JP3062745B1 - Material capable of selectively adsorbing and desorbing protein and method for producing the same - Google Patents
Material capable of selectively adsorbing and desorbing protein and method for producing the sameInfo
- Publication number
- JP3062745B1 JP3062745B1 JP11012070A JP1207099A JP3062745B1 JP 3062745 B1 JP3062745 B1 JP 3062745B1 JP 11012070 A JP11012070 A JP 11012070A JP 1207099 A JP1207099 A JP 1207099A JP 3062745 B1 JP3062745 B1 JP 3062745B1
- Authority
- JP
- Japan
- Prior art keywords
- protein
- desorbing
- material capable
- monomer
- fine particles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Peptides Or Proteins (AREA)
Abstract
【要約】
【課題】 特定のタンパク質のみを分子識別することが
でき、多種類のタンパク質混合物から特定のタンパク質
を高い精度で吸着し、脱離することができるタンパク質
吸脱着材料及びその製造方法。
【課題を解決する手段】 タンパク質にタンパク質と相
互作用をする官能基を有する単量体を加え、混合接触さ
せて相互作用を行い、相互作用に関与しなかった単量体
をほぼ完全に除去した後、タンパク質と相互作用する官
能基をもたない単量体と多孔質無機微粒子を加えて重合
させて複合ポリマーとし、該複合ポリマーを粉砕して微
粒子状とし、該微粒子よりタンパク質を除去することに
より作られる分子形状鋳型をもつ特定のタンパク質を吸
着脱離できる材料。Kind Code: A1 Abstract: A protein adsorbing / desorbing material capable of molecularly identifying only a specific protein, adsorbing and desorbing a specific protein from a mixture of various kinds of proteins with high accuracy, and a method for producing the same. [Means for Solving the Problems] A monomer having a functional group that interacts with a protein is added to the protein, and the mixture is brought into contact with each other to perform an interaction, and a monomer that has not been involved in the interaction is almost completely removed. Thereafter, a monomer having no functional group interacting with the protein and a porous inorganic fine particle are added and polymerized to form a composite polymer, and the composite polymer is pulverized into fine particles, and the protein is removed from the fine particles. A material capable of adsorbing and desorbing a specific protein having a molecular template formed by the method.
Description
【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION
【０００１】[0001]
【産業上の利用分野】本発明は、特定のタンパク質のみ
を選択的に吸着及び脱着する機能を有する材料及びその
製造方法に関するものである。本発明のタンパク質吸脱
着材料は、特定のタンパク質のみを分子識別することが
でき、多種類のタンパク質混合物から特定のタンパク質
を吸着分離精製するためのアフィニティクロマトグラフ
用担体として用いることができ、また、特定のタンパク
質を検出分析するための試薬あるいはセンサ用感応物質
として、さらに、医療、食品産業において有害タンパク
質を吸着除去するための吸着剤として利用することがで
きる。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a material having a function of selectively adsorbing and desorbing only a specific protein and a method for producing the same. The protein adsorbing and desorbing material of the present invention can identify only a specific protein, and can be used as an affinity chromatographic carrier for adsorptive separation / purification of a specific protein from a mixture of various proteins, It can be used as a reagent for detecting and analyzing a specific protein or a sensitive substance for a sensor, and as an adsorbent for adsorbing and removing harmful proteins in the medical and food industries.
【０００２】[0002]
【従来技術】病気診断のためのセンサ開発において、ま
た、病気治療のために用いるタンパク質の分離・精製過
程において、所望するタンパク質のみを選択的に吸着、
脱着する材料が望まれている。このようなタンパク質分
子識別機能をもつ材料の合成法がAngew. Chem. Int. E
d. Engl. 1995年, 34巻, pp.1812-1832に記されてい
る。この報告によればタンパク質共存下でモノマーと架
橋剤をラジカル重合することによりタンパク質鋳型をも
つ物質を形成させる方法が示されている。しかしなが
ら、ある特定のタンパク質に対して最適な分子識別能を
もつ物質を合成するためにはどのようなモノマーをどれ
だけ用いるかについては目安がなく、試行錯誤を繰り返
すことになるから目安となる物理化学的性質が所望され
ていた。そこで、所望するタンパク質のゼータ電位を目
安としてモノマー量を決めることにより、同じモノマー
を用いても所望するタンパク質の選択的吸着能の高い材
料を得ることができる方法が開発されている（特願平１
０−７８３４１）。しかしながら、この方法においても
タンパク質や、合成した材料のゼータ電位値を測定して
モノマーの最適量を知る必要が生じる。そこで所望する
タンパク質に対しては他のタンパク質に比べて高い選択
性をもつと同時に高い吸着能をもつ材料の開発とその簡
単な製造法の開発が望まれていた。2. Description of the Related Art In the development of sensors for disease diagnosis and in the process of separating and purifying proteins used for disease treatment, only the desired protein is selectively adsorbed.
A material that can be desorbed is desired. Angew. Chem. Int. E
d. Engl. 1995, 34, pp. 1812-1832. According to this report, a method is disclosed in which a substance having a protein template is formed by radical polymerization of a monomer and a crosslinking agent in the presence of a protein. However, there is no guide as to which monomer to use and how much to use in order to synthesize a substance with the optimal molecular discrimination ability for a specific protein. The chemistry was desired. Therefore, a method has been developed in which the amount of a monomer is determined by using the zeta potential of a desired protein as a guide to obtain a material having a high selective adsorption ability of a desired protein even when the same monomer is used (Japanese Patent Application No. Hei. 1
0-78341). However, also in this method, it is necessary to measure the zeta potential value of the protein or the synthesized material to know the optimum amount of the monomer. Therefore, it has been desired to develop a material having a high selectivity to a desired protein as compared with other proteins and a high adsorptivity and a simple production method thereof.
【０００３】[0003]
【発明が解決しようとする課題】本発明は、所望するタ
ンパク質のみに対して特異的に分子識別機能をもつタン
パク質吸脱着材料及びその製造方法を提供することをそ
の課題とする。SUMMARY OF THE INVENTION It is an object of the present invention to provide a protein adsorbing / desorbing material having a function of specifically discriminating only a desired protein and a method for producing the same.
【０００４】[0004]
【課題を解決するための手段】本発明者は、今までに得
られていなかった、所望するタンパク質のみに対して特
異的に分子識別機能をもつタンパク質吸脱着材料を経済
的に得るべく、鋭意研究を重ねた結果、本研究を完成す
るに至った。即ち、本発明は、タンパク質吸脱着材料及
びその製造方法を提供する。本明細書におけるタンパク
質吸脱着材料は、表面にビニル基などの反応性基を坦持
させたシリカゲルに、単量体と相互作用させたタンパク
質、単量体、重合性架橋剤を共存させ、重合させること
により得られる複合ポリマーを粉砕して微粒子状とした
後、微粒子よりタンパク質を除去してタンパク質分子形
状鋳型を表面に作り出した複合体である。本明細書にお
ける多孔質無機微粒子としては、シリカゲルやゼオライ
ト、酸化チタン等があるがシリカゲルが望ましい。多孔
質無機微粒子は、その表面積が大きい方が望ましく、多
孔質無機微粒子の孔径は目的とするタンパク質が十分に
内部に侵入できるほど大きいのが望ましい。Means for Solving the Problems The present inventors have been keen to economically obtain a protein adsorbing / desorbing material having a molecular discriminating function specifically for only a desired protein, which has not been obtained until now. As a result of repeated research, this research was completed. That is, the present invention provides a protein adsorption / desorption material and a method for producing the same. The protein adsorbing / desorbing material in the present specification is obtained by coexisting a protein, a monomer, and a polymerizable cross-linking agent, which are made to interact with a monomer, on silica gel having a reactive group such as a vinyl group on the surface. The resulting composite polymer is pulverized into fine particles, and then the protein is removed from the fine particles to form a protein molecular template on the surface. Examples of the porous inorganic fine particles in the present specification include silica gel, zeolite, and titanium oxide, but silica gel is preferable. The surface area of the porous inorganic fine particles is desirably large, and the pore diameter of the porous inorganic fine particles is desirably large enough to allow a target protein to sufficiently enter the inside.
【０００５】本発明では、タンパク質と相互作用する官
能基をもつ機能性単量体の選択が重要であるが、本発明
でいう相互作用とは、正−負荷電間の静電的相互作用、
酵素−基質間類似相互作用、酵素−阻害剤間相互作用、
酵素−補酵素間相互作用などタンパク質となんらかの形
で強い影響力を及ぼし合う作用を総称して相互作用とい
う。ここで、正-負荷電間の静電的相互作用としては、
タンパク質のもつアミノ基、イミダゾール基などの正荷
電に対してカルボキシル基、リン酸基などの負荷電など
が対応し、一方、タンパク質のもつカルボキシル基など
の負荷電基に対してアミノ基、イミノ基、３級アミノ基
などの正荷電が対応する。すなわち、この相互作用を利
用するためには、アクリル酸、メタクリル酸、Ｎ，Ｎ−
ジメチルアミノプロピルアクリルアミド、などのビニル
基を有する機能性単量体などが選ばれ、水溶性であるこ
とが望ましい。酵素−基質間類似相互作用の例として
は、酵素グルコースオキシダーゼに対してその基質であ
るグルコースにビニル基などの重合性官能基を導入した
化合物、例えばメタクリル酸グルコシルオキシエチル、
があげられる。また、酵素−阻害剤間相互作用の例とし
て、酵素トリプシンに対してその阻害剤であるパラアミ
ノベンズアミジンにビニル基などの反応性官能基を導入
した化合物があげられる。酵素−補酵素間相互作用の例
として、グルタミン酸−オキサロ酢酸トランスアミナー
ゼに対してその補酵素であるピリドキサルリン酸にビニ
ル基などの反応性官能基を導入した化合物が挙げられ
る。本発明において、精度の高いタンパク質の分子形状
鋳型を作り出せるのは、相互作用に関与しなかった単量
体を注意深くほぼ完全に除去するのに加えて、正−負荷
電間の静電的相互作用のみならず、酵素−基質間類似相
互作用、酵素−阻害剤間相互作用によるタンパク質特有
の形を鋳型にとることができるからである。したがっ
て、本発明の分子形状鋳型は、タンパク質の特徴点に相
互作用を有する物質を残存させることができ、精密なタ
ンパク質の分子形状鋳型を形成することができる。これ
らの相互作用を１種類あるいは多種類含んでもよい。タ
ンパク質と機能性単量体とを相互作用させる方法として
は、タンパク質と過剰量の機能性単量体とを適当なｐＨ
及びイオン強度の緩衝液に溶かすことにより達成され、
条件によっては、タンパク質と機能性単量体との複合体
が沈殿として生じる。相互作用に関与しなかった機能性
単量体を除く方法としては、上記の複合体を含む溶液を
透析膜を用いて緩衝液に対して透析するか、あるいは限
外濾過膜を用いて加圧濾過除去する方法があげられる。In the present invention, it is important to select a functional monomer having a functional group that interacts with a protein. The term “interaction” as used in the present invention refers to an electrostatic interaction between positive and negative charges,
Enzyme-substrate analogous interaction, enzyme-inhibitor interaction,
Actions that have a strong influence on proteins in some way, such as enzyme-coenzyme interactions, are collectively referred to as interactions. Here, as the electrostatic interaction between the positive and negative charges,
Negative charges such as carboxyl groups and phosphate groups correspond to positive charges such as amino groups and imidazole groups of proteins, while amino groups and imino groups correspond to negative charges such as carboxyl groups of proteins. Positive charges such as tertiary amino groups correspond. That is, in order to utilize this interaction, acrylic acid, methacrylic acid, N, N-
A functional monomer having a vinyl group, such as dimethylaminopropylacrylamide, is selected, and is preferably water-soluble. Examples of the enzyme-substrate analogous interaction include compounds obtained by introducing a polymerizable functional group such as a vinyl group into glucose, which is a substrate of the enzyme glucose oxidase, such as glucosyloxyethyl methacrylate.
Is raised. Examples of the enzyme-inhibitor interaction include compounds in which a reactive functional group such as a vinyl group is introduced into paraaminobenzamidine, which is an inhibitor of the enzyme trypsin. An example of the enzyme-coenzyme interaction is a compound in which a reactive functional group such as a vinyl group is introduced into pyridoxal phosphate, which is a coenzyme of glutamic acid-oxaloacetate transaminase. In the present invention, it is possible to create a high-precision protein molecular shape template by carefully and almost completely removing monomers that did not participate in the interaction, in addition to electrostatic interaction between positive and negative charges. This is because not only a similar form between the enzyme and the substrate but also a specific form of the protein due to the interaction between the enzyme and the inhibitor can be used as a template. Therefore, the molecular shape template of the present invention can leave a substance that interacts with a characteristic point of a protein, and can form a precise protein molecular shape template. One or more of these interactions may be included. As a method of interacting a protein and a functional monomer, a protein and an excess amount of a functional monomer are mixed at an appropriate pH.
And by dissolving in ionic strength buffer,
Under some conditions, a complex of the protein and the functional monomer forms as a precipitate. As a method for removing the functional monomer not involved in the interaction, a solution containing the above complex is dialyzed against a buffer using a dialysis membrane, or pressurized using an ultrafiltration membrane. There is a method of removing by filtration.
【０００６】タンパク質と相互作用する官能基をもたな
い水溶性単量体としては、アクリルアミド、ヒドロキシ
エチルメタクリレートなどが選ばれる。一方、架橋剤と
しては、Ｎ，Ｎ′−（１，２−ジヒドロキシエチレン）
−ビスアクリルアミド、Ｎ，Ｎ′−メチレンビスアクリ
ルアミドなどの重合性官能基を２つ以上有する化合物が
選ばれ、水溶性の高い化合物が望ましい。重合反応で
は、通常、タンパク質が水に溶けているため、水溶性の
重合開始剤、例えば過硫酸アンモニウム、などが用いら
れる。重合温度はタンパク質の変性を防ぐためできるだ
け低い温度が望ましく、室温あるいはそれ以下に設定す
る。さらに、重合促進剤、例えばＮ，Ｎ, Ｎ′,Ｎ′−
テトラメチルエチレンジアミンなどが重合溶液中に添加
される。シリカゲル基材表面に硬い重合体層を薄く形成
させるために、架橋剤の量をできるだけ多くすることが
望ましい。[0006] Acrylamide, hydroxyethyl methacrylate and the like are selected as water-soluble monomers having no functional group that interacts with proteins. On the other hand, as a crosslinking agent, N, N '-(1,2-dihydroxyethylene)
Compounds having two or more polymerizable functional groups such as -bisacrylamide and N, N'-methylenebisacrylamide are selected, and compounds having high water solubility are desirable. In the polymerization reaction, since a protein is usually dissolved in water, a water-soluble polymerization initiator such as ammonium persulfate is used. The polymerization temperature is desirably as low as possible to prevent protein denaturation, and is set to room temperature or lower. Further, a polymerization accelerator such as N, N, N ', N'-
Tetramethylethylenediamine or the like is added to the polymerization solution. In order to form a thin hard polymer layer on the surface of the silica gel substrate, it is desirable to increase the amount of the crosslinking agent as much as possible.
【０００７】[0007]
【実施の形態】本発明の実施の形態は以下の通りであ
る。 （１） 多孔質無機微粒子の表面のポリマーに、タンパ
ク質の特徴点に相互作用を有する物質を残存させた精度
の高いタンパク質の分子形状鋳型をもつタンパク質を選
択的に吸着脱離できる材料。 （２） 分子形状鋳型を作るポリマーが架橋されている
上記１記載のタンパク質を選択的に吸着脱離できる材
料。 （３） 表面に予めビニル基を配位させた多孔質無機微
粒子を用いる上記１または上記２記載のタンパク質を選
択的に吸着脱離できる材料 （４） タンパク質にタンパク質と相互作用をする官能
基を有する単量体を加え、混合接触させて相互作用を行
い、相互作用に関与しなかった単量体をほぼ完全に除去
した後、タンパク質と相互作用する官能基をもたない単
量体と多孔質無機微粒子を加えて重合させて複合ポリマ
ーとし、該複合ポリマーを粉砕して微粒子状とし、該微
粒子よりタンパク質を除去することにより作られる精度
の高いタンパク質の分子形状鋳型をもつタンパク質を選
択的に吸着脱離できる材料の製造方法。 （５） タンパク質と相互作用する官能基をもたない単
量体と共に重合性架橋剤を加える上記４記載のタンパク
質を選択的に吸着脱離できる材料の製造方法。 （６） 表面に予め反応性官能基を配位させた多孔質無
機微粒子を用いる上記４または上記５記載のタンパク質
を選択的に吸着脱離できる材料の製造方法。 （７） 反応性官能基がビニル基である上記４ないし上
記６記載のタンパク質を選択的に吸着脱離できる材料の
製造方法。Embodiments of the present invention are as follows. (1) A material capable of selectively adsorbing and desorbing a protein having a high-precision protein molecular shape template in which a substance having an interaction with a characteristic point of the protein is left in the polymer on the surface of the porous inorganic fine particles. (2) A material capable of selectively adsorbing and desorbing the protein according to 1 above, wherein the polymer forming the molecular template is crosslinked. (3) A material capable of selectively adsorbing and desorbing the protein according to the above (1) or (2), which uses porous inorganic fine particles having a vinyl group coordinated on the surface in advance. (4) The protein has a functional group that interacts with the protein. After adding monomers that have a functional group that does not have a functional group that interacts with the protein, the monomer that has not participated in the interaction is almost completely removed. A polymer having a high-precision protein molecular shape template produced by removing the protein from the fine particles by selectively pulverizing the composite polymer into fine particles, and selectively polymerizing the composite polymer. A method for producing a material that can be adsorbed and desorbed. (5) The method for producing a material capable of selectively adsorbing and desorbing a protein according to the above (4), wherein a polymerizable crosslinking agent is added together with a monomer having no functional group that interacts with the protein. (6) The method for producing a material capable of selectively adsorbing and desorbing the protein according to the above (4) or (5), using porous inorganic fine particles having a reactive functional group coordinated in advance on the surface. (7) A method for producing a material capable of selectively adsorbing and desorbing the protein according to the above items 4 to 6, wherein the reactive functional group is a vinyl group.
【０００８】[0008]
【実施例】実施例１ （イ）多孔性シリカゲルの調製 アミノ基を表面にもつ粒径０.０１ｍｍの多孔性シリカ
ゲル(LiChrosorb-NH２）に、氷冷下シクロヘキサン中に
て塩化アクリロイルを反応させてビニル基を表面にもつ
多孔性シリカゲル[LiChrosorb-NH-COCHCH２］を得た。 （ロ）タンパク質と相互作用する官能基をもつ機能性単
量体の調製 これとは別に、１２ｍＭリン酸二水素ナトリウム水溶液
（１０ｍＬ）に、負荷電基をもつアクリル酸（ＡＡｃ）
（１５．９ｍｇ)、メタクリル酸グルコシルオキシエチ
ル（ＧＥＭＡ）（１．８６ｍｇ）、及び正荷電基をもつ
Ｎ，Ｎ′−ジメチルアミノプロピルアクリルアミド（Ｄ
ＭＡＰＡＡｍ）（２０．９ｍｇ）を加えてｐＨを５．６
に調節した。この溶液に酵素グルコースオキシダーゼ
（ＥＣ１．１．３．４)(ＧＯＤ)(３０．４ｍｇ)を加え
て冷蔵庫中に１２時間放置したところ沈殿物が得られ
た。 （ハ）相互作用に関与しなかった機能性単量体の除去 この沈殿物を含む溶液を透析チューブに入れ、１２ｍＭ
リン酸二水素ナトリウム水溶液（ｐＨ５．６）に対して
２０時間透析した。透析チューブ中の溶液が２．５ｍＬ
になるまで透析チューブをそのまま風乾した。この沈殿
物含有溶液（２．５ｍＬ）にアクリルアミド（ＡＡｍ）
（２５０ｍｇ）と、２種類の架橋剤Ｎ，Ｎ′−（１,２
−ジヒドロキシエチレン）−ビスアクリルアミド（Ｄｉ
Ｂｉｓ）（３０ｍｇ）、Ｎ，Ｎ′−メチレンビスアクリ
ルアミド（Ｂｉｓ）（３２．５ｍｇ）とを混合してｐＨ
を５.６に調節したモノマー混合溶液を作成した。 （ニ）重合 このモノマー混合溶液（３ｍＬ）に、ビニル基を表面に
もつ多孔性シリカゲル（１ｇ）を混合させ、その溶液中
に窒素ガスを導入して溶存酸素を追い出した。この溶液
に重合開始剤として過硫酸アンモニウムの水溶液（濃度
２００ｍｇ／０．３００ｍＬを０.０１５ｍＬ）と重合
促進剤としてＮ，Ｎ, Ｎ′,Ｎ′−テトラメチルエチレ
ンジアミン（０.００５ｍＬ）とを加えて、この混合溶
液を直ちに遠心器にて室温下で毎分３０００回転で１０
分間遠心した。遠心終了後、静置して重合反応をさらに
３時間継続させた。 （ホ）タンパク質の除去による鋳型の生成 重合反応した固体から多孔性シリカゲルを含む部分を破
砕して取り出した。このLiChrosorb-重合物複合体から
なる微粒子を２４０ｍＭリン酸二水素ナトリウム（７ｍ
Ｌ,ｐＨ５.６）で５回洗浄し、さらに１２ｍＭリン酸二
水素ナトリウム（７ｍＬ，ｐＨ５．６）で２回洗浄して
酵素グルコースオキシダーゼを抜き出した。この微粒子
をタンパク質形状鋳型を表面に有する活性化複合体、つ
まりタンパク質吸脱着材料と称する。Example 1 (a) Preparation of porous silica gel Acryloyl chloride was reacted with porous silica gel (LiChrosorb-NH 2 ) having amino groups on the surface and having a particle size of 0.01 mm in cyclohexane under ice cooling. Thus, porous silica gel [LiChrosorb-NH-COCHCH 2 ] having a vinyl group on the surface was obtained. (B) Preparation of a functional monomer having a functional group that interacts with a protein Separately, an acrylic acid (AAc) having a negatively charged group was added to a 12 mM aqueous solution of sodium dihydrogen phosphate (10 mL).
(15.9 mg), glucosyloxyethyl methacrylate (GEMA) (1.86 mg), and N, N'-dimethylaminopropylacrylamide (D
MAPAAm) (20.9 mg) and added to pH 5.6.
Was adjusted to The enzyme glucose oxidase (EC 1.1.3.4) (GOD) (30.4 mg) was added to this solution, and the mixture was allowed to stand in a refrigerator for 12 hours to obtain a precipitate. (C) Removal of functional monomer not involved in the interaction The solution containing the precipitate was placed in a dialysis tube, and 12 mM
Dialysis was performed for 20 hours against an aqueous solution of sodium dihydrogen phosphate (pH 5.6). 2.5 mL of solution in dialysis tube
The dialysis tubing was air-dried until it reached. Acrylamide (AAm) was added to the precipitate-containing solution (2.5 mL).
(250 mg) and two kinds of cross-linking agents N, N '-(1,2
-Dihydroxyethylene) -bisacrylamide (Di)
Bis) (30 mg) and N, N'-methylenebisacrylamide (Bis) (32.5 mg).
Was adjusted to 5.6 to prepare a monomer mixed solution. (D) Polymerization Porous silica gel (1 g) having a vinyl group on the surface was mixed with the monomer mixture solution (3 mL), and nitrogen gas was introduced into the solution to drive out dissolved oxygen. To this solution was added an aqueous solution of ammonium persulfate (0.015 mL at a concentration of 200 mg / 0.300 mL) as a polymerization initiator and N, N, N ', N'-tetramethylethylenediamine (0.005 mL) as a polymerization accelerator. This mixed solution was immediately centrifuged at room temperature at 3000 rpm for 10 minutes at room temperature.
Centrifuge for minutes. After completion of the centrifugation, the mixture was allowed to stand and the polymerization reaction was continued for another 3 hours. (E) Formation of template by removal of protein A portion containing porous silica gel was crushed and taken out from the polymerized solid. The fine particles composed of the LiChrosorb-polymer composite were treated with 240 mM sodium dihydrogen phosphate (7 m
L, pH 5.6) and washed twice with 12 mM sodium dihydrogen phosphate (7 mL, pH 5.6) to extract the enzyme glucose oxidase. These fine particles are called an activated complex having a protein-shaped template on the surface, that is, a protein adsorbing / desorbing material.
【０００９】実施例２ 表１に記載したように４種類の微粒子を調製し、実施例
１と同様にしてタンパク質吸脱着材料を製造した。Example 2 Four types of fine particles were prepared as shown in Table 1, and a protein adsorbing / desorbing material was produced in the same manner as in Example 1.
【表１】 ここで、ＧＯＤ，ＧＥＭＡ，ＡＡｃ，ＤＭＡＰＡＡｍ，
ＡＡｍは実施例１と同じであり、架橋剤も実施例１と同
様に、ＤｉＢｉｓとＢｉｓの２種類であった。[Table 1] Here, GOD, GEMA, AAc, DMAPAAm,
The AAm was the same as in Example 1, and two types of crosslinking agents, DiBis and Bis, as in Example 1.
【００１０】実施例３ 実施例１及び実施例２で調製した微粒子に対する酵素グ
ルコースオキシダーゼの吸着実験を行った結果を図１に
示す。吸着量は、１２ｍＭのリン酸二水素ナトリウム溶
液１．５ｍＬに酵素０．０１５ｍｇを溶かした溶液に、
湿潤した微粒子を２時間撹拌しながら接触させた後、こ
の上澄溶液の酵素活性と元液の酵素活性を測定すること
により算出した。吸着量は試料Ｎｏ．２、Ｎｏ．１、Ｎ
ｏ．３、Ｎｏ．４、Ｎｏ．５の順に減少した。Ｎｏ．５
は酵素の鋳型を形成させてない試料であり、この吸着量
は非特異的吸着量とみなすことができる。したがって、
酵素と相互作用する機能性単量体を含む微粒子、Ｎｏ．
１及びＮｏ．２はＮｏ．５と比べ、３倍以上多くの酵素
を吸着することがわかる。Example 3 FIG. 1 shows the results of an adsorption experiment of the enzyme glucose oxidase on the fine particles prepared in Examples 1 and 2. The adsorption amount is a solution obtained by dissolving 0.015 mg of enzyme in 1.5 mL of 12 mM sodium dihydrogen phosphate solution.
After the wet fine particles were contacted with stirring for 2 hours, the enzyme activity of the supernatant solution and the enzyme activity of the original solution were measured to calculate the value. The amount of adsorption is the same as for the sample No. 2, No. 1, N
o. 3, No. 4, no. It decreased in the order of 5. No. 5
Is a sample on which no enzyme template is formed, and the amount of adsorption can be regarded as a non-specific adsorption amount. Therefore,
Fine particles containing a functional monomer that interacts with the enzyme,
1 and No. 1 No. 2 is No. It can be seen that 3 times or more of the enzyme is adsorbed as compared with 5.
【００１１】実施例４ 実施例１及び実施例２で調製した微粒子に対して、２種
類のタンパク質の混合物競争的吸着試験を行った。タン
パク質として、濃度１．０ｍｇ／１００ｍＬのグルコー
スオキシダーゼ（ＧＯＤ）と濃度０．３０８ｍｇ／１０
０ｍＬのグルコース−６−フォスフェートデヒドロゲナ
ーゼ（Ｇ６ＰＤ）（ＥＣ１.１.１.４９）との混合溶液
に上記の微粒子を２時間攪拌しながら接触させた後、そ
の上澄溶液を採取した。いずれの酵素が特異的に微粒子
に再結合したかを知るため、この上澄溶液のそれぞれの
酵素の活性を測定し、吸着量を求めた。表２に示すよう
に、Ｇ６ＰＤの吸着量に対するＧＯＤの吸着量の比とし
て定義した吸着選択性は、ＧＯＤ鋳型をもつ試料Ｎｏ．
１では大きな値が見い出された。ＧＯＤに対して静電的
相互作用だけを利用して調製した試料Ｎo．２では吸着
選択性が低い値である。これらの結果から、選択性を高
めるためにはできるだけ多くの相互作用を用いる方がよ
いことが確かめられた。Example 4 The fine particles prepared in Examples 1 and 2 were subjected to a competitive adsorption test of a mixture of two types of proteins. As a protein, glucose oxidase (GOD) at a concentration of 1.0 mg / 100 mL and a concentration of 0.308 mg / 10
The fine particles were brought into contact with a mixed solution of 0 mL of glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) with stirring for 2 hours, and the supernatant solution was collected. In order to know which enzyme specifically recombined with the microparticles, the activity of each enzyme in this supernatant solution was measured to determine the amount of adsorption. As shown in Table 2, the adsorption selectivity, defined as the ratio of the amount of GOD adsorbed to the amount of G6PD adsorbed, was determined for sample no.
At 1 a large value was found. Sample No. prepared using only electrostatic interaction with GOD In the case of 2, the adsorption selectivity is a low value. These results confirm that it is better to use as many interactions as possible to increase the selectivity.
【００１２】[0012]
【表２】 [Table 2]
【００１３】[0013]
【本発明の効果】本発明のタンパク質を選択的に吸着脱
離できる材料は、選択性が高く、多種類のタンパク質混
合物から特定のタンパク質を吸着分離精製するためのア
フィニティクロマトグラフ用担体、特定のタンパク質を
検出分析するための試薬あるいはセンサ用感応物質とし
て、さらに、医療、食品産業において有害タンパク質を
吸着除去するための吸着剤として用いることができる。The material of the present invention capable of selectively adsorbing and desorbing the protein has a high selectivity, a carrier for affinity chromatography for adsorbing, separating and purifying a specific protein from a mixture of various proteins, and a specific material. It can be used as a reagent for detecting and analyzing proteins or as a sensitive substance for sensors, and as an adsorbent for adsorbing and removing harmful proteins in the medical and food industries.
【図１】 ＧＯＤ吸着量ｂ（湿潤した試料１グラム当たりに吸着し
たＧＯＤのモル 数）と溶液のＧＯＤ濃度［Ｓ］との関
係を表す説明図 ●：試料Ｎｏ．１、 ○：試料Ｎｏ．２、◆：試料Ｎ
ｏ．３、◇：試料Ｎｏ．４、 △：試料Ｎｏ．５FIG. 1 is an explanatory diagram showing the relationship between the GOD adsorption amount b (the number of moles of GOD adsorbed per gram of wet sample) and the GOD concentration [S] of the solution. 1, ：: Sample No. 2, △: Sample N
o. 3, Δ: Sample No. 4, Δ: Sample No. 5
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ Ｇ０１Ｎ 30/48 Ｇ０１Ｎ 30/48 Ｌ Ｒ Ｚ 30/88 30/88 Ｊ (58)調査した分野(Int.Cl.7，ＤＢ名) B01J 20/00 - 20/34 C07K 1/14 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 identification code FI G01N 30/48 G01N 30/48 L R Z 30/88 30/88 J (58) Investigated field (Int. Cl. 7 , DB Name) B01J 20/00-20/34 C07K 1/14
Claims (7)
タンパク質の特徴点に相互作用を有する物質を残存させ
たタンパク質の分子形状鋳型をもつ、タンパク質を選択
的に吸着脱離できる材料。1. The polymer on the surface of the porous inorganic fine particles,
A material capable of selectively adsorbing and desorbing a protein, which has a protein molecular template in which a substance having an interaction with a characteristic point of the protein remains.
ている請求項１記載のタンパク質を選択的に吸着脱離で
きる材料。2. The material capable of selectively adsorbing and desorbing a protein according to claim 1, wherein the polymer forming the molecular shape template is crosslinked.
無機微粒子を用いる請求項１または請求項２記載のタン
パク質を選択的に吸着脱離できる材料3. A material capable of selectively adsorbing and desorbing a protein according to claim 1 or 2, wherein porous inorganic fine particles having a vinyl group coordinated in advance on the surface are used.
る官能基を有する単量体を加え、混合接触させて相互作
用を行い、相互作用に関与しなかった単量体をほぼ完全
に除去した後、タンパク質と相互作用する官能基をもた
ない単量体と多孔質無機微粒子を加えて重合させて複合
ポリマーとし、該複合ポリマーを粉砕して微粒子状と
し、該微粒子よりタンパク質を除去することにより作ら
れる精度の高いタンパク質の分子形状鋳型をもつタンパ
ク質を選択的に吸着脱離できる材料の製造方法。4. After adding a monomer having a functional group that interacts with the protein to the protein and mixing and contacting the protein to remove the monomer not involved in the interaction almost completely, A polymer is prepared by adding a monomer having no functional group that interacts with a protein and a porous inorganic fine particle and polymerizing it to form a composite polymer, pulverizing the composite polymer into fine particles, and removing the protein from the fine particles. A method for producing a material capable of selectively adsorbing and desorbing a protein having a high-precision protein molecular template.
ない単量体と共に重合性架橋剤を加える請求項４記載の
タンパク質を選択的に吸着脱離できる材料の製造方法。5. The method according to claim 4, wherein a polymerizable crosslinking agent is added together with a monomer having no functional group that interacts with the protein.
孔質無機微粒子を用いる請求項４または請求項５記載の
タンパク質を選択的に吸着脱離できる材料の製造方法。6. The method for producing a material capable of selectively adsorbing and desorbing a protein according to claim 4 or 5, wherein porous inorganic fine particles having a reactive functional group previously coordinated on the surface are used.
ないし請求項６記載のタンパク質を選択的に吸着脱離で
きる材料の製造方法。7. The reactive functional group is a vinyl group.
A method for producing a material capable of selectively adsorbing and desorbing a protein according to claim 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11012070A JP3062745B1 (en) | 1999-01-20 | 1999-01-20 | Material capable of selectively adsorbing and desorbing protein and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11012070A JP3062745B1 (en) | 1999-01-20 | 1999-01-20 | Material capable of selectively adsorbing and desorbing protein and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP3062745B1 true JP3062745B1 (en) | 2000-07-12 |
| JP2000202285A JP2000202285A (en) | 2000-07-25 |
Family
ID=11795349
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11012070A Expired - Lifetime JP3062745B1 (en) | 1999-01-20 | 1999-01-20 | Material capable of selectively adsorbing and desorbing protein and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3062745B1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002122579A (en) * | 2000-10-12 | 2002-04-26 | Nippon Laser & Electronics Lab | Adsorbent for chromatography |
| JP4058517B2 (en) * | 2003-04-08 | 2008-03-12 | 独立行政法人産業技術総合研究所 | Photoresponsive molecular identification material |
| JP3835439B2 (en) | 2003-08-20 | 2006-10-18 | トヨタ自動車株式会社 | Concentration detector |
| JP4541715B2 (en) * | 2004-01-23 | 2010-09-08 | 北越紀州製紙株式会社 | Sheet with molecular adsorption function |
| JP4852743B2 (en) * | 2005-08-17 | 2012-01-11 | 国立大学法人 宮崎大学 | Adsorbent and production method thereof |
| JP5099074B2 (en) * | 2009-05-11 | 2012-12-12 | 株式会社豊田中央研究所 | Adsorption carrier having selective adsorption region of target molecule and method for producing the same |
-
1999
- 1999-01-20 JP JP11012070A patent/JP3062745B1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000202285A (en) | 2000-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4443762B2 (en) | Improved magnetic particles, their production method, and their use for separating molecules | |
| Qin et al. | Macroporous thermosensitive imprinted hydrogel for recognition of protein by metal coordinate interaction | |
| US6753396B2 (en) | Method for producing template-textured materials with high binding specificity and selectivity and utilization of said materials | |
| Khumsap et al. | Epitope-imprinted polymers: applications in protein recognition and separation | |
| ES2557577T3 (en) | Preparation of polymers with molecular imprint | |
| JPH08502534A (en) | Pore size selective modification method for porous materials | |
| US7122381B2 (en) | Selective affinity material, preparation thereof by molecular imprinting, and use of the same | |
| CA1223859A (en) | Surface-grafted particulate support, process for its preparation and adsorbants for affinity chromatography incorporating such support, and their use, particularly in biology | |
| US20120029154A1 (en) | Grafting method to improve chromatography media performance | |
| JP6063529B2 (en) | Pretreatment cartridge for substance separation and pretreatment method using the same | |
| JP3062745B1 (en) | Material capable of selectively adsorbing and desorbing protein and method for producing the same | |
| EP0669942A1 (en) | Artificial antibodies, method of producing the same and use thereof | |
| CN108003287B (en) | Preparation method of protein affinity imprinted hydrogel polymer based on acrylamide metal chelating monomer | |
| US20040241753A1 (en) | Device for producing polymer support materials in the form of porous polymer beads | |
| JP4793816B2 (en) | Method for producing macromolecular identification polymer | |
| JP2003524680A (en) | Affinity control type material using stimulus-responsive polymer and separation / purification method using the material | |
| US7015281B2 (en) | Alkali-stable hydrophilic sorbents for ion-exchange chromatography | |
| JPH09127116A (en) | Substance with protein molecule discriminating function | |
| JP3543107B2 (en) | Protein identification method | |
| JP2946036B2 (en) | Substance having protein molecule identification function and method for producing the same | |
| CN112898624B (en) | Alternative template imprinted polymer for specifically recognizing exosomes and application thereof | |
| Yilmaz et al. | The noncovalent approach | |
| JPH04503408A (en) | High-speed affinity chromatography columns containing non-porous monodisperse polymer packing materials | |
| JP3970319B2 (en) | Separation materials and methods for producing them | |
| CA2806930A1 (en) | Polymeric complements to b-amyloid peptides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| EXPY | Cancellation because of completion of term |