JP2008308472A - Gel for crystallization of water-soluble polymer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide water-soluble polymer gel which enables to secure gelation while holding a polymer crystallizing agent (precipitant) in a crystallization technique using gel, to provide a method for making a microarray using the same, and to provide a method for crystallization using the gel or the microarray. <P>SOLUTION: Disclosed are gel for water-soluble polymer crystallization composed of a polymer of a crosslinkable monomer having a polyethylene glycol skeleton represented by formula I: R<SP>1</SP>-O-(CH<SB>2</SB>CH<SB>2</SB>O)<SB>n</SB>-R<SP>2</SP>(wherein R<SP>1</SP>and R<SP>2</SP>are each independently a crosslinking group; and n is an integer of 9 or greater) and a crystallizing agent held therein; a microarray in which the gel is immobilized; and a method for crystallizing a water-soluble polymer comprising bringing a water-soluble polymer into contact with the gel and depositing crystals of the water-soluble polymer. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a water-soluble polymer crystallization gel capable of precipitating crystals of each water-soluble polymer from a sample containing proteins, peptides, amino acids, nucleic acids, and other water-soluble polymers. The present invention also relates to a microarray on which the gel is fixed, a method for using the microarray, and the like.

  In recent years, a so-called structural genomics science has been activated to comprehensively analyze protein structures and to search for the mechanism of life phenomena based on the analysis. Here, in order to analyze the structure of the protein, it is necessary to have a good crystal. Therefore, optimization of the crystallization conditions is an important technique. In addition, in the purification process of amino acid or peptide synthesis, it is necessary to obtain high-purity crystals in order to obtain high-purity products, and optimization of crystallization conditions for that purpose is important. . In the case of proteins, various crystallization methods including the vapor diffusion method have been devised, but the problem remains that the experimental operation is complicated and the study cannot be performed with a small amount of sample.

  In order to reduce the burden of the complicated processes of these conventional methods and to reduce the amount of necessary samples, new crystallization methods and apparatuses have been developed, and research on full automation of the crystallization apparatus is also actively conducted. However, as the performance of the equipment increases, the price of the equipment also rises, and crystallization studies using these equipment are limited to a few laboratories with sufficient funds. It is.

  Under such circumstances, as a crystallization technique using a gel, a method of growing a crystal in a gel by suppressing the convection seen in a solution by containing each of a crystallization agent and a protein in the gel and laminating them. Has been proposed (Patent Document 1: Japanese Patent Laid-Open No. 6-321700). However, this method is not a practical method because it is difficult to retain the protein in the gel.

  In recent years, by holding a crystallization agent in the gel and bringing the gel into contact with the sample, the crystallization agent component in the gel is gradually released into the sample and the protein solubility is gradually lowered to promote crystallization. (Patent Document 2: International Publication No. 03/053998 pamphlet) and its array device (Patent Document 3: Japanese Patent Application Laid-Open No. 2005-58889) have been developed, whereby crystallization conditions can be easily adjusted with a small amount of sample. It became possible to explore. Furthermore, a technology for retaining a plurality of types of crystallization agents in a gel (Patent Document 4: Japanese Patent Application Laid-Open No. 2005-60284) has been developed. Many crystallization conditions can be searched by a simple method.

In the above-mentioned Patent Document 4 (Japanese Patent Application Laid-Open No. 2005-60284), the conditions for allowing the gelation reaction of the monomer in the state of holding the crystallization agent are as follows: Gel-like product containing at least one monomer selected from the group of 2-methylpropanesulfonic acid and methacryldimethylaminoethylmethyl chloride salt; (2) In 2-methyl-2,4-pentanediol, dimethylacrylamide is used. (3) Gel phosphate containing 2-acrylamido-2-methylpropanesulfonic acid for Na / K phosphate; (4) Gel containing methacryldimethylaminoethyl methyl chloride salt for AmSO ₄ etc. (5) In the case of sodium malonate or the like, gel-like material containing acrylamide; (6) P In G6000, it found that gel containing polyoxyethylene monoacrylate is suitable, thereby, it becomes possible to hold the crystallization agent into the gel. However, since the type of gel that can be selected differs depending on the type of crystallization agent, a complex manufacturing process is required for mixing reagents in arraying.
Many crystallization agents contain a polymer precipitating agent such as a high molecular weight polyethylene glycol. This polymer crystallization agent (precipitating agent) destabilizes the gelation reaction and prevents gelation. . For this reason, even if it reacts on the same gelation conditions, it may not be surely gelled with sufficient reproducibility, and it was difficult to surely make many of these crystallizing agents on the array at once.
Japanese Patent Laid-Open No. 6-321700 International Publication No. 03/053998 Pamphlet JP 2005-58889 A Japanese Patent Laying-Open No. 2005-60284

  The gel-based crystallization technology has succeeded in reducing the cost and space by reducing enormous pipette operations, reducing the amount of necessary samples, and making the device compact. However, the type of gel to be selected differs depending on the type of crystallization agent, and the reagent mixing process has become complicated in the array manufacturing process. In addition, in the case of a crystallization agent containing a polymer precipitating agent such as a polymer polyethylene glycol, it is difficult to reliably form a gel and it is difficult to produce an array. In order to solve these problems, it has been necessary to search for gelation conditions that can ensure gelation while retaining the polymer crystallization agent (precipitating agent).

  As a result of intensive studies to solve the above problems, the present inventors have found that when a crystallization agent containing a polymer precipitating agent such as a polymer polypolyethylene glycol is gelled, the crosslinkability having polyethylene glycol in the skeleton It has been found that by using one monomer or two or more monomers as necessary, a transparent gel-like product that does not cause cloudiness or the like can be reliably formed with good reproducibility, and the present invention has been completed.

（式中、ｎは９以上の整数を表す。）
で示されるものである（１）に記載のゲル。
（３）架橋性モノマーは、ポリエチレングリコールの分子量が異なる少なくとも２種類のモノマーである（１）に記載のゲル。
（４）結晶化剤が分子量1000以上の高分子を含むものである（１）に記載のゲル。
（５）次式Ｉ：
Ｒ¹−Ｏ−（ＣＨ₂ＣＨ₂Ｏ）_n−Ｒ² （Ｉ）
（式中、Ｒ¹及びＲ²は、それぞれ独立して架橋性基を表し、ｎは９以上の整数を表す。）
で示されるポリエチレングリコール骨格を有する架橋性モノマーと単官能モノマーとの重合物に結晶化剤が保持された水溶性高分子結晶化用ゲル。
（６） 架橋性モノマーが次式II：

（式中、ｎは９以上の整数を表す。）
で示されるものである（５）に記載のゲル。
（７）架橋性モノマーは、ポリエチレングリコールの分子量が異なる少なくとも２種類のモノマーである（５）に記載のゲル。
（８）結晶化剤が分子量1000以上の高分子を含むものである（５）に記載のゲル。
（９）次式Ｉ：
Ｒ¹−Ｏ−（ＣＨ₂ＣＨ₂Ｏ）_n−Ｒ² （Ｉ）
（式中、Ｒ¹及びＲ²は、それぞれ独立して架橋性基を表し、ｎは９以上の整数を表す。）
で示されるポリエチレングリコール骨格を有する架橋性モノマーと、該架橋性モノマーとは異なる他の架橋性モノマーと、単官能モノマーとの重合物に結晶化剤が保持された水溶性高分子結晶化用ゲル。
（１０） 架橋性モノマーが次式II：

（式中、ｎは９以上の整数を表す。）
で示されるものである（９）に記載のゲル。
（１１） 架橋性モノマーは、ポリエチレングリコールの分子量が異なる少なくとも２種類のモノマーである（９）に記載のゲル。
（１２）結晶化剤が分子量1000以上の高分子を含むものである（９）に記載のゲル。
（１３）（１）〜（１２）のいずれか１項に記載のゲルが固定されたマイクロアレイ。
（１４）（１）〜（１２）のいずれか１項に記載のゲルに水溶性高分子を接触させ、水溶性高分子の結晶を析出させることを特徴とする水溶性高分子の結晶化方法。
（１５）（１３）に記載のマイクロアレイに固定されたゲルに水溶性高分子を接触させ、水溶性高分子の結晶を析出させることを特徴とする水溶性高分子の結晶化方法。
（１６）水溶性高分子が、タンパク質、ペプチド、アミノ酸及び核酸からなる群から選ばれる少なくとも１種である（１４）又は（１５）に記載の方法。 That is, the present invention relates to a water-soluble polymer crystallization gel as shown below, a microarray using the gel, a crystallization method, and the like.
(1) The following formula I:
R ¹ —O— (CH ₂ CH ₂ O) _n —R ² (I)
(In the formula, R ¹ and R ² each independently represent a crosslinkable group, and n represents an integer of 9 or more.)
A water-soluble polymer crystallization gel in which a crystallization agent is retained in a polymer of a crosslinkable monomer having a polyethylene glycol skeleton represented by
(2) The crosslinkable monomer is represented by the following formula II:

(In the formula, n represents an integer of 9 or more.)
The gel as described in (1) which is shown by these.
(3) The gel according to (1), wherein the crosslinkable monomer is at least two types of monomers having different molecular weights of polyethylene glycol.
(4) The gel according to (1), wherein the crystallization agent comprises a polymer having a molecular weight of 1000 or more.
(5) Formula I:
R ¹ —O— (CH ₂ CH ₂ O) _n —R ² (I)
(In the formula, R ¹ and R ² each independently represent a crosslinkable group, and n represents an integer of 9 or more.)
A water-soluble polymer crystallization gel in which a crystallization agent is retained in a polymer of a crosslinkable monomer having a polyethylene glycol skeleton and a monofunctional monomer.
(6) The crosslinkable monomer is represented by the following formula II:

(In the formula, n represents an integer of 9 or more.)
The gel according to (5), which is represented by
(7) The gel according to (5), wherein the crosslinkable monomer is at least two types of monomers having different molecular weights of polyethylene glycol.
(8) The gel according to (5), wherein the crystallization agent comprises a polymer having a molecular weight of 1000 or more.
(9) Formula I:
R ¹ —O— (CH ₂ CH ₂ O) _n —R ² (I)
(In the formula, R ¹ and R ² each independently represent a crosslinkable group, and n represents an integer of 9 or more.)
A water-soluble polymer crystallization gel in which a crystallization agent is retained in a polymer of a crosslinkable monomer having a polyethylene glycol skeleton represented by the formula, another crosslinkable monomer different from the crosslinkable monomer, and a monofunctional monomer .
(10) The crosslinkable monomer is represented by the following formula II:

(In the formula, n represents an integer of 9 or more.)
The gel according to (9), which is represented by
(11) The gel according to (9), wherein the crosslinkable monomer is at least two types of monomers having different molecular weights of polyethylene glycol.
(12) The gel according to (9), wherein the crystallization agent comprises a polymer having a molecular weight of 1000 or more.
(13) A microarray on which the gel according to any one of (1) to (12) is fixed.
(14) A method for crystallizing a water-soluble polymer, comprising bringing the water-soluble polymer into contact with the gel according to any one of (1) to (12) to precipitate a crystal of the water-soluble polymer. .
(15) A method for crystallizing a water-soluble polymer, comprising bringing the water-soluble polymer into contact with the gel fixed to the microarray according to (13), thereby precipitating a crystal of the water-soluble polymer.
(16) The method according to (14) or (15), wherein the water-soluble polymer is at least one selected from the group consisting of proteins, peptides, amino acids, and nucleic acids.

  ADVANTAGE OF THE INVENTION According to this invention, the water-soluble polymer crystallization gel which can hold | maintain the crystallization agent containing polymeric precipitating agents, such as high molecular polyethylene glycol, can be provided with reproducibility. By using the gel of the present invention, it is not necessary to select a gel composition depending on the type of crystallization agent, so that a complicated process is not required for arraying. In addition, when the gel of the present invention is fixed to the microarray, the gel can be surely formed. Therefore, even when a large number of crystallization agents are mounted on the chip, the chip having the gel that holds all the crystallization agents securely is mounted. Manufacture is possible. The microarray to which the gel of the present invention is immobilized can be quickly conducted with a simple method, and with a small amount of sample, many crystallization experiments or search experiments for crystallization conditions. In addition, the microarray of the present invention can be manufactured at low cost and is economical.

  Hereinafter, the present invention will be described more specifically. The present invention relates to a water-soluble polymer crystallization gel, a microarray on which the gel is fixed, a method for using them, and the like.

1. Water-soluble polymer crystallization gel One embodiment of the water-soluble polymer crystallization gel of the present invention (hereinafter referred to as the gel of the present invention) is as follows.

(1) Water-soluble polymer crystallization gel in which a crystallization agent is held in a polymer (gel-like product) containing a repeating unit derived from a crosslinkable monomer having a structure having a polyethylene glycol skeleton. A crosslinkable monomer having a glycol skeleton structure (hereinafter also referred to as a PEG structure crosslinkable monomer) is represented by the following formula I.
R ¹ —O— (CH ₂ CH ₂ O) _n —R ² (I)
In the above formula I, R ¹ and R ² each independently represent a crosslinkable group, and n represents an integer of 9 or more.

本発明のゲルは、ＰＥＧ構造架橋性モノマーから誘導される繰り返し単位を含むことにより、種々の結晶化剤を保持することができる。「保持」とは、ゲルの網目構造中に結晶化剤を取り込んでゲルに固定すること（ゲルが結晶化剤を捕捉すること）を意味し、物理的に保持されるほか、化学的に結合する場合も含まれる。結晶化剤の保持は、例えば結晶化剤と上記ＰＥＧ構造架橋性モノマーを混合し、重合過程を経てゲルを形成させることにより行うことができる。
また、本発明のゲルを用いることにより、蛋白質、ペプチド、アミノ酸、核酸、その他の水溶性高分子を含有する試料から各水溶性高分子の結晶を析出させることができる。 The crosslinkable group used in the present invention is not particularly limited as long as it is a functional group that can generally form a gel in the presence of a crosslinking agent. For example, those having an ethylenically unsaturated functional group are preferable. For example, R ¹ and R ² are the same or different and include a methacryloyl group, an acryloyl group, and the like.
As such a monomer having a crosslinkable group, a monomer having a polyethylene glycol skeleton represented by the following formula II is preferable.

The gel of this invention can hold | maintain various crystallizing agents by including the repeating unit induced | guided | derived from a PEG structure crosslinkable monomer. “Holding” means that the crystallization agent is taken into the gel network structure and fixed to the gel (the gel traps the crystallization agent), and is physically held or chemically bonded. It is also included when doing. The crystallizing agent can be retained, for example, by mixing the crystallizing agent and the PEG structure crosslinking monomer and forming a gel through a polymerization process.
Moreover, by using the gel of the present invention, crystals of each water-soluble polymer can be precipitated from a sample containing proteins, peptides, amino acids, nucleic acids, and other water-soluble polymers.

  In the present invention, a polymer containing a repeating unit derived from a PEG structure crosslinking monomer can be produced by performing a polymerization reaction in an aqueous medium containing a PEG structure crosslinking monomer and a crystallization agent. The PEG structure-crosslinking monomer used in the present invention is not particularly limited as long as the compound having a polyethylene glycol structure can form a gel by polymerization in an aqueous medium.

Preferably, the gel is a water-soluble polymer crystallization gel obtained by polymerizing a crosslinkable monomer represented by the formula I, more preferably a crosslinkable monomer represented by the formula II and a polymer crystallization agent in an aqueous medium. . The molecular weight of the polymer crystallization agent is preferably 1000 or more. In the present invention, if necessary, a water-soluble polymer crystallization gel in which a crystallization agent is held in a polymer of two or more kinds of crosslinkable monomers and monofunctional monomers having different molecular weights of polyethylene glycol is used. Is possible.
Therefore, the gel of the present invention can have the following aspects in addition to the aspect (1).

(2) A gel comprising a polymer of two or more types of crosslinkable monomers having different molecular weights of polyethylene glycol as the crosslinkable monomer represented by formula I. (3) Crosslinkability represented by formula I A gel formed by holding a polymer crystallization agent in a polymer of a monomer and one or more types of monofunctional monomers. (4) Two or more types of crosslinkers having different molecular weights of polyethylene glycol, a crosslinkable monomer represented by formula I A gel formed by holding a polymer crystallization agent in a polymer of a monomer and a monofunctional monomer (5) A crosslinkable monomer represented by Formula I, another crosslinkable monomer different from the crosslinkable monomer, and a monofunctional (6) Two or more kinds of crosslinkable monomers having different molecular weights of polyethyleneglycol, which is a crosslinkable monomer represented by formula I, and the crosslinkable monomer A gel obtained by holding a polymer crystallizing agent in a polymer of a monofunctional monomer and another crosslinkable monomer different from the polymer

  The gel of the present invention can be obtained by polymerizing each of the above monomers in an aqueous medium in the presence of a polymer crystallization agent. The monomer used is represented by the formula I (preferably the formula II). A combination of the crosslinkable monomer shown and one or more monofunctional monomers (embodiment (3) above), or two or more types of crosslinks having different molecular weights of the crosslinkable monomer shown by Formula I (preferably Formula II) It is preferable that the combination is a combination of a functional monomer and a monofunctional monomer (embodiment (4) above). The molecular weight of the polymer crystallization agent is not particularly limited, but is preferably 1000 or more.

  In particular, when a polymer crystallization agent having a molecular weight of 1000 or more is contained, it is possible to reliably gel by the present invention. In addition, when two or more kinds of crosslinkable monomers having different molecular weights are used, a polymer crystallization agent and a monofunctional monomer are used to further promote gelation, and a polymer crystallization agent having a higher molecular weight of 4000 or more. It is characterized by enabling reliable gelation. In addition, it is possible to reliably produce a microarray in which these gels are filled in each spot of the microarray and a stable gel is fixed. The production of the microarray will be described later.

  The monofunctional monomer used in the present invention is not particularly limited as long as it can form a gel by polymerization in an aqueous medium, and two or more kinds can be used as necessary. Examples include (meth) acrylamide monomers and (meth) acrylic monomers.

  Examples of (meth) acrylamide monomers include (i) N, N-dialkylamino (meth) acrylamides such as (meth) acrylamide, N, N-dimethylacrylamide, N, N-diethylacrylamide, and (ii) (meth) acrylamide. (Meth) acrylamide alkylsulfonic acid such as methanesulfonic acid, (meth) acrylamide ethanesulfonic acid, 2- (meth) acrylamide-2-methylpropanesulfonic acid, (iii) dimethylaminopropyl (meth) acrylamide, diethylaminopropyl (meta) ) Dialkylaminoalkyl (meth) acrylamide such as acrylamide, dimethylaminoethyl (meth) acrylamide, (iv) Dimethylaminopropyl (meth) acrylamide methyl chloride salt, Diethylaminopropyl (meth) acrylic De-methylethyl chloride salt, dimethylaminoethyl (meth) dialkylamino (meth) acrylamide quaternary ammonium salt such as acrylamide methyl chloride salt is preferably used.

  As (meth) acrylic monomers, (i) 2-hydroxyethyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate, 6-hydroxyhexyl (meth) acrylate, diethylene glycol mono (meth) acrylate, triethylene glycol mono (Meth) acrylate, hydroxyl group-containing (meth) acrylate such as polyethylene glycol mono (meth) acrylate, (ii) dialkylaminoalkyl (meth) acrylate such as dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, (iii) ) Quaternary ammonia of dialkylaminoalkyl (meth) acrylates such as dimethylaminoethyl (meth) acrylate methyl chloride salt and ethyl chloride salt of diethylaminoethyl (meth) acrylate Salts and, glycosyl oxyethyl (meth) acrylate is preferably used.

  In addition, if necessary, another crosslinkable monomer different from the crosslinkable monomer represented by the formula I, which can be copolymerized with the monofunctional monomer, can be used in combination. Such other crosslinkable monomer is not particularly limited as long as it has a bifunctional or higher radical polymerizable group. For example, N, N′-methylenebis (meth) acrylamide, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, trimethylolpropane tri ( And (meth) acrylate, trimethylolpropane ethylene oxide-modified tri (meth) acrylate, and the like. Among these, N, N′-methylenebis (meth) acrylamide, ethylene glycol di (meth) acrylate, and polyethylene glycol di (meth) acrylate are preferable. When the crystallization agent is a polymer having a molecular weight of 1000 or more, gelation is promoted by using these crosslinkable monomers having different molecular weights in combination. Furthermore, when the crystallizing agent has a molecular weight of 4000 or more, a PEG-structure crosslinkable monomer having a molecular weight of 500 or more (for example, Bremer PDE-400 (n = 9): manufactured by NOF Corporation) and the other crosslinkable monomers described above. Alternatively, a combination of blemer PDE-400 (n = 9) and blemer PDE-1000 (n = 23) having a molecular weight of 1100 or more is preferred as a combination of PEG structure crosslinkable monomers from the viewpoint of increasing the certainty of gelation. The addition amount when this crosslinkable monomer is used in combination is 0.001 to 0.5 parts by mass, preferably 0.05 to 0.1 parts by mass with respect to the amount of the PEG structure crosslinkable monomer used.

  In the present invention, the concentration of the monofunctional monomer is preferably 1 to 50% by mass and more preferably 1 to 20% by mass with respect to 100% by mass of the crystallization agent solution. For example, in the case of a protein crystallization gel, 0.1 to 30% by mass is preferable and 1 to 20% by mass is more preferable with respect to 100% by mass of the protein crystallization agent solution. Here, the protein crystallizing agent solution is an aqueous solution containing a precipitating agent and optionally dissolving a salt and / or a buffering agent.

  The crystallizing agent used in the present invention is not particularly limited as long as it lowers the solubility of not only polymer precipitants such as polyethylene glycol but also proteins, peptides, amino acids, nucleic acids, and other water-soluble polymers. is not. For example, in the case of a protein, there is no particular limitation as long as the solubility of the protein in the protein solution can be lowered.

  Examples of the precipitant include metal salts, ammonium salts, alcohols, phosphates, polyethylene glycols and derivatives thereof, and other water-soluble organic compounds. These may be used alone or in combination of two or more.

  Examples of the metal salt include sodium chloride, calcium chloride, magnesium chloride hexahydrate, zinc acetate dihydrate, lithium sulfide monohydrate, or nickel chloride pentahydrate.

  Examples of ammonium salts include ammonium sulfate, ammonium acetate, and ammonium sulfide.

  Examples of alcohols include 2-methyl-2,4-pentanediol, isopropanol, 1,6-hexanediol, tetrabutanol, ethanol, imidazole, glycerol, or ethylene glycol, methanol, n-propanol, and the like.

  Examples of the phosphate include sodium phosphate and potassium phosphate.

Polyethylene glycols and derivatives thereof include polyethylene glycol, polyethylene glycol monoalkyl ether, polyethyleneimine, and the like. For example, polyethylene glycol 400, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 8000, polyethylene glycol 20000, polyethylene glycol monoethyl ether 550, polyethylene glycol monoethyl ether 5000, polyethylene glycol monoethyl ether 20000, and ortho- (2-amino Propyl) -ortho- (2-methoxyethyl) polypropylene glycol 500 and the like. In this specification, polyethylene glycol (derivatives) refers to HO (CH ₂ CH ₂ O) _n H (n is an arbitrary integer) and derivatives thereof. Hereinafter, polyethylene glycol is referred to as PEG.

  The other water-soluble organic compound is not particularly limited as long as it can act as a crystallization agent for the water-soluble polymer when contacted with a sample containing the water-soluble polymer. Examples include dioxane, tartaric acid ester trihydrate, sodium cacodylate, lithium chloride, sodium malonate, sodium citrate, magnesium sulfate, lithium sulfate, sodium nitrate, cadmium sulfate, sodium sulfate and the like.

  These precipitants can be used singly or in combination of two or more. Among these, ammonium sulfate, sodium chloride, sodium potassium phosphate, lithium chloride, sodium malonate, MPD (2-methyl-2,4-pentanediol), and PEG are particularly preferable. Commercially available products include "WIZARD I / II" by Emerald BioStructures, "Crystal screen" by "Hampton Research", "Crystal screen II", "Grid Screen Ammonium Sulfate", "Grid Screen PEG600", "Grid Screen MPD", "Grid Screen" The same crystallization agent component as PEG / LiCl, “Grid Screen NaCl”, “Quik Screen”, “Salt RX”, “MembFac”, “Natrix”, etc. can be retained.

  The use concentration of the precipitating agent is preferably 0.1 to 5.0 mol / L in an aqueous medium for salts such as metal salts, ammonium salts or phosphates, and 1 to 80% by volume in organic solvents such as alcohols and pH buffering agents. In the case of polyethylene glycols and derivatives thereof, or other water-soluble polymer compounds, 1 to 50% by weight is preferable.

The crystallization with the precipitating agent is preferably performed in a specific pH range, and the crystallization agent preferably contains a buffer. Examples of the buffer include 2-amino-2-hydroxymethyl-1,3-propanediol Tris (hydroxymethyl) aminomethane, 2-amino-2-hydroxymethyl-1,3-propanediol Tris hydrochloride, 2- [ 4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid, 2-morpholioethanesulfonic acid monohydrate, or N, N-bis (2-hydroxyethyl) glycine, citric acid and the like.
These can also be used individually by 1 type and can also use 2 or more types together. The buffer solution is used after neutralizing with acid or alkali as necessary to adjust to a predetermined pH. The pH is preferably in the range of 3.0 to 10.0, and more preferably in the range of 4.0 to 9.0.

  The water-soluble polymer crystallization gel of the present invention can be obtained as a transparent gel. When the gel is transparent, it is preferable because observation of the produced protein crystal becomes extremely easy and automation by an optical detection system becomes easier. Here, “transparent” means that it is transparent to the extent that crystallization can be observed, and does not require that the light transmittance be 100%.

  The gel of the present invention thermally polymerizes a monomer component containing at least a PEG structure crosslinking monomer in the presence of (1) a crystallizing agent and an aqueous medium and, if necessary, an aqueous solution containing a buffer. Alternatively, (2) in the presence of a crystallization agent and an aqueous medium, polymerization can be carried out using a heat and / or radical photopolymerization initiator and gelled. According to these methods, the crystallization agent can be uniformly held in the gel. At this time, the polymerization is preferably performed in the presence of a radical polymerization initiator.

  In the present invention, examples of the aqueous medium used for gel formation include water or a mixed liquid of water and an aqueous organic solvent. Here, examples of the aqueous organic solvent include methanol and ethanol. When using the liquid mixture of water and an aqueous organic solvent, it is preferable to contain 60% or more of water in terms of mass in the liquid mixture. The preferred aqueous medium used in the present invention is water.

  Examples of the radical polymerization initiator suitably used in an aqueous solution include peroxides such as tert-butyl hydroperoxide, hydrogen peroxide, ammonium persulfate, and potassium persulfate, and 2,2′-azobis (2-amidino Propane) dihydrochloride, 2,2'-azobis (2-amidinobutane) dihydrochloride, 2,2'-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, etc. Agents. These radical polymerization initiators can be used singly or as a mixture of two or more.

  In addition, a redox polymerization initiator in which a reducing agent such as a tertiary amine, sulfite or ferrous salt is combined with the peroxide, and a combination in which a redox polymerization initiator and an azo polymerization initiator are combined. A polymerization initiator may be used.

  In the present invention, polymerization can also be carried out using a radical photopolymerization initiator under a light source that provides light of a specific wavelength. At that time, the radical photopolymerization initiator used is not particularly limited as long as it is decomposed by light irradiation within a specific wavelength range and generates radicals. Preferable examples of usable initiators include, for example, initiators usually used for photopolymerization of acylphosphine oxide, benzoin, benzoin alkyl ether, benzyl, benzophenone, anthraquinone and the like, and 2,2′-azobis (2-methylpropion) Amidine) hydrochloride, 4,4′-azobis (4-cyanovaleric acid) sodium salt, 2,2′-azobis [2-methyl-N- (2-hydroxyethyl) propionamide] and other azo polymerization initiators Is mentioned. From these, one or more arbitrary types can be selected and used according to the light wavelength to be used.

  In consideration of two points of light absorption by the monomer itself contained in the reaction solution and photon energy used for radical generation, the specific wavelength should use light having a wavelength of 200 to 650 nm. Is desirable. Typical examples of light sources that can be used for light irradiation in a wavelength region of 200 to 650 nm include a high-pressure mercury lamp, a low-pressure mercury lamp, a metal halide lamp, a fluorescent chemical lamp, and a fluorescent blue lamp.

  Further, the present invention includes a water-soluble polymer crystallization gel obtained by polymerizing a PEG structure crosslinking monomer and a crystallization agent in an aqueous medium. The types and amounts of the PEG structure crosslinking monomer, the crystallization agent, and the aqueous medium are as described above.

2. Crystallization of water-soluble polymer The crystallization method of the present invention is carried out by bringing a water-soluble polymer into contact with the gel of the present invention and precipitating a crystal of the water-soluble polymer, or fixed to the microarray of the present invention described later. Contacting the water-soluble polymer with the gel and precipitating crystals of the water-soluble polymer.
That is, in the method of the present invention, by applying a water-soluble polymer to the gel holding the crystallization agent, the water-soluble polymer diffuses in the gel and comes into contact with the crystallization agent. Is to be precipitated.

Examples of the water-soluble polymer include proteins, peptides, amino acids, nucleic acids, and other water-soluble polymers, and for bringing a sample containing at least one selected from these groups into contact with a crystallization agent. Can be used. In addition, using the microarray of the present invention described later, the gel fixed to the microarray and a sample containing the water-soluble polymer can be brought into contact with each other to precipitate a water-soluble polymer crystal.
In the present invention, it is possible to obtain crystals of good quality suitable for X-ray analysis, purification and the like simply by bringing the sample into contact with the gel.

Since the gel of the present invention can hold various crystallization agents as described above, it contains at least one selected from the group consisting of proteins, peptides, amino acids, nucleic acids, and other water-soluble polymers. By bringing it into contact with the sample, crystals of water-soluble polymer of good quality can be easily and efficiently precipitated.
The method of bringing the gel prepared as described above into contact with the water-soluble polymer-containing sample can be performed by any method. For example, a sample and a crystal can be crystallized by dropping a polymer-containing sample on a gel holding a crystallization agent, manually or mechanically filling the sample with a syringe, or immersing the gel in a polymer-containing sample. Contact with the agent is possible. The amount of the sample can be set according to the purpose in consideration of crystallization conditions and the like.

In the crystallization method of the present invention, the crystallization agent gradually moves from the gel to the part of the sample containing the water-soluble polymer or into the gel in which the sample holds the crystallization agent, and the crystallization reaction is little. It happens one by one. As a result, the interaction between the water-soluble polymer, which is a crystallization mechanism, and the surrounding environment (presence of the crystallizing agent, concentration, pH, etc.) gradually proceeds, so that high-quality crystals are precipitated.
After contacting the water-soluble polymer-containing sample with the crystallizing agent, the gel is kept in a sealed state under an appropriate temperature condition for a time sufficient for the water-soluble polymer to precipitate, or Or leave it in the atmosphere.

The time sufficient for the precipitation of the water-soluble polymer is about 1 hour to 10 days, although it depends on the substance, concentration, crystallization conditions, and the like. Further, an appropriate temperature condition is about 4 ° C. to 30 ° C., although it varies depending on the type, concentration, crystallization condition and the like of the water-soluble polymer.
After a sufficient time has elapsed for the water-soluble polymer to precipitate, the state of crystal precipitation is observed, for example, with an optical microscope, an X-ray diffractometer or the like. In the crystallization method of the present invention, a known system for monitoring crystallization in a gel can be used in combination. For example, it is possible to determine the success or failure of crystallization at high speed by photographing and recording the state of crystal precipitation with a CCD camera mounted on a microscope and performing image processing.

3. Microarray In the present invention, it is possible to produce a microarray in which the gel is fixed by fixing the gel of the present invention to a substrate.
The gel of the present invention can be used, for example, by being fixed to a microarray. By using the gel of the present invention, there is an advantage that it is not necessary to select a gel composition depending on the kind of crystallization agent as in the prior art, and a complicated manufacturing process is not required for arraying. In addition, in the case of a crystallization agent containing a polymer precipitant such as polymer polyethylene glycol, it is difficult to reliably gel (hold on the gel), and it is difficult to produce a reliable chip. In the present invention, since the gelling of the polymer precipitant is reliable, it is possible to produce a microarray having a large number of gels.

  The microarray of the present invention thus obtained is a gel on which various crystallization agents can be retained, so that even a small amount of sample can be used for crystallization experiments of water-soluble polymers or search for crystallization conditions. It can be implemented quickly and economically.

  The microarray of the present invention is not particularly limited as long as the gel of the present invention is fixed, but preferably has a gel holding part for holding the water-soluble polymer crystallization gel. In addition, the microarray of the present invention preferably has a crystallization section for filling a water-soluble polymer-containing sample.

  When the sample containing the water-soluble polymer filled in the crystallization section comes into contact with the gel of the present invention held in the gel holding section, the crystallization agent held in the gel enters the crystallization section. It can move and promote the crystallization of the water-soluble polymer. In the present invention, since the crystallizing agent is retained in the gel, the movement of the crystallizing agent to the water-soluble polymer-containing sample is controlled at a moderately moderate speed, and the crystallization can be performed stably. it can.

  In the present invention, the material, shape and the like of the microarray are not particularly limited as long as they do not hinder the observation of water-soluble polymer crystal precipitation. Examples of the material for the microarray include glass, resin, metal, and the like, and a composite produced by combining these materials may be used. However, in order to easily confirm the presence or absence of protein crystal precipitation, it is preferable to use a material having high light transmittance.

Examples of the shape of the microarray include a circular shape, a square shape, and a rectangular shape. The thickness can be arbitrarily selected in consideration of improvement in crystallization efficiency and ease and speed of observation of crystal precipitation. For example, the thickness is 0.1 to 5 mm, preferably 0.2 to 2 mm. can do.
The microarray used in the present invention can be produced, for example, by spotting a solution containing a monomer, a cross-linking agent, and a crystallizing agent on a predetermined section on a flat substrate. With regard to a method for producing such a microarray, reference can be made to JP-A-6-507486, US Pat. No. 5,770,721, and the like. When compartments are formed by grooves or through-holes, a solution containing a monomer, a crosslinking agent and a crystallization agent is added to the grooves or through-holes, and a microarray is produced by carrying out a polymerization reaction in the compartments. (See JP 2000-60554 A).

Further, in the present invention, a plurality of hollow tubular bodies holding the gel are converged (that is, a plurality of bundles are bundled and fixed with a resin or the like), and the converged object is cut in a direction intersecting the longitudinal direction of the tubular body. By repeating the above, a microarray can be produced. For the production of such a microarray, the description of WO03 / 53998 and WO00 / 53736 can be referred to, for example.
After converging a plurality of tubular bodies, the gel may be held in each hollow part of the converging object. In that case, the microarray can be manufactured, for example, by performing the following steps (a) to (c).

(A) A step of converging a plurality of tubular bodies so that their longitudinal directions coincide.
(B) A step of filling a hollow part of each tubular body of the converged product with a solution containing a monomer, a crosslinking agent and a crystallization material, and performing a polymerization reaction at a predetermined polymerization temperature in the hollow part.
(C) The process of cut | disconnecting a converging object in the direction which cross | intersects the longitudinal direction.
The produced microarray is preferably stored in a well-sealed container that prevents contact with outside air. Examples of the material of the storage container include a polymer material having a low gas or water permeability, glass, or metal. It is preferable to store at a low temperature. In particular, in the case of long-term storage, it can also be stored frozen.

Hereinafter, the present invention will be described more specifically with reference to examples. However, it goes without saying that the present invention is not limited to these examples. In addition, the monomer and crosslinking agent which were used for the Example and the comparative example are described as follows.
N, N'-Methylenebisacrylamide: MBAAm
Tetraethylene diacrylate: NKester
Dimethylacrylamide: DMAAm
Glycosyloxyethyl methacrylate: GEMA
2-methyl-2,4-pentanediol: MPD
Polyethylene glycol dimethacrylate n ≒ 9: PDE400
Polyethylene glycol dimethacrylate n ≒ 23: PDE1000

A hollow fiber holding the gel was prepared according to the following procedure, and the gel containing the prepared crystallization agent was evaluated. The composition of the crystallization agent and the gel is shown in Table 1 (Examples 1 to 7, Comparative Examples 1 to 6).
<Gel holding hollow fiber preparation process>
1. Mix liquid A (crystallizing agent) into a sample tube.
2. Mixture B (16 μL) and water-soluble polymerization initiator (1 μL).
3. Mixture A (83 μL) was added in the above 2. In addition to the solution prepared in, stir well to create mixture C.
4). Mixture C was heated in a 55 ° C warm bath for 3 hours under a nitrogen atmosphere and filled into a polycarbonate hollow fiber (inner diameter 300 µm).
5. Heat the hollow fiber in a 55 ° C warm bath for 3 hours under a nitrogen atmosphere.

<Gel evaluation method>
The prepared hollow fiber is transversely cut in the longitudinal direction of the fiber, and the transparency of the gel held in the hollow fiber is observed with a transmission optical microscope. The results are shown in Table 1.

Claims (16)

Formula I:
R ¹ —O— (CH ₂ CH ₂ O) _n —R ² (I)
(In the formula, R ¹ and R ² each independently represent a crosslinkable group, and n represents an integer of 9 or more.)
A water-soluble polymer crystallization gel in which a crystallization agent is retained in a polymer of a crosslinkable monomer having a polyethylene glycol skeleton represented by The crosslinkable monomer is represented by the following formula II:

(In the formula, n represents an integer of 9 or more.)
The gel according to claim 1, which is represented by:   The gel according to claim 1, wherein the crosslinkable monomer is at least two types of monomers having different molecular weights of polyethylene glycol.   The gel according to claim 1, wherein the crystallization agent contains a polymer having a molecular weight of 1000 or more. Formula I:
R ¹ —O— (CH ₂ CH ₂ O) _n —R ² (I)
(In the formula, R ¹ and R ² each independently represent a crosslinkable group, and n represents an integer of 9 or more.)
A water-soluble polymer crystallization gel in which a crystallization agent is retained in a polymer of a crosslinkable monomer having a polyethylene glycol skeleton and a monofunctional monomer. The crosslinkable monomer is represented by the following formula II:

(In the formula, n represents an integer of 9 or more.)
The gel according to claim 5, wherein   The gel according to claim 5, wherein the crosslinkable monomer is at least two types of monomers having different molecular weights of polyethylene glycol.   The gel according to claim 5, wherein the crystallization agent comprises a polymer having a molecular weight of 1000 or more. Formula I:
R ¹ —O— (CH ₂ CH ₂ O) _n —R ² (I)
(In the formula, R ¹ and R ² each independently represent a crosslinkable group, and n represents an integer of 9 or more.)
A water-soluble polymer crystallization gel in which a crystallization agent is retained in a polymer of a crosslinkable monomer having a polyethylene glycol skeleton represented by the formula, another crosslinkable monomer different from the crosslinkable monomer, and a monofunctional monomer . The crosslinkable monomer is represented by the following formula II:

(In the formula, n represents an integer of 9 or more.)
The gel according to claim 9, which is represented by:   The gel according to claim 9, wherein the crosslinkable monomer is at least two types of monomers having different molecular weights of polyethylene glycol.   The gel according to claim 9, wherein the crystallization agent contains a polymer having a molecular weight of 1000 or more.   A microarray on which the gel according to any one of claims 1 to 12 is fixed.   A method for crystallizing a water-soluble polymer, comprising bringing the water-soluble polymer into contact with the gel according to any one of claims 1 to 12 to precipitate crystals of the water-soluble polymer.   A method for crystallizing a water-soluble polymer, comprising bringing the water-soluble polymer into contact with the gel fixed to the microarray according to claim 13 to precipitate crystals of the water-soluble polymer.   The method according to claim 14 or 15, wherein the water-soluble polymer is at least one selected from the group consisting of proteins, peptides, amino acids and nucleic acids.