/C72G-C was 1043 mg/L-wet carrier. As for the particle size distribution, the wet state particle size range was 300 to 600 μm, and the proportion of particles less than 300 μm was <5.0%. Fig. 1 shows the microscopic images of the carrier after classification and the cell adsorbent 127Q39L/C72G-C prepared by immobilizing the rBC2LCN lectin in a wet state, and Fig. 2 shows the particle size distribution.

Preparation Example 5 Production of Adsorbent 127Q39L/C72G-D Preparation Example 5 is a preparation example of adsorbent 127Q39L/C72G-D in which the particle size of Toyopearl HW-40EC (manufactured by Tosoh) used in Preparation Example 2 is changed. Specifically, Toyopearl HW-40EC was classified with a stainless steel standard sieve having an opening of 300 μm equipped with an ultrasonic oscillator of 35 kHz, and the fraction that passed through the sieve was collected. This recovered suspension was carefully classified again with a stainless steel standard sieve with an opening of 212 μm equipped with an ultrasonic oscillator, and the particle size distribution of the fraction remaining on the sieve was measured using a laser diffraction particle size distribution meter (Microtrac Bell). MT-3200 II type, using pure water as a dispersion medium), and confirmed that the mixing ratio of particles of less than 212 μm was less than 5.0%, and was collected as a carrier. Using this carrier, a reaction from epichlorohydrin to N-succinimidyl 3-maleimidopropionate was carried out in the same manner as in Preparation Example 2. 40.0 g of this maleimide group-introduced Toyopearl HW-40EC suction gel was charged into a 500 mL Teflon (registered trademark) container, and the BC2LCN lectin prepared in advance at a concentration of 1.33 mg / mL in Preparation Example 1 (7) was immobilized. 61 mL of the reaction solution was added, and the mixture was slowly shaken at 35° C. for 15 hours. A washing operation was performed with D-PBS(-) in the same manner as in Preparation Example 2, 40 g of suction gel was collected, and this was dispersed in D-PBS(-) to obtain cell adsorbent 127Q39L/C72G-C (suspension Volume of cell adsorbent in suspension is 52 mL). The amount of rBC2LCN lectin immobilized on the cell adsorbent 127Q39L/C72G-C was 447 mg/L-wet carrier. As for the particle size distribution, the wet state particle size range was 212 to 300 μm, and the proportion of particles less than 212 μm was <5.0%. Fig. 3 shows the microscopic images of the carrier after classification and the cell adsorbent 127Q39L/C72G-D prepared by immobilizing the rBC2LCN lectin in a wet state, and Fig. 4 shows the particle size distribution.

Preparation Example 6 Preparation of Adsorbent 127Q39L/C72G-E Preparation Example 6 is a preparation example of adsorbent 127Q39L/C72G-E in which the particle size of Toyopearl HW-40EC (manufactured by Tosoh) used in Preparation Example 2 is changed. Specifically, the Toyopearl HW-40EC was filtered from the dispersion medium and dried in a hot air dryer at 100°C until the water content was less than 1%. Dry classification was performed using a stainless steel standard sieve with an opening of 212 μm, and the fraction that passed through the sieve was collected. This recovered fraction is carefully classified again with a stainless steel standard sieve with an opening of 150 μm equipped with an ultrasonic oscillator, and the particle size distribution of the fraction remaining on the sieve is measured by a laser diffraction particle size distribution analyzer (Microtrack Bell). , MT-3200II type, using pure water as a dispersion medium), and confirmed that the mixed ratio of particles of less than 176 μm was less than 6.0%, and was collected as a carrier. Note that Toyopearl HW-40EC has a property that the particle size becomes smaller when dried, and when the particles of 150 μm in the dry state are wetted with pure water, the particle size swells to 192 μm. Similarly, particles of 212 μm in dry state swell to a particle diameter of 263 μm when wetted with pure water. In other words, dry classification of Toyopearl HW-40EC in a dry state with a 212 μm/150 μm sieve has the same meaning as wet classification with a 263 μm/192 μm sieve.

Using this carrier, a reaction from epichlorohydrin to N-succinimidyl 3-maleimidopropionate was carried out in the same manner as in Preparation Example 2.
40.0 g of this maleimide group-introduced Toyopearl HW-40EC suction gel was charged into a 500 mL Teflon (registered trademark) container, and the BC2LCN lectin prepared in advance at a concentration of 1.33 mg / mL in Preparation Example 1 (7) was immobilized. 61 mL of the reaction solution was added, and the mixture was slowly shaken at 35° C. for 15 hours. A washing operation was performed with D-PBS(-) in the same manner as in Preparation Example 2, 40 g of suction gel was collected, and this was dispersed in D-PBS(-) to obtain cell adsorbent 127Q39L/C72G-C (suspension Volume of cell adsorbent in suspension is 52 mL). The amount of rBC2LCN lectin immobilized on the cell adsorbent 127Q39L/C72G-C was 300 mg/L-wet carrier. As for the particle size distribution, the wet state particle size range was 176 to 300 μm, and the proportion of particles less than 176 μm was <6.0%. FIG. 5 shows microscopic images of the carrier after classification and the cell adsorbent 127Q39L/C72G-E prepared by immobilizing the rBC2LCN lectin in a wet state, and FIG. 6 shows the particle size distribution.

Preparation Example 7 Preparation of Adsorbent 127Q39L/C72G-F Preparation Example 7 is a preparation example of adsorbent 127Q39L/C72G-F in which the particle size of Toyopearl HW-40EC (manufactured by Tosoh) used in Preparation Example 2 is changed. Specifically, Toyopearl HW-40EC was classified with a stainless steel standard sieve having an opening of 212 μm equipped with an ultrasonic oscillator of 35 kHz, and the fraction that passed through the sieve was collected. This recovered suspension was carefully classified again with a stainless steel standard sieve with an opening of 100 μm equipped with an ultrasonic oscillator, and the particle size distribution of the fraction remaining on the sieve was measured using a laser diffraction particle size distribution analyzer (Microtrac Bell). MT-3200II type, using pure water as a dispersion medium), and confirmed that the mixed ratio of particles of less than 100 μm was less than 5.0%, and collected as a carrier. Using this carrier, a reaction from epichlorohydrin to N-succinimidyl 3-maleimidopropionate was carried out in the same manner as in Preparation Example 2. 40.0 g of this maleimide group-introduced Toyopearl HW-40EC suction gel was charged into a 500 mL Teflon (registered trademark) container, and the BC2LCN lectin prepared in advance at a concentration of 1.33 mg / mL in Preparation Example 1 (7) was immobilized. 61 mL of the reaction solution was added, and the mixture was slowly shaken at 35° C. for 15 hours. A washing operation was performed with D-PBS(-) in the same manner as in Preparation Example 2, 40 g of suction gel was collected, and this was dispersed in D-PBS(-) to obtain cell adsorbent 127Q39L/C72G-C (suspension Volume of cell adsorbent in suspension is 52 mL).

The amount of rBC2LCN lectin immobilized on the cell adsorbent 127Q39L/C72G-C was 338 mg/L-wet carrier. As for the particle size distribution, the wet state particle size range was 100 to 212 μm, and the proportion of particles less than 100 μm was <5.0%. FIG. 7 shows microscopic images of the carrier after classification and the cell adsorbent 127Q39L/C72G-F prepared by immobilizing the rBC2LCN lectin in a wet state, and FIG. 8 shows the particle size distribution.

Example 1 Separation and Recovery of 201B7 Cells and Human Mesenchymal Stem Cells from a Mixture of 201B7 Cells and Human Mesenchymal Stem Cells Using an Adsorbent in Which a Fucose-Binding Protein is Immobilized on a Water-Insoluble Carrier with a Particle Size of 300 μm or More and Less than 600 μm Implementation Example 1 is the separation of 201B7 cells and human mesenchymal stem cells from a mixture of 201B7 cells and human mesenchymal stem cells using the adsorbents 127Q39L/C72G-A, B, and C produced in Preparation Example 1 above. It relates to the collection method.
(1) Preparation of Column Filled with Adsorbent A column was prepared by attaching a 40 μM polyester mesh filter (manufactured by BioLab) between a 2.5 mL syringe (manufactured by Terumo) and an injection needle (manufactured by Terumo, 22G). .
Next, the adsorbents 127Q39L/C72G-A, B, and C, which are adsorbents in which the fucose-binding proteins prepared in Preparation Examples 2, 3, and 4 are immobilized on a water-insoluble carrier having a particle size of 300 μm or more and less than 600 μm, are subjected to MACS. After replacement with a buffer solution, a 50% suspension of the adsorbent was prepared so that the sedimentation volume of the adsorbent after standing for 12 hours or more was 50%, and 4.0 mL was added to the prepared column. , each adsorbent was packed in a column (adsorbent capacity: 2.0 mL). Two identical columns were prepared for each adsorbent. Columns packed with adsorbent 127Q39L/C72G-A are referred to as column No. 1 below. 1, 2, a column packed with adsorbent 127Q39L/C72G-B was designated as column NO. 3, 4, the column packed with adsorbent 127Q39L/C72G-A was designated as column NO. 5 and 6.
(2) Culture of 201B7 Cells and Preparation of Cell Suspension As human iPS cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, licensed from iPS Academia Japan, Purchased human iPS cell strain 201B7 (hereinafter sometimes abbreviated as 201B7 cells) was used.

The culture of 201B7 cells was performed using a petri dish for adhesion culture (manufactured by Corning) by the following method. iMatrix-511 (manufactured by Nippi) prepared in advance was diluted to 3 μg/mL in D-PBS (−), added to the petri dish, and left at 4° C. overnight or longer to spread iMatrix-511 onto the petri dish culture surface. was coated. After discarding the iMatrix-511 solution in the coated petri dish, StemFit AK02N medium (manufactured by Ajinomoto Co., Ltd.), which is an iPS cell culture medium, was added and washed. 27632: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was suspended in the same medium to which 10 μM was added and seeded. After culturing overnight at 37°C in a 5% CO2 atmosphere, the StemFit AK02N medium containing Y-27632 was discarded, and the medium was replaced with StemFit AK02N medium not containing Y-27632. , cell harvesting and passaging were performed.

Next, cell collection and cell suspension preparation were performed by the following methods. After washing the cells by adding D-PBS (-) to the petri dish and rinsing the cells, discarding the D-PBS (-) was repeated twice, and then the cells were washed with CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific). and Versene Solution (manufactured by Thermo Fisher Scientific) were added at a ratio of 1:1 and left at 37° C. for 10 minutes in a 5% CO 2 atmosphere. After confirming that the cells were detaching in a round shape, the cells were detached by repeating pipetting in the detachment solution and collected in a 50 mL tube. After centrifuging and sedimenting the collected cells, the cells were suspended in MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells. After repeating the cell washing operation twice, the cells were suspended in a MACS buffer and filtered using a cell strainer to prepare a cell suspension of 201B7 cells. A portion of the obtained 201B7 cell solution was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Hereinafter, based on this cell density, the amount of cells to be added to the column was calculated from cell density×amount of cell solution applied to the column.

(3) Culture of Human Mesenchymal Stem Cells and Preparation of Cell Suspension Human mesenchymal stem cells UE6E7T-2 (hereinafter referred to as human mesenchymal stem cells) were obtained from the JCRB cell bank. In addition, culture was performed according to the following procedure. Human mesenchymal stem cells, which are adherent cells, were prepared with 10% FBS (manufactured by Biological Industries), 4 mM L-glutamine (manufactured by Fujifilm Wako Pure Chemical Industries) and an antibiotic solution (penicillin-streptomycin solution, manufactured by Fujifilm Wako Pure Chemical Industries). Using D-MEM medium (Low Glucose, manufactured by Sigma-Aldrich) supplemented with, cells are seeded in a 6 cm diameter adherent culture petri dish (Corning) or a 10 cm diameter adherent culture petri dish (Corning), 5% CO2 The cells were cultured at 37°C in an atmosphere. Next, fluorescent staining of human mesenchymal stem cells using Cell Tracker Red (manufactured by Thermo Fisher Scientific) was performed by the following method. After discarding the medium in the petri dish in which the human mesenchymal stem cells were being cultured, serum-free RPMI 1640 medium was added to wash the cells, and then the serum-free RPMI 1640 medium was discarded. Next, Cell Tracker Red dissolved in serum-free RPMI 1640 medium to a final concentration of 10 μM was added and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the fluorescent reagent solution, D-MEM medium supplemented with 10% FBS, 4 mM L-glutamine and an antibiotic solution was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. Next, after discarding the medium, fresh D-MEM medium supplemented with 10% FBS, 4 mM L-glutamine and an antibiotic solution was added, and cultured overnight at 37° C. in a 5% CO 2 atmosphere.

Next, cell collection and cell suspension preparation were performed by the following methods. After the medium in the petri dish during cell culture was discarded and D-PBS(-) was added, the cells were washed and D-PBS(-) was discarded. Next, an appropriate amount of Accutase (manufactured by Innovative Cell Technology) was added and left for several minutes to detach the human mesenchymal stem cells, which were collected in a 50 mL tube. After centrifuging and sedimenting the collected cells, the cells were suspended in MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells. After repeating the cell washing operation twice, the cells were suspended in MACS buffer and filtered using a cell strainer to prepare a cell suspension of human mesenchymal stem cells stained with Cell Tracker Red. A portion of the obtained human mesenchymal stem cell solution was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Hereinafter, based on this cell density, the amount of cells to be added to the column was calculated from cell density×amount of cell solution applied to the column.

(4) Adsorption and desorption of cells using a column filled with adsorbent The 201B7 cells prepared by the above method and human mesenchymal stem cells are mixed and diluted with MACS buffer to obtain a cell suspension volume of 1 mL. , The number of added cells was 2.2 x 10^6 for 201B7 cells, and 7.1 x 10^5 for human mesenchymal stem cells. It was added from the top of the column while standing. Next, 4 mL of MACS buffer solution was added from the top of the column, and a total of 5 mL of effluent cell fluid was fractionated from the 22G syringe needle at the bottom of the column. (Hereinafter, it describes as an outflow cell fluid). Next, after replacing the receiver, 4 mL of MACS buffer containing 0.2 M fucose and 10 mM EDTA was added from the top of the column, and a total of 4 mL of cell solution was collected from the syringe needle at the bottom of the column (hereinafter referred to as described as desorbed cell sap).

(5) Measurement of cell recovery rate of 201B7 cells and human mesenchymal stem cells After adding MACS buffer to each of the effluent cell sap and desorbed cell sap obtained by the above operation and diluting them, 2 mL of each cell strainer - Dispensed into 5 mL polystyrene round tubes with caps (manufactured by BD Japan). After adding 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as an internal standard bead for counting cells and 50 μL of 7-AAD as a cell viability determination reagent, the number of cells was measured using a cell sorter BD FACSAria (manufactured by BD Japan). gone. The number of 201B7 cells and human mesenchymal stem cells contained in each cell solution was calculated by proportional calculation based on the number of particles of internal standard beads obtained by dot plot (this cell number is the number of outflow cells and the number of cells, respectively). the number of detached cells). The recovery rate of each cell in each effluent cell sap and desorbed cell sap was calculated by dividing the number of effluent cells and the number of desorbed cells by the number of each cell applied to the column.

Table 8, FIG. 5 and FIG. 6 show the results of calculating the recovery rate of each cell. The cell recovery rate in the effluent cell fluid was measured by column No. 1 packed with adsorbent 127Q39L/C72G-A. 1, 201B7 cells 3.4%, human mesenchymal stem cells 84.6%, NO. Column No. 2 packed with 3.0% 201B7 cells, 90.5% human mesenchymal stem cells, and adsorbent 127Q39L/C72G-B. 3, 1.2% of 201B7 cells, 81.9% of human mesenchymal stem cells, NO. Column No. 4 packed with 1.1% 201B7 cells, 70.7% human mesenchymal stem cells, and adsorbent 127Q39L/C72G-C. 5, 1.2% of 201B7 cells, 78.2% of human mesenchymal stem cells, NO. 6, 201B7 cells were 1.1% and human mesenchymal stem cells were 77.3%. In addition, the cell recovery rate in the desorbed cell sap was measured by column No. 1 filled with adsorbent 127Q39L/C72G-A. 1, 93.0% of 201B7 cells, 1.2% of human mesenchymal stem cells, NO. Column No. 2 packed with 85.9% 201B7 cells, 2.0% human mesenchymal stem cells, and adsorbent 127Q39L/C72G-B. 3, 80.2% of 201B7 cells, 2.8% of human mesenchymal stem cells, NO. Column No. 4 packed with 92.4% 201B7 cells, 2.7% human mesenchymal stem cells, and adsorbent 127Q39L/C72G-C. 5, 201B7 cells 97.1%, human mesenchymal stem cells 2.5%, NO. 6 was 96.9% 201B7 cells and 2.9% human mesenchymal stem cells.

From this result, when the mixed cell sap of 201B7 cells and human mesenchymal stem cells was treated with an adsorbent in which a fucose-binding protein was immobilized on a water-insoluble carrier having a particle size of 300 μm or more and less than 600 μm, the effluent cell sap was Among them, it was found that human mesenchymal stem cells were obtained at a high recovery rate. In addition, it was confirmed that the recovery rate of 201B7 cells in the effluent cells was lower than around 3%. When used, the recovery rate of 201B7 cells is around 1%, and the adsorbent 632 mg/L, which is the amount of fucose-binding protein immobilized on the adsorbent 127Q39L/C72G-B, has sufficient 201B7 cell adsorption performance. It became clear. Furthermore, most of the 201B7 cells in the desorbed cell sap were recovered at a high recovery rate of 80% or more, and the human mesenchymal stem cell recovery rate in the desorbed cell sap was found to be as low as less than 3%. .

These results show that when using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier having a particle size of 300 μm or more and less than 600 μm, human mesenchymal stem cells, which are cells that do not adsorb to the adsorbent, and It was considered that the detached 201B7 cells could smoothly pass through the gaps between the adsorbent particles.

From the above, by treating the mixed cell sap of 201B7 cells and human mesenchymal stem cells using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier having a particle size of 300 μm or more and less than 600 μm, the effluent cell sap can be obtained. It was proved that human mesenchymal stem cells in the medium and 201B7 cells in the desorbed cell solution could be separated and recovered with high recovery and purity.

Comparative Example 1 Separation and Recovery of 201B7 Cells and Human Mesenchymal Stem Cells from a Mixture of 201B7 Cells and Human Mesenchymal Stem Cells Using an Adsorbent in Which a Fucose-Binding Protein is Immobilized on a Water-Insoluble Carrier with a Particle Size of Less Than 300 μm Comparative Example 1 201B7 cells and human mesenchymal stem cells from a mixture of 201B7 cells and human mesenchymal stem cells using the adsorbents 127Q39L/C72G-D, E, and F prepared in Preparation Examples 5, 6, and 7 above. It relates to the separation and recovery method of.

Adsorbent 127Q39L/C72G-D, which is an adsorbent in which the fucose-binding protein prepared in Preparation Example 5 is immobilized on a water-insoluble carrier having a particle size of 212 μm or more and less than 300 μm, and the fucose-binding protein prepared in Preparation Example 6 is 176 μm. Adsorbent 127Q39L/C72G-E, which is an adsorbent immobilized on a water-insoluble carrier with a particle size of 300 μm or more, and the fucose-binding protein prepared in Preparation Example 7 is immobilized on a water-insoluble carrier with a particle size of 100 μm or more and less than 212 μm. It was carried out in the same manner as in Example 1, except that the adsorbent 127Q39L/C72G-F, which is a modified adsorbent, was used. Columns packed with adsorbents 127Q39L/C72G-D are referred to as column NO. 7, 8, a column packed with adsorbent 127Q39L/C72G-E was designated as column NO. 9, 10, a column packed with adsorbent 127Q39L/C72G-F was designated as column NO. 11 and 12.

Table 8, FIG. 9, and FIG. 10 show the results of calculating the recovery rate of each cell. The cell recovery in the effluent cell fluid was measured by column No. 1 packed with adsorbent 127Q39L/C72G-D. 7, 0.4% of 201B7 cells, 83.0% of human mesenchymal stem cells, NO. Column No. 8 packed with 0.4% 201B7 cells, 84.5% human mesenchymal stem cells, and adsorbent 127Q39L/C72G-E. 9, 0.3% of 201B7 cells, 66.6% of human mesenchymal stem cells, NO. Column No. 10 packed with 0.2% 201B7 cells, 53.9% human mesenchymal stem cells, and adsorbent 127Q39L/C72G-F. 11, 0.3% of 201B7 cells, 20.8% of human mesenchymal stem cells, NO. 12, 0.2% of 201B7 cells and 21.7% of human mesenchymal stem cells. In addition, the cell recovery rate in the desorbed cell sap was measured by column No. 1 filled with adsorbent 127Q39L/C72G-D. 7, 80.7% of 201B7 cells, 10.6% of human mesenchymal stem cells, NO. Column No. 8 packed with 88.5% 201B7 cells, 13.8% human mesenchymal stem cells, and adsorbent 127Q39L/C72G-E. 9, 58.0% of 201B7 cells, 15.2% of human mesenchymal stem cells, NO. Column No. 10 packed with 51.0% 201B7 cells, 13.4% human mesenchymal stem cells, and adsorbent 127Q39L/C72G-F. 11, 65.9% of 201B7 cells, 32.3% of human mesenchymal stem cells, NO. 12 was 67.0% 201B7 cells and 32.6% human mesenchymal stem cells.

From this result, when the mixed cell solution of 201B7 cells and human mesenchymal stem cells was treated with an adsorbent in which a fucose-binding protein was immobilized on a water-insoluble carrier having a particle size of 212 μm or more and less than 300 μm, the outflow cells Human mesenchymal stem cells are obtained in the liquid at a high recovery rate, and an adsorbent in which the fucose-binding protein is immobilized on a water-insoluble carrier with a particle size of 176 μm or more and less than 300 μm or 100 μm or more and less than 212 μm is used. When used, it was found that the recovery of human mesenchymal stem cells in the effluent cells was reduced. In addition, the recovery rate of 201B7 cells in the desorbed cell fluid is about 80% when using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier having a particle size of 212 μm or more and less than 300 μm. It was found that the recovery rate of 201B7 cells in the desorbed cell sap was reduced when using an adsorbent in which the biological protein was immobilized on a water-insoluble carrier having a particle size of 176 µm or more and less than 300 µm or 100 µm or more and less than 212 µm. In both cases, human mesenchymal stem cells are mixed in the desorbed cell solution at a recovery rate of about 10 to 30%, and it is difficult to obtain 201B7 cells with a high recovery rate and purity during cell desorption. It became clear that it was not possible. These results show that human mesenchymal stem cells, which are cells that do not adsorb to the adsorbent when the cells and the adsorbent are in contact with each other, when using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier with a particle size of less than 300 μm. is likely to be caught in the gaps between the adsorbent particles, and when the 201B7 cells adsorbed to the adsorbent are desorbed with the fucose solution, the desorbed 201B7 cells are likely to be stuck in the gaps between the adsorbent particles.

From the above, when the mixed cell sap of 201B7 cells and human mesenchymal stem cells was treated using an adsorbent in which a fucose-binding protein was immobilized on a water-insoluble carrier with a particle size of less than 300 μm, human It was proved that the mesenchymal stem cells and the 201B7 cells in the desorbed cell sap could not be separated and recovered with high recovery and purity.

Reference Example 1 Separation and recovery of 201B7 cells and human mesenchymal stem cells from a mixture of 201B7 cells and human mesenchymal stem cells using an adsorbent with a particle size of 176 μm or more and less than 300 μm without immobilizing a fucose-binding protein on a water-insoluble carrier In Reference Example 1, 201B7 cells from a mixture of 201B7 cells and human mesenchymal stem cells and human mesenchymal stem cells were used as adsorbents using particles with a particle size of 176 μm or more and less than 300 μm without immobilizing a fucose-binding protein on a water-insoluble carrier. The present invention relates to a method for separating and collecting mesenchymal stem cells.

In the same manner as in Example 1, except that Toyopearl HW-40EC having a particle size of 176 μm or more and less than 300 μm, which was prepared by classifying Toyopearl HW-40EC (manufactured by Tosoh) according to the method of Preparation Example 6, was used as an adsorbent. carried out. A column filled with Toyopearl HW-40EC classified to a particle size of 176 μm or more and less than 300 μm is referred to as Column No. 1 below. 13 and 14.

Table 8, FIG. 9, and FIG. 10 show the results of calculating the recovery rate of each cell. The cell recovery rate in the effluent cell fluid was about 90% for 201B7 cells and about 50% for human mesenchymal stem cells. It was found that the recovery rate of human mesenchymal stem cells, which are large cells, was low. In addition, the cell recovery rate in the desorbed cells was less than 2% for 201B7 cells and less than 8% for human mesenchymal stem cells. It was found that a small amount of leaf stem cells were recovered.

From the above, when the mixed cell solution of 201B7 cells and human mesenchymal stem cells was treated using an adsorbent with a particle size of 176 μm or more and less than 300 μm without immobilizing fucose-binding protein, each cell was recovered at a high recovery rate. It was proved that it could not be separated and recovered in terms of purity and purity.

Claims (6)

A method for separating and collecting cells using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier having a wet particle size of 300 µm or more and less than 600 µm, wherein cell populations with different degrees of undifferentiation are separated. A step of contacting the cell suspension containing the adsorbent with an adsorbent to bind the cells that bind to the fucose-binding protein to the adsorbent, and then recovering the cells that did not bind to the fucose-binding protein; A method for separating and recovering cells with different degrees of undifferentiation, comprising the step of contacting with a fucose-containing aqueous solution and recovering the detached cells. 2. The method according to claim 1, wherein the cell population with different degrees of undifferentiation is a mixture of heterologous cells with different expression of sugar chains containing structures consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. 3. The method according to any one of claims 1 and 2, wherein the cells highly expressing sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc are human iPS cells. 4. The method according to any one of claims 1 to 3, wherein 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 X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, wherein X is an integer of 120 or more. sex protein.
(b) 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 in the amino acid sequence shown by SEQ ID NO: 1 A fucose-binding protein comprising fucose and having binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more binding protein.
(c) any one of the amino acid substitutions described in (1) to (3) below in the 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 Fucose-binding protein (1) substitution of leucine residue for glutamine residue at position 39 in the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or more, comprising an amino acid sequence containing the above (2) Substitution of the 72nd cysteine residue in the amino acid sequence shown by SEQ ID NO: 1 with one type of amino acid residue selected from glycine residues and alanine residues (3) 65th amino acid sequence shown in SEQ ID NO: 1 (d) in the amino acid sequence of the fucose-binding protein of (c), one or more in regions other than 39th, 65th and 72nd of SEQ ID NO: 1 and has binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, Fucose-binding protein. 4. The method according to any one of claims 1 to 3, wherein the fucose-binding protein is any of the following (e) to (h).
(e) to the amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is A fucose-binding protein consisting of an amino acid sequence to which a cysteine-containing oligopeptide is added, wherein X is an integer of 120 or more.
(f) 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 in the amino acid sequence shown by SEQ ID NO: 1 On the other hand, a sugar comprising an amino acid sequence to which a polyhistidine sequence is added to the N-terminus and an oligopeptide containing cysteine is added to the C-terminus, and which has a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. A fucose-binding protein having binding affinity to a chain, wherein X is an integer of 120 or greater.
(g) to the amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is In the amino acid sequence to which an oligopeptide containing cysteine is added, an amino acid sequence containing any one or more of the amino acid substitutions described in (4) to (6) below, wherein X is an integer of 120 or more, fucose-binding protein (4) replacement of the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (5) substitution of the 72nd cysteine residue in the amino acid sequence shown in SEQ ID NO: 1, Substitution of one amino acid residue selected from glycine residues and alanine residues (6) Substitution of the 65th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (h) The above ( An amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added to regions other than the 39th, 65th and 72nd regions of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of g) to which an oligopeptide containing a polyhistidine sequence is added at the N-terminus and an oligopeptide containing a cysteine sequence is added at the C-terminus, comprising an amino acid sequence, Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc A fucose-binding protein having binding affinity to sugar chains comprising a structure of 6. The method according to any one of claims 1 to 5, wherein an adsorbent packed in a column is used.

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