JP5045920B2 - Protein capturing material, protein capturing cartridge and manufacturing method - Google Patents

Description

  The present invention relates to a protein capturing material, a protein capturing cartridge, and a manufacturing method.

  In recent years, it has become possible to synthesize proteins in microorganisms and animal cells in large quantities using genetic recombination techniques. For example, in research on the structure and properties of proteins, separation and purification of proteins that can easily increase the purity is required.

Examples of protein separation and purification techniques include ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and the like.
In ion exchange chromatography, a resin with an ion exchange group introduced is used as a capture unit for capturing a protein. In hydrophobic interaction chromatography, a hydrophobic functional group is introduced as a capture unit for capturing a protein. In affinity chromatography, a resin into which an antibody, a receptor, a metal ion, or the like has been introduced is used as a capturing part for capturing a protein. Specifically, the resin is separated and purified by filling the resin into a column and allowing the protein solution in which the protein is dissolved to pass through the column.
Since these resins are in the form of beads, it takes time for the protein to diffuse to the trapping portion inside the resin. Therefore, if the protein solution is permeated at a high speed, the protein will flow out without being captured by the capture part inside the resin, so that the protein solution cannot be permeated at a high speed and it takes time to separate and purify the protein. There is.

Then, the protein capture material produced by applying the radiation graft polymerization method was proposed as a protein capture material which can perform separation and refinement | purification of protein at high speed (for example, refer patent documents 1-4). The radiation graft polymerization method is known as a technique capable of imparting graft (grafting) polymer chains at high density to polymer base materials of various shapes and materials.
Specifically, in Patent Documents 1 to 3, a graft polymer chain is bonded to a porous membrane (a porous hollow fiber membrane or a porous film) by applying a radiation graft polymerization method, and captured by the graft polymer chain. A protein capture material prepared by introducing an ion exchange group (anion exchange group or cation exchange group) as a part has been proposed.
Further, in Patent Document 4, a graft polymer chain is bonded to a porous membrane (porous hollow fiber membrane or porous film) by applying a radiation graft polymerization method, and a hydrophobic functional group is used as a trapping portion for the graft polymer chain. Protein capture materials made by introducing groups have been proposed.
Japanese Patent Laid-Open No. 11-12300 JP-A-11-266862 JP 2000-83659 A JP-A-8-141392

However, the protein-capturing material of Patent Documents 1 to 3 is a protein capable of ion exchange (electrostatic interaction) with the capturing unit, that is, a protein having a positive charge or a negative charge. Therefore, there is a problem of poor selectivity.
Further, the protein capturing material of Patent Document 4 has a problem of poor selectivity because it captures any protein that can interact with the capturing part in a hydrophobic manner, that is, a protein having a hydrophobic part.

  An object of the present invention is to provide a protein capture material having high selectivity, a cartridge for protein capture, and a method for producing the protein capture material.

In order to solve the above-mentioned problem, the invention described in claim 1
In protein capture materials,
A substrate;
A graft polymer chain bonded to the substrate;
Metal ions immobilized on the graft polymer chain and having affinity for a given protein;
With
The predetermined protein is captured using the affinity between the predetermined protein and the metal ion.

The invention of claim 1 further prior Symbol metal ions, characterized in that there cysteine, protein affinity comprising histidine and / or tryptophan.

The invention according to claim 1, further pre Kimotozai is characterized in that it is made of a porous material having continuous pores.

The invention according to claim 1, further pre Kimotozai has a thickness of 1 to 10 mm, an average pore diameter of the communication hole is 0.5~5μm and volume fraction of the communication hole 70 to 85% It is characterized by being in the form of a sheet.

The invention of claim 1 further prior Symbol graft polymer chain is bonded to a surface portion of the substrate, the interior of the base material, characterized in that it is non-binding.

The invention described in claim 2
The protein capture material according to claim 1 ,
The base material is composed of polyolefin.

The invention according to claim 3
In the protein capture cartridge,
A protein capturing material according to claim 1 or 2 is filled in a cartridge.

The invention according to claim 4
In the method for producing a protein capturing material according to claim 1 or 2 ,
An irradiation step of irradiating the substrate with ionizing radiation;
Next, a graft polymerization step of bonding a graft polymer chain having the predetermined functional group to the substrate by graft polymerization of a polymerizable monomer having the predetermined functional group to the substrate;
Next, by converting at least a part of the predetermined functional group into an adsorption functional group for adsorbing the metal ion, the graft polymer chain having the predetermined functional group is converted into the adsorption functional group. A conversion step for converting to a graft polymer chain having;
Next, a fixing step of fixing the metal ion to the graft polymer chain bonded to the base material by adsorbing the metal ion to the functional group for adsorption;
It is characterized by providing.

The invention described in claim 5
In the method for producing a protein capturing material according to claim 1 or 2 ,
In the state where the substrate and a polymerizable monomer having a predetermined functional group coexist, the substrate is irradiated with ionizing radiation, and the polymerizable monomer having the predetermined functional group is applied to the substrate. An irradiation / graft polymerization step in which a graft polymer chain having the predetermined functional group is bonded to the substrate by graft polymerization;
Next, by converting at least a part of the predetermined functional group into an adsorption functional group for adsorbing the metal ion, the graft polymer chain having the predetermined functional group is converted into the adsorption functional group. A conversion step for converting to a graft polymer chain having;
Next, a fixing step of fixing the metal ion to the graft polymer chain bonded to the base material by adsorbing the metal ion to the functional group for adsorption;
It is characterized by providing.

The invention described in claim 6
In the manufacturing method of Claim 4 or Claim 5 ,
It comprises a hydrophilization step of hydrophilizing the graft polymer chain bonded to the substrate by converting the predetermined functional group of the graft polymer chain into a hydrophilic functional group.

The invention described in claim 7
In the method for producing a protein capturing material according to claim 1 or 2 ,
An irradiation step of irradiating the substrate with ionizing radiation;
Subsequently, the graft polymer chain having the adsorption functional group is bonded to the substrate by graft polymerization of the polymerizable monomer having the adsorption functional group for adsorbing the metal ion to the substrate. A graft polymerization step;
Next, a fixing step of fixing the metal ion to the graft polymer chain bonded to the base material by adsorbing the metal ion to the functional group for adsorption;
It is characterized by providing.

The invention according to claim 8 provides:
In the method for producing a protein capturing material according to claim 1 or 2 ,
In the coexistence of the base material and a polymerizable monomer having an adsorption functional group for adsorbing the metal ions, the base material is irradiated with ionizing radiation, and the polymerization property having the adsorption functional group is obtained. An irradiation / graft polymerization step in which a graft polymer chain having the adsorption functional group is bonded to the substrate by graft polymerization of the monomer to the substrate;
Next, a fixing step of fixing the metal ion to the graft polymer chain bonded to the base material by adsorbing the metal ion to the functional group for adsorption;
It is characterized by providing.

  According to the first aspect of the present invention, the protein capturing material comprises a graft polymer chain bonded to a base material, and a metal ion that is fixed to the graft polymer chain and has an affinity for a predetermined protein. The predetermined protein is captured using the affinity between the predetermined protein and the metal ion. Therefore, it has higher selectivity than conventional protein capture materials that capture proteins using electrostatic interaction or hydrophobic interaction.

Further, according to the invention of claim 1, the metal ions comprise protein capture material, cysteine, an affinity with proteins having histidine and / or tryptophan. That is, the protein can be captured by utilizing the fact that the metal ion specifically reacts with the protein having a portion having an unshared electron pair. Therefore, the protein-capturing material of the present invention can be used, for example, when the desired protein has cysteine, histidine and / or tryptophan, or by fusing cysteine, histidine and / or tryptophan with the desired protein. By providing the desired protein with cysteine, histidine and / or tryptophan, the desired protein can be selectively captured.

Further, according to the invention described in claim 1, the substrate is made of a porous material having continuous pores. Therefore, for example, the desired protein can be captured only by permeating the protein capturing material of the present invention through a protein solution in which the desired protein is dissolved.
Furthermore, the desired protein can easily access the metal ions fixed to the graft polymer chain disposed in the communication hole while passing through the communication hole. Therefore, a desired protein is rarely passed through the protein capturing material without being captured, and a protein capturing material having a high capturing performance even during high-speed processing can be configured.

Further, according to the invention described in claim 1, the substrate is intended thickness sheet of 1 to 10 mm, conventional protein capture material a porous hollow fiber membrane and a porous film as a base material There is thickness compared with. Therefore, a protein capturing material having a high physical strength can be configured, and a protein capturing material having excellent handleability can be configured.
Furthermore, the base material is a sheet-like material having an average pore diameter of communication holes of 0.5 to 5 μm and a volume fraction of communication holes of 70 to 85%, and has a large number of fine communication holes. Therefore, since the diffusibility is good, the filter efficiency is good, and a protein capturing material having high capturing performance even during high-speed processing can be configured.

Further, according to the invention described in claim 1, grayed rafts polymer chain is coupled to the surface portion of the substrate, the inside of the base material is non-binding. That is, the portion where the graft polymer chain is not bonded (inside the substrate) maintains the physical and chemical characteristics of the substrate itself, and the portion where the graft polymer chain is bonded (the surface of the substrate) Part), the protein-capturing material having excellent physical and chemical stability can be constructed.

According to the invention described in claim 2 , it is needless to say that the same effect as that of the invention described in claim 1 can be obtained, and the substrate is made of polyolefin. Therefore, it is possible to construct a protein capture material excellent in physical and chemical stability and to construct a protein capture material capable of selective capture by metal ions because the reactivity of the substrate itself is low. it can.

According to the invention described in claim 3 , since the protein capturing cartridge is configured by filling the protein capturing material described in claim 1 or 2 into the cartridge, it is easy to use.

According to the invention described in claim 4 , the protein capturing material according to claim 1 or 2 is obtained by a simple method including only the irradiation step, the graft polymerization step, the conversion step, and the fixing step. Can be manufactured.

According to the invention described in claim 5 , the protein-capturing material according to claim 1 or 2 is manufactured by a simple method including only the irradiation / graft polymerization step, the conversion step, and the fixing step. be able to.

According to the invention described in claim 6 , it is needless to say that the same effect as that of the invention described in claim 4 or 5 can be obtained, and a protein capturing material having a hydrophilic graft polymer chain is produced. can do. Therefore, since the reactivity of the graft polymer chain itself can be reduced, a protein capturing material that can be selectively captured by metal ions can be produced.

According to the invention described in claim 7 , the protein capture material according to claim 1 or 2 can be produced by a simple method including only the irradiation step, the graft polymerization step, and the fixing step. it can.

According to the invention described in claim 8 , the protein capturing material according to claim 1 or 2 can be produced by a simple method including only the irradiation / graft polymerization step and the fixing step.

  Hereinafter, the best mode for carrying out the present invention will be described in detail with reference to the drawings. The scope of the invention is not limited to the illustrated example.

<Protein Capture Material, Protein Capture Cartridge, and Protein Capture Material Manufacturing Method>
FIG. 1 shows an example of a production path for the protein capturing material 10, and FIG. 2 shows an example of a protein capturing cartridge 100.
The protein capturing material 10 is, for example, an immobilized metal affinity material for capturing and separating a predetermined protein from a protein solution in which the predetermined protein is dissolved by using the affinity between the predetermined protein and the metal ion 5. It is.
The protein capturing material 10 includes, for example, a base material 1 made of a porous body having communication holes, and a graft polymer chain 4 having a functional group for adsorption for adsorbing metal ions 5 bonded to the base material 1. And metal ions 5 fixed to the graft polymer chain 4 and having affinity for a predetermined protein. That is, the protein capturing material 10 has a structure in which, for example, a graft polymer chain 4 in which metal ions 5 are fixed is bonded to a base material 1 made of a porous body having communication holes.

Here, the predetermined protein is not particularly limited as long as the protein has affinity for the metal ion 5.
Specifically, examples of the predetermined protein include a protein having a portion having an unshared electron pair, preferably a protein having a portion having an unshared electron pair on the surface.
Examples of the protein having a portion having an unshared electron pair include a protein having an amino acid residue having an unshared electron pair, and a protein to which a tag (label) having an unshared electron pair is attached.

Examples of the protein having an amino acid residue having an unshared electron pair include a protein having a sulfur-containing amino acid residue such as cysteine and / or a heterocyclic amino acid residue such as histidine and tryptophan.
Examples of proteins to which a tag having an unshared electron pair is attached include, for example, sulfur-containing amino acids such as cysteine and / or proteins to which a heterocyclic amino acid such as histidine and tryptophan is attached as a tag, and sulfur-containing amino acid residues such as cysteine. And / or a protein to which a polypeptide having a heterocyclic amino acid residue such as histidine or tryptophan is attached as a tag.
That is, examples of the predetermined protein include a protein comprising cysteine, histidine and / or tryptophan.

The metal ion 5 is not particularly limited as long as it is a metal ion having an affinity for a predetermined protein (an ion of a metal element having an affinity for a predetermined protein).
Specifically, examples of the metal ion 5 include a metal ion having affinity for a portion having a non-shared electron pair included in a predetermined protein.
Examples of metal ions having affinity for a portion having a non-shared electron pair of a predetermined protein include divalent metal ions (Mg ²⁺ , Ca ²⁺ , Cr ²⁺ , Mn ²⁺ , Co ²⁺ , Ni ²⁺ , Cu ^2+). , Zn ²⁺ ), divalent and trivalent iron ions (Fe ²⁺ and Fe ³⁺ ), ions of rare earth elements (Sc, Y and lanthanoids), and the like.

The metal ion 5 captures the predetermined protein by, for example, affinity interaction with a portion having a non-shared electron pair included in the predetermined protein to form a bond (electrical bond or chelate bond).
In addition, the protein capture | acquisition material 10 may be provided with one type of metal ion as the metal ion 5, and may be provided with multiple types of metal ion.

The protein capturing material 10 is manufactured by applying, for example, a graft polymerization method.
Specifically, for example, as shown in FIG. 1, the protein capturing material 10 is manufactured by an irradiation step, a graft polymerization step, a conversion step, a hydrophilization step, and a fixing step.

In the irradiation step, the substrate 1 is irradiated with ionizing radiation (for example, an electron beam). That is, radicals are generated on the substrate 1.
In the graft polymerization step, a polymerizable monomer having a predetermined functional group (for example, an epoxy group) (for example, glycidyl methacrylate (GMA)) is graft-polymerized on the substrate 1, thereby increasing the graft height having the predetermined functional group. The molecular chain 2 is bonded to the substrate 1. That is, the graft polymer chain 2 having a predetermined functional group is grown using a radical as a starting point.
In the conversion step, at least a part of the predetermined functional group of the graft polymer chain 2 is converted into an adsorption functional group (for example, iminodiacetic acid group (—N (CH ₂ COOH) ₂ )). The graft polymer chain 2 having a functional group is converted into a graft polymer chain 3 having a functional group for adsorption.
In the hydrophilization step, a predetermined functional group remaining in the graft polymer chain 3 having a functional group for adsorption is converted into a hydrophilic functional group (for example, a diol group ((—OH) ₂ )), whereby a base material is obtained. The graft polymer chain bonded to 1 is hydrophilized.
In the fixing step, the metal ion 5 (for example, Ni ²⁺ ) is adsorbed on the adsorption functional group of the grafted polymer chain 4 that has been hydrophilized, thereby grafting the polymer chain in which the metal ion 5 is bonded to the substrate 1. Fix to 4.
FIG. 1 is an example of a manufacturing path for the protein capturing material 10, and the manufacturing path for the protein capturing material 10 is not limited to FIG. 1.

The protein capturing cartridge 100 is configured, for example, by filling a cartridge 20 with a protein capturing material 10 as shown in FIG.
Then, for example, a protein solution in which a predetermined protein is dissolved is supplied to the protein capturing cartridge 100 using a syringe pump or a liquid feed pump, and the other surface (for example, the lower surface) of the protein capturing material 10 is For example, the protein capturing material 10 captures a predetermined protein by allowing the protein solution to permeate toward the upper surface).
The cartridge 20 may be filled with one protein capturing material 10 or a plurality of protein capturing materials 10.

The shape of the substrate 1 is not particularly limited as long as it is a porous body having communication holes.
Examples of a preferable shape of the substrate 1 include a sheet shape having a thickness of 1 to 10 mm, an average pore diameter of communication holes of 0.5 to 5 μm, and a volume fraction of communication holes of 70 to 85%.

When the thickness of the base material 1 is less than 1 mm, the thickness becomes too thin. For this reason, it tends to be difficult to handle, for example, it is difficult to fill the cartridge 20, and the physical strength is low, for example, it tends to be unsuitable for repeated use.
On the other hand, when the thickness of the base material 1 exceeds 10 mm, the thickness becomes too thick. Therefore, it tends to be difficult to handle, for example, it is difficult to fill the cartridge 20.

Further, when the average pore diameter of the communication holes is less than 0.5 μm, the pore diameter becomes too small. Therefore, the solution permeability at the time of allowing the protein solution to permeate from one surface of the protein capturing material 10 to the other surface is lowered, and high-speed processing tends to be difficult.
On the other hand, when the average pore diameter of the communication holes exceeds 5 μm, the pore diameter becomes too large. Therefore, when the protein solution is permeated from one surface of the protein capturing material 10 to the other surface to capture a predetermined protein, if the protein solution is permeated at a high speed, the predetermined protein is not captured and the protein capturing material is captured. The tendency to pass through 10 is shown.
Here, the average pore diameter of the communication holes is an average flow pore diameter obtained by using a predetermined evaluation method such as a bubble point method.

In addition, when the volume fraction of the communication holes is less than 70%, the ratio of the communication holes is too small. Therefore, the solution permeability at the time of allowing the protein solution to permeate from one surface of the protein capturing material 10 to the other surface is lowered, and high-speed processing tends to be difficult.
On the other hand, when the volume fraction of the communication holes exceeds 85%, the ratio of the communication holes becomes too large. For this reason, the physical strength is low, and for example, it tends to be unsuitable for repeated use.

The material for forming the substrate 1 is not particularly limited as long as the protein capturing material 10 can be produced by applying a graft polymerization method.
Preferred materials for forming the substrate 1 include, for example, polyolefins having characteristics such as excellent physical and chemical stability and low reactivity of the substrate 1 itself (for example, polyethylene and polypropylene). It is done. In addition, as a material which forms the base material 1, the combination (mixture and copolymer) etc. of fluororesins, such as polytetrafluoroethylene (PTFE), polyolefin, and a fluororesin, etc. are mentioned, for example. Moreover, for example, ethylene-vinyl acetate copolymer, polystyrene and the like can be mentioned.

The graft polymerization method is not particularly limited as long as the graft polymer chain 2 having a predetermined functional group can be grafted to the substrate 1.
As a preferable graft polymerization method, for example, a polymerization initiator is not required, the shape of the base material 1 is not limited, and radicals can be generated on the entire base material 1 (ionizing radiation (electron beam, And X-ray, α-ray, β-ray, γ-ray, etc.). In addition, examples of the graft polymerization method include a polymerization method using ultraviolet rays and a polymerization method using a polymerization initiator.

The polymerizable monomer that forms the graft polymer chain 2 having a predetermined functional group is not particularly limited as long as the predetermined functional group can be converted into an adsorption functional group.
Preferred examples of the predetermined functional group include an epoxy group having characteristics such as easy conversion to an adsorption functional group. Therefore, preferable polymerizable monomers include, for example, glycidyl methacrylate (GMA) and glycidyl acrylate which are polymerization monomers having an epoxy group.

The adsorption functional group of the graft polymer chains 3 and 4 is not particularly limited as long as the metal ion 5 can be adsorbed.
Specifically, examples of the functional group for adsorption include an ion exchange group and a chelate forming group. Examples of ion exchange groups include cation exchange groups such as sulfonic acid groups, phosphoric acid groups, and carboxyl groups. Examples of the chelate-forming group include iminodiacetic acid group and iminodiethanol group. The graft polymer chains 3 and 4 may have one or a plurality of types of ion exchange groups or one or a plurality of types of chelate-forming groups as an adsorption functional group. It may have one or more types of ion exchange groups and one or more types of chelate-forming groups.
As a preferable functional group for adsorption, for example, a chelate-forming group having characteristics such as binding to the metal ion 5 more firmly can be mentioned. It is known that the bond between the metal ion 5 and the chelate-forming group (chelate bond) is stronger than the bond between the metal ion 5 and the ion exchange group (bond due to electrostatic interaction).

The hydrophilic functional group possessed by the graft polymer chain 4 is not particularly limited as long as it is more hydrophilic than the predetermined functional group possessed by the graft polymer chains 2 and 3.
Preferred hydrophilic functional groups include, for example, diol groups (two hydroxyl groups) having characteristics such as easy conversion from predetermined functional groups (for example, epoxy groups). In addition, examples of the hydrophilic functional group include a hydroxyl group, a hydroxyalkyl group (an alkyl group is preferably a lower alkyl group), a pyrrolidonyl group, and the like.

  The graft polymer chains 4 (graft polymer chains 2, 3 and 4) are grafted (grafted) on the surface of the communication hole to form a “polymer brush” in the communication hole. That is, at least a part of the graft polymer chain 4 and the metal ion 5 fixed to the graft polymer chain 4 is disposed in the communication hole.

The graft polymer chain 4 (graft polymer chains 2, 3, and 4) may be bonded to the entire base material 1, or may be bonded to the surface portion of the base material 1 so as not to be present inside the base material 1. It may be combined.
The protein-capturing material 10 in which the graft polymer chain 4 is bonded to the entire base material 1 is, for example, controlled by controlling the concentration of polymerizable monomer, type of solvent, reaction temperature, reaction time, etc. in the graft polymerization. It is obtained by bonding the graft polymer chain 2 having a functional group to the entire base material 1.
On the other hand, the protein-capturing material 10 in which the graft polymer chain 4 is bonded to the surface portion of the base material 1 and is not bonded to the inside of the base material 1 includes, for example, the concentration of the polymerizable monomer in the graft polymerization, It is obtained by binding the graft polymer chain 2 having a predetermined functional group only to the surface portion of the substrate 1 by controlling the type, reaction temperature, reaction time and the like.

  When the graft polymer chain 4 is bonded to the whole substrate 1, the graft polymer chain 4 is bonded to the surface portion of the substrate 1 and not bonded to the inside of the substrate 1. And since the fixed amount of the metal ion 5 in the protein capture | acquisition material 10 can be increased, there exists an advantage that a larger amount of predetermined proteins can be captured, capture efficiency is good, etc.

On the other hand, when the graft polymer chain 4 is bonded to the surface portion of the substrate 1 and is not bonded to the inside of the substrate 1, that is, the protein capturing material 10 is a surface to which the graft polymer chain 4 is bonded. Part and the interior where the graft polymer chain 4 is unbound, the interior where the graft polymer chain 4 is unbound is bound to the graft polymer chain 4. Since it plays a role as a support for the surface portion, it can suppress swelling and has higher physical strength than the case where the graft polymer chain 4 is bonded to the entire substrate 1. There is.
Here, the surface part to which the graft polymer chain 4 is bonded refers to the surface part (surface layer) of the substrate 1 in the film thickness direction of the substrate 1, and the inside to which the graft polymer chain 4 is not bonded is the group The inside (inner layer) of the base material 1 in the film thickness direction of the material 1 is indicated.

  When the graft polymer chain 4 (graft polymer chains 2, 3 and 4) is bonded to the base material 1, the structure of the base material 1 is divided at that portion, and the substrate 1 is not dense and the rigidity of the base material 1 is lowered. Therefore, the portion to which the graft polymer chain 4 is bonded increases the degree of swelling with respect to the solvent and decreases the physical strength. On the other hand, when the ratio of the portion where the graft polymer chain 4 is not bonded is large, the amount of the fixed metal ions 5 in the protein capturing material 10 is decreased, and the amount of the predetermined protein that can be captured is decreased. Problems such as bad. Therefore, the ratio of the thickness of the surface portion (the total in the case of both surfaces) to which the graft polymer chain 4 is bonded in the film thickness direction of the protein capturing material 10 is 40-60% of the thickness of the protein capturing material 10; When the ratio of the internal thickness where the graft polymer chains 4 are not bonded in the film thickness direction of the protein capturing material 10 is set to 60 to 40% of the thickness of the protein capturing material 10, the capturing performance (predetermined to be captured) The balance between the amount of protein and the capture efficiency, etc.) and the degree of swelling and physical strength (including deformation resistance) can be kept good.

<Protein separation and purification method>
Next, a protein separation and purification method using the protein capture material 10 will be described.

  The protein capture material 10 can separate and purify a predetermined protein by, for example, a capture step and an elution step.

  First, a predetermined protein is captured by the protein capturing material 10 in the capturing step. Specifically, for example, the protein capturing material 10 captures the predetermined protein by supplying a protein solution in which the predetermined protein is dissolved to the protein capturing cartridge 100.

Next, in the elution step, the predetermined protein is eluted from the protein capturing material 10 by using an eluate for eluting the predetermined protein captured by the protein capturing material 10. Specifically, for example, a predetermined protein is eluted by supplying an eluate to the protein capturing cartridge 100.
Here, the eluate is appropriately selected depending on, for example, the combination of the metal ions 5 included in the protein capturing material 10 and the predetermined protein to be eluted.
Specifically, examples of the eluent include a solution that weakens the affinity interaction between the protein and the metal ion 5, and a solution that shifts the equilibrium relationship of the affinity interaction. More specifically, for example, a solution in which a competitive reagent dissolves, a solution in which a chelating agent dissolves, a buffer solution having a predetermined pH, and the like can be given.

<Example>
EXAMPLES Next, although an Example demonstrates this invention concretely, this invention is not limited to these.

(Manufacture of protein capture materials)
First, the protein capturing material 10 was manufactured.
In the examples, GAM is selected as a polymerizable monomer having a predetermined functional group, an iminodiacetic acid (IDA) group is selected as a functional group for adsorption, a diol group is selected as a hydrophilic functional group, and metal ions 5 Ni ²⁺ was selected as Then, for example, the protein capturing material 10 (nickel-fixed sheet) to which Ni ²⁺ is fixed is manufactured by the manufacturing route shown in FIG.

  Specifically, the substrate 1 was irradiated with 200 kGy of an electron beam at room temperature in a nitrogen atmosphere (irradiation step). The base material 1 is a sheet-like polyethylene porous body having communication holes (thickness is 2.0 mm, average pore diameter of communication holes is 1 μm, porosity (volume fraction of communication holes) is 75%) It was used.

Next, the substrate 1 irradiated with the electron beam was immersed in a 20% by volume methanol solution (GMA solution) of GMA at 40 ° C. for a predetermined time, thereby grafting GMA to the substrate 1 (graft polymerization step). . The obtained sheet is called a GMA sheet.
The graft ratio [%] (= 100 × (weight of grafted GMA) / (weight of substrate 1)) of the GMA sheet increases as the time for immersing the substrate 1 in the GMA solution increases. confirmed. In the examples, a graft rate of 150% was employed.

Next, the grafted graft polymer was obtained by immersing the GMA sheet in a 0.425 M disodium iminodiacetate (NH (CH ₂ COONa) ₂ ) solution using a 50% by volume aqueous solution of dioxane as a solvent at 80 ° C. for a predetermined time. The epoxy group of chain 2 was converted to an IDA group (conversion step). Then, it was conditioned using 1M hydrochloric acid. The obtained sheet is called an IDA sheet.
Molar conversion rate [%] of IDA sheet (= 100 × (number of moles of introduced IDA group) / (number of moles of epoxy group before introduction of IDA group)) is the time for immersing the GMA sheet in disodium iminodiacetate solution It has been confirmed that it increases as the number increases. In the examples, a molar conversion of 60% was employed.

Subsequently, the epoxy group remaining in the graft polymer chain 3 having the IDA group is converted into a diol group by immersing the IDA sheet in 0.5 M sulfuric acid at 80 ° C. for 4 hours, and the graft polymer chain having the IDA group 3 was hydrophilized (hydrophilization step). The obtained sheet is called IDA-diol sheet.
The number of moles of IDA group per IDA-diol sheet wet volume, that is, the IDA group density of the IDA-diol sheet was 0.76 mmol / mL.

  Next, for example, a cylindrical cartridge 20 was filled with one IDA-diol sheet cut into a disk shape in accordance with the inner diameter (for example, 13 mm in diameter) of the cartridge 20 to produce an IDA-diol cartridge.

Next, a 10 mM nickel sulfate solution containing 20 mM acetate buffer (pH 7.5) as a solvent is supplied to the IDA-diol cartridge at a flow rate of 120 mL / h to permeate the IDA-diol sheet. Metal ions 5 (Ni ²⁺ ) were fixed to the graft polymer chains 4 provided (fixing step). Thereafter, the sheet was washed with a buffer solution. The obtained sheet is called a nickel fixing sheet.
In this way, the protein capturing material 10 (nickel fixing sheet) was manufactured, and the protein capturing cartridge 100 filled with the protein capturing material 10 was manufactured.

Specifically, in the fixing step, for example, the effluent from the outlet of the IDA-diol cartridge was continuously collected, and the concentration of Ni ²⁺ in the effluent was measured by absorptiometry. Then, the concentration of Ni ²⁺ in effluent, feed to reach a concentration of Ni ²⁺ of (nickel sulfate solution) and allowed to transmit nickel sulfate solution.
When the volume of the nickel sulfate solution permeated through the IDA-diol sheet was in the range of 0 to about 13 mL, the concentration of Ni ^{2+ in} the effluent was 0 (zero) mM.
When the volume of the nickel sulfate solution permeated through the IDA-diol sheet was in the range of about 13 to about 65 mL, the concentration of Ni ^{2+ in} the effluent increased as the supply amount of the nickel sulfate solution increased.
When the volume of the nickel sulfate solution permeated through the IDA-diol sheet is about 65 mL or more, the concentration of Ni ^{2+ in} the effluent is equivalent to the concentration of Ni ^{2+ in} the feed solution (nickel sulfate solution), and the IDA-diol It was found that the adsorption reached equilibrium when about 65 mL of nickel sulfate solution was permeated through the sheet.
The Ni ²⁺ equilibrium adsorption amount per wet volume of the IDA-diol sheet, that is, the Ni ²⁺ fixing density of the nickel fixing sheet was 0.59 mmol / mL.

Here, the Ni ²⁺ fixation density of a commercially available immobilized metal affinity material (column for purifying histidine tag fusion protein) into which an IDA group has been introduced as an adsorption functional group is 0.030 to 0.037 mmol / mL. Therefore, the protein-capturing material 10 (nickel-fixed sheet) of the present invention has a Ni ²⁺ fixing density 16 to 20 times that of a commercially available immobilized metal affinity material, and has a higher density than that of a commercially available immobilized metal affinity material. It was found that ions 5 could be fixed.

  Moreover, the base material 1 and the nickel fixed sheet were observed using SEM (scanning electron microscope). As a result, there is almost no difference between the porous structure of the base material 1 and the porous structure of the nickel fixing sheet, and even if the graft polymer chain 4 is bonded to the base material 1 to fix the metal ions 5, the porous structure is obtained. It turns out that it can be maintained.

(Water permeability)
Next, the water permeability of the protein capturing material 10 was measured.
For example, pure water was allowed to permeate through a nickel fixing sheet at a constant pressure and temperature, and the water permeability of the nickel fixing sheet was measured.

Specifically, for example, pure water is supplied to the protein capturing cartridge 100 filled with one nickel fixing sheet at a constant pressure and a constant temperature, and a pure water permeation flux [m / h] (= ( The permeation flow rate of pure water under a constant pressure (ΔP = 0.1 MPa) and a constant temperature (25 ° C.) / (The cross-sectional area of the nickel fixed sheet (π (radius of the nickel fixed sheet) ² )) was determined.
The pure water permeation flux of the nickel fixed sheet having an IDA group density of 0.76 mmol / mL and Ni ²⁺ fixed density of 0.59 mmol / mL was 36 m / h (ΔP = 0.1 MPa, 25 ° C.).

Here, pure water of a commercially available immobilized metal affinity material (column for purifying histidine tag fusion protein) having an Ni ²⁺ immobilization density of 0.030 to 0.037 mmol / mL and an IDA group introduced as a functional group for adsorption. The permeation flux is 7.0 m / h (ΔP = 0.1 MPa, 25 ° C.). Therefore, the protein capture material 10 (nickel-fixed sheet) of the present invention has a pure water permeation flux 5 times or more that of a commercially available immobilized metal affinity material, and has a higher water permeability than a commercially available immobilized metal affinity material. I understood that. That is, it was found that the protein capture material 10 (nickel-fixed sheet) of the present invention can separate and purify proteins at a higher speed than a commercially available immobilized metal affinity material.

(Protein separation and purification)
Next, the protein capture material 10 was used to separate and purify a predetermined protein.
In the examples, a green fluorescent protein to which histidine is added as a tag (hereinafter referred to as “histidine tag fusion protein”) is selected as a predetermined protein, and a solution (250 mM) in which a competitive reagent (imidazole) is dissolved as an eluent. Imidazole solution) was selected.

  First, for example, a protein solution in which a histidine tag fusion protein and other proteins (such as lysozyme) were dissolved was prepared (protein solution preparation step).

  Next, for example, by supplying 45 mL of the protein solution to the protein capturing cartridge 100 filled with one nickel fixing sheet at 120 mL / h at room temperature, and allowing the protein solution to permeate the nickel fixing sheet, the histidine tag The fusion protein was captured (capture step).

  Next, for example, 5 mL of 30 mM phosphate buffer (300 mM NaCl, pH 7.8) is supplied to the protein-capturing cartridge 100 filled with the nickel-fixing sheet capturing the histidine tag fusion protein, and the nickel-fixing sheet is phosphated. Washing was done by permeating the buffer (washing step).

  Next, for example, 2 mL of a 250 mM imidazole solution is supplied to the protein capturing cartridge 100 filled with the nickel fixing sheet that has captured the histidine tag fusion protein, and the 250 mM imidazole solution is allowed to permeate the nickel fixing sheet. The histidine-tagged fusion protein that was captured by was eluted (eg, elution step).

The protein distribution in the protein solution adjusted in the protein solution preparation step, and the protein distribution in the effluent (hereinafter referred to as “outflow protein solution”) flowing out from the outlet of the protein capturing cartridge 100 in the capturing step. In the elution step, the protein distribution in the effluent flowing out from the outlet of the protein capturing cartridge 100 (hereinafter referred to as “effluent eluate”) was measured by gel filtration analysis.
Specifically, chromatograms of the protein solution, the effluent protein solution, and the effluent eluate were obtained by liquid chromatography. The result is shown in FIG.

  FIG. 3A is a chromatogram of the protein solution (solid line) and the effluent protein solution (dashed line). According to FIG. 3A, the peak of the histidine tag fusion protein in the effluent protein solution was reduced as compared with the protein solution. Thereby, it was found that the histidine tag fusion protein was captured by the nickel fixing sheet.

  FIG. 3 (b) is a chromatogram of the effluent eluate. According to FIG. 3B, the peak of the histidine tag fusion protein was detected in the effluent eluate. Thus, it was found that the histidine tag fusion protein captured by the nickel fixed sheet can be eluted by permeating the 250 mM imidazole solution as an eluent through the nickel fixed sheet.

  Furthermore, according to FIG. 3, it was found from the ratio of peak areas that the concentration of the histidine tag fusion protein in the effluent eluate was 10 times that of the histidine tag fusion protein in the protein solution. Thus, it was found that when the protein capturing material 10 (nickel-fixed sheet) is used, a predetermined protein (histidine tag fusion protein) can be separated and purified at a concentration of 10 times.

Further, the protein distribution in the protein solution prepared in the protein solution preparation step and the protein distribution in the effluent eluate were measured by electrophoresis analysis.
Specifically, protein solution and effluent eluate bands were obtained by polyacrylamide gel electrophoresis (SDS-PAGE). The result is shown in FIG.

  According to FIG. 4, the histidine tag fusion protein band and other protein bands appear in the protein solution band, whereas the effluent eluate band contains the histidine tag fusion protein band and the other protein bands. And a band of degradation product of histidine tag fusion protein appeared. Thereby, it was found that the histidine tag fusion protein was captured by the nickel-fixed sheet, and the histidine tag fusion protein captured by the nickel-fixed sheet could be eluted by permeating the nickel-fixed sheet with a 250 mM imidazole solution as an eluent.

Furthermore, from the color depth and area of the band obtained by the SDS-PAGE method, the purity of the histidine tag fusion protein in the protein solution (= 100 × (the histidine tag in the protein solution) was determined using a predetermined image analysis software. (Weight of fusion protein) / (weight of total protein in protein solution)) and purity of histidine-tagged fusion protein in effluent eluate (= 100 × (weight of histidine tag fusion protein in effluent eluate) / (outflow) The total protein weight)) in the eluate was calculated. The amount of degradation product of histidine tag fusion protein was included in the amount of histidine tag fusion protein.
As a result, the purity of the protein solution and the effluent eluate was found to be 30 and 98, respectively. Thus, it was found that when the protein capturing material 10 (nickel-fixed sheet) is used, a predetermined protein (histidine tag fusion protein) can be separated and purified with a purity of 98%.

  According to the protein capturing material 10 of the present invention described above, the graft polymer chain 4 bonded to the base material 1, the metal ion 5 having an affinity for a predetermined protein fixed to the graft polymer chain 4, and The predetermined protein is captured by utilizing the affinity between the predetermined protein and the metal ion 5. Therefore, it has higher selectivity than conventional protein capture materials that capture proteins using electrostatic interaction or hydrophobic interaction.

  Moreover, according to the protein capturing material 10 of the present invention, the metal ion 5 has an affinity for a protein comprising cysteine, histidine and / or tryptophan. That is, the protein can be captured by utilizing the fact that the metal ion 5 specifically reacts with a protein having a portion having an unshared electron pair. Therefore, the protein-capturing material of the present invention can be used, for example, when the desired protein has cysteine, histidine and / or tryptophan, or by fusing cysteine, histidine and / or tryptophan with the desired protein. By providing the desired protein with cysteine, histidine and / or tryptophan, the desired protein can be selectively captured.

Moreover, according to the protein capture | acquisition material 10 of this invention, the base material 1 consists of a porous body which has a communicating hole. Therefore, for example, the desired protein can be captured only by allowing the protein capturing material 10 of the present invention to permeate a protein solution in which the desired protein is dissolved.
Furthermore, the desired protein can easily access the metal ion 5 fixed to the graft polymer chain 4 disposed in the communication hole while passing through the communication hole. Therefore, the protein capturing material 10 having a high capturing performance can be configured even when a desired protein is rarely captured and hardly passes through the protein capturing material 10 even during high-speed processing.

Moreover, according to the protein capture | acquisition material 10 of this invention, the base material 1 is a sheet-like thing with a thickness of 1-10 mm, The conventional protein capture which used the porous hollow fiber membrane and the porous film as the base material Thick compared to the material. Therefore, the protein capturing material 10 having high physical strength can be configured, and the protein capturing material 10 having excellent handleability can be configured.
Furthermore, the base material 1 is a sheet-like thing with the average pore diameter of a communicating hole of 0.5-5 micrometers, and the volume fraction of a communicating hole of 70-85%, and has many fine communicating holes. Therefore, since the diffusibility is good, the filter efficiency is good, and the protein capture material 10 having high capture performance even during high-speed processing can be configured.

  In addition, according to the protein capturing material 10 of the present invention, the graft polymer chain 4 to which the metal ion 5 is immobilized and the graft polymer chain 4 to which the metal ion 5 is immobilized and the graft polymer chain 4 to which the metal ion 5 is immobilized are non-bonded. It can be configured with a two-layer structure including a joined portion (inside). That is, the portion (inside) where the graft polymer chain 4 is not bonded maintains the physical and chemical characteristics of the substrate itself, and the portion (surface portion) where the graft polymer chain 4 is bonded is supported. Since it plays a role as a body, the protein capturing material 10 excellent in physical and chemical stability can be constituted.

  Moreover, according to the protein capture | acquisition material 10 of this invention, the base material 1 is comprised from polyolefin. Therefore, the protein capturing material 10 having excellent physical and chemical stability can be configured, and the reactivity of the base material 1 itself is low, so that the protein capturing material 10 capable of selective capturing with the metal ions 5 is provided. Can be configured.

  Further, according to the protein capturing cartridge 100 of the present invention described above, since the protein capturing material 10 is filled in the cartridge 20, it is easy to use.

  Moreover, according to the manufacturing method of the protein capture | acquisition material 10 of this invention demonstrated above, it is a simple method provided only with an irradiation step, a graft polymerization step, a conversion step, a hydrophilization step, and a fixation step, The capture material 10 can be manufactured.

  Moreover, according to the manufacturing method of the protein capture | acquisition material 10 of this invention, the graft | grafting polymer chain couple | bonded with the base material 1 can be hydrophilized by a hydrophilization step. Therefore, since the reactivity of the graft polymer chain 4 itself can be reduced, the protein capturing material 10 that can be selectively captured by the metal ions 5 can be manufactured.

  The present invention is not limited to the above-described embodiment, and can be appropriately changed without departing from the gist thereof.

<Modification 1>
In the above-described embodiment, the protein capturing material 10 is manufactured by the irradiation step, the graft polymerization step, the conversion step, the hydrophilization step, and the fixing step. For example, the protein capturing material 10 has a predetermined functional group. When a polymerizable monomer having an adsorption functional group is graft-polymerized to the substrate 1 instead of the polymerizable monomer, the graft polymer chain 4 having the adsorption functional group is converted into the substrate 1 only by the graft polymerization step. Therefore, since the adsorption functional group is hydrophilic, the hydrophilization step can be omitted.
Here, the polymerizable monomer having a functional group for adsorption is a polymerizable monomer having an ion exchange group for adsorbing the metal ion 5 or a polymerization having a chelate forming group for adsorbing the metal ion 5. It is optional if it is a sex monomer.
Specifically, examples of the polymerizable monomer having a functional group for adsorption include acrylic acid having an ion exchange group (cation exchange group). In addition, for example, a polymerizable produced by converting a predetermined functional group of a polymerizable monomer (for example, glycidyl methacrylate, glycidyl acrylate, etc.) having a predetermined functional group (for example, epoxy group) into an adsorption functional group. And monomers.

  According to the method for producing the protein-capturing material 10 of Modification 1 described above, the protein-capturing material 10 can be produced by a simple method including only the irradiation step, the graft polymerization step, and the fixing step.

<Modification 2>
In the above-described embodiment, the protein capturing material 10 is manufactured by the irradiation step, the graft polymerization step, the conversion step, the hydrophilization step, and the fixing step. When the substrate 1 is irradiated with ionizing radiation in the coexistence of a polymerizable monomer having a functional group (irradiation / graft polymerization step), the irradiation step and the graft polymerization step in the above-described embodiment are performed simultaneously. be able to.
Here, the state in which the substrate 1 and the polymerizable monomer having a predetermined functional group coexist is, for example, in a solution (GMA solution or the like) in which the polymerizable monomer having the predetermined functional group is dissolved. The substrate 1 is immersed in the substrate 1 or the substrate 1 is impregnated with a solution in which a polymerizable monomer having a predetermined functional group is dissolved.

  According to the method for producing the protein-capturing material 10 of Modification 2 described above, the protein-capturing material 10 is a simple method including only the irradiation / graft polymerization step, the conversion step, the hydrophilization step, and the fixing step. Can be manufactured.

<Modification 3>
In the above-described embodiment, the protein capturing material 10 is manufactured by the irradiation step, the graft polymerization step, the conversion step, the hydrophilization step, and the fixing step. When the base material 1 is irradiated with ionizing radiation in the coexistence of a polymerizable monomer having a functional group (irradiation / graft polymerization step), the conversion step and the hydrophilization step in the above-described embodiment can be omitted. The irradiation step and the graft polymerization step can be performed simultaneously.
Here, the state in which the base material 1 and the polymerizable monomer having a functional group for adsorption coexist is, for example, the base material 1 in a solution in which the polymerizable monomer having a functional group for adsorption is dissolved. For example, the substrate 1 is immersed, or the substrate 1 is impregnated with a solution in which a polymerizable monomer having a functional group for adsorption is dissolved.

  According to the method for producing the protein capturing material 10 of Modification 3 described above, the protein capturing material 10 can be produced by a simple method including only the irradiation / graft polymerization step and the fixing step.

In the above-described embodiment, the hydrophilization step is performed after the conversion step, but there is no limitation on the timing of performing the hydrophilization step, which may be performed after the graft polymerization step, or after the fixing step. Also good. The same applies to the second modification.
Further, the hydrophilization step is not necessarily performed. The hydrophilization step is effective when the predetermined functional group is a hydrophobic functional group such as an epoxy group, but it is not particularly necessary when the predetermined functional group is a hydrophilic functional group.

In the above-described embodiment and modifications 1 to 3, the shape of the substrate 1 is not limited to a sheet shape, and may be, for example, a disk shape or a square plate shape. It may be in the form of a hollow fiber or a film as in the base material.
Further, the substrate 1 does not have to be made of a porous body having communication holes, and a graft polymer chain (side chain) is added to a polymer material (main chain) forming the substrate 1 by a graft polymerization method. ) Can be applied, and may be, for example, a fiber, a nonwoven fabric, a non-porous polymer base material, or the like.

In the above-described embodiment and Modifications 1 to 3, the protein capturing material 10 is filled in the cartridge 20 when the protein capturing material 10 captures a predetermined protein. However, the present invention is not limited to this. For example, A predetermined amount of protein-capturing material 10 may be accommodated in a container, and a protein solution may be injected into the protein-capturing material 10 to cause the protein-capturing material 10 to capture a predetermined protein.
In the embodiment, the material (IDA-diol sheet) is filled in the cartridge 20 and the fixing step is performed. However, there is no restriction on the timing of filling the material into the cartridge 20, and for example, the substrate 1 is placed in the cartridge. 20 may be filled, and the fixing step may be performed from the irradiation step, or the base material 1 irradiated with the electron beam may be filled into the cartridge 20 and the fixing step may be performed from the graft polymerization step. The cartridge 20 may be filled and the fixing step may be performed from the conversion step, or the IDA sheet may be filled into the cartridge 20 and the hydrophilic step may be performed, or the fixing step may be performed. The cartridge 20 may be filled with the protein capturing material 10 (nickel fixing sheet).

  In the above-described embodiment and Modifications 1 to 3, the protein capturing material 10 captures and separates and purifies a predetermined protein from a protein solution in which the predetermined protein dissolves, but is not limited thereto. As long as it is a substance (gas, liquid, sol, gel, etc.) that can pass through the communication holes of the protein capture material 10, the protein capture material 10 can capture and isolate and purify a predetermined protein from the substance.

It is a figure which shows an example of the manufacture path | route of the protein capture material of this invention. It is a figure which shows an example of the cartridge for protein capture | acquisition of this invention. It is a figure which shows the chromatogram (a) of the protein solution and the outflow protein solution, and the chromatogram (b) of the outflow eluate obtained by the liquid chromatography. It is a figure which shows the band of the protein solution and effluent eluate obtained by the polyacrylamide gel electrophoresis (SDS-PAGE) method.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Base material 2 Graft polymer chain 3, 4 which has a predetermined functional group Graft polymer chain 5 which has a functional group for adsorption 5 Metal ion 10 Protein capture material 20 Cartridge 100 Cartridge for protein capture

Claims (8)

A substrate;
A graft polymer chain bonded to the substrate;
A metal ion having affinity for a protein comprising cysteine, histidine and / or tryptophan, which is fixed to the graft polymer chain;
With
The base material is made of a porous body having communication holes, has a thickness of 1 to 10 mm, an average pore diameter of the communication holes of 0.5 to 5 μm, and a volume fraction of the communication holes of 70 to 85% sheet-like,
The graft polymer chain is bonded to the surface portion of the base material and is not bonded to the inside of the base material.
By utilizing the affinity of the previous northern protein and the metal ion, protein-capture material characterized by capturing those 該Ta protein. The protein capture material according to claim 1 ,
The protein-capturing material, wherein the base material is made of polyolefin. A protein-capturing cartridge, wherein the protein-capturing material according to claim 1 or 2 is filled in a cartridge. In the method for producing a protein capturing material according to claim 1 or 2 ,
An irradiation step of irradiating the substrate with ionizing radiation;
Next, a graft polymerization step of bonding a graft polymer chain having the predetermined functional group to the substrate by graft polymerization of a polymerizable monomer having the predetermined functional group to the substrate;
Next, by converting at least a part of the predetermined functional group into an adsorption functional group for adsorbing the metal ion, the graft polymer chain having the predetermined functional group is converted into the adsorption functional group. A conversion step for converting to a graft polymer chain having;
Next, a fixing step of fixing the metal ion to the graft polymer chain bonded to the base material by adsorbing the metal ion to the functional group for adsorption;
A manufacturing method comprising: In the method for producing a protein capturing material according to claim 1 or 2 ,
In the state where the substrate and a polymerizable monomer having a predetermined functional group coexist, the substrate is irradiated with ionizing radiation, and the polymerizable monomer having the predetermined functional group is applied to the substrate. An irradiation / graft polymerization step in which a graft polymer chain having the predetermined functional group is bonded to the substrate by graft polymerization;
Next, by converting at least a part of the predetermined functional group into an adsorption functional group for adsorbing the metal ion, the graft polymer chain having the predetermined functional group is converted into the adsorption functional group. A conversion step for converting to a graft polymer chain having;
Next, a fixing step of fixing the metal ion to the graft polymer chain bonded to the base material by adsorbing the metal ion to the functional group for adsorption;
A manufacturing method comprising: In the manufacturing method of Claim 4 or Claim 5 ,
Production comprising the step of hydrophilizing the graft polymer chain bonded to the substrate by converting the predetermined functional group of the graft polymer chain into a hydrophilic functional group. Method. In the method for producing a protein capturing material according to claim 1 or 2 ,
An irradiation step of irradiating the substrate with ionizing radiation;
Subsequently, the graft polymer chain having the adsorption functional group is bonded to the substrate by graft polymerization of the polymerizable monomer having the adsorption functional group for adsorbing the metal ion to the substrate. A graft polymerization step;
Next, a fixing step of fixing the metal ion to the graft polymer chain bonded to the base material by adsorbing the metal ion to the functional group for adsorption;
A manufacturing method comprising: In the method for producing a protein capturing material according to claim 1 or 2 ,
In the coexistence of the base material and a polymerizable monomer having an adsorption functional group for adsorbing the metal ions, the base material is irradiated with ionizing radiation, and the polymerization property having the adsorption functional group is obtained. An irradiation / graft polymerization step in which a graft polymer chain having the adsorption functional group is bonded to the substrate by graft polymerization of the monomer to the substrate;
Next, a fixing step of fixing the metal ion to the graft polymer chain bonded to the base material by adsorbing the metal ion to the functional group for adsorption;
A manufacturing method comprising: