JP2005060284A - Protein crystallizing agent and its preparation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein crystallizing agent capable of performing crystallization operation of protein by a simpler method, by previously incorporating a precipitant for decreasing the solubility of protein into a gel. <P>SOLUTION: The protein crystallizing agent, homogeneously contained in a gel, is formed by gelatinizing a solution containing a protein precipitant and an unsaturated monomer. The protein crystallizing agent can be a transparent gel formed by combining a specific precipitant with an unsaturated monomer. Since the gel is transparent, the observation of formed protein crystals is made very easy, and so is the automation with an optical detection system. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a protein crystallizing agent used for the purpose of precipitating protein crystals from a protein-containing sample.

  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.

  In order to analyze the three-dimensional structure of proteins, good crystals are required, and various crystallization methods such as a vapor diffusion method have been devised, but there are still many problems such as complicated experimental operations. Development of new crystallization methods and apparatuses that replace these conventional methods is underway. For example, Patent Document 1 proposes a method in which a precipitating agent and a protein are contained in a gel and these are laminated to suppress convection in a solution and grow crystals in the gel. In addition to this, attempts have been made to use gel-like materials in order to simplify the crystallization method.

Furthermore, various precipitating agents have been proposed for searching for crystallization conditions.
Japanese Patent Laid-Open No. 6-321700

  However, when a gel-like material is used, depending on the combination of the gel-like material and the precipitant, there is a problem that the gel becomes cloudy or the gelation reaction does not proceed.

  If the gel becomes cloudy, the presence or absence of the crystallization state cannot be observed with a microscope, which is a problem.

  In order to solve the above-described problems, the present inventors diligently studied. As a result, the gelation reaction is completed by the combination of the protein precipitant and the components constituting the gel-like material, and further, a gel-like material that does not cause cloudiness or the like It was found that it was important to form, and the present invention was completed.

  That is, according to the present invention, (1) sodium chloride is held in a gel-like material containing at least one monomer selected from the group of acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and methacryldimethylaminoethylmethyl chloride salt. Protein crystallization gels, (2) gel crystallization gels containing MPD in gels containing dimethylacrylamide, (3) gels containing 2-acrylamido-2-methylpropanesulfonic acid A protein crystallization agent in which Na / K phosphate is retained, (4) a gel for protein crystallization in which ammonium sulfate is retained in a gel-like material containing methacryldimethylaminoethylmethyl chloride salt, and (5) acrylamide. A gel for protein crystallization in which sodium malonate is retained in the gel-containing material, (6) poly A gel for protein crystallization, in which PEG6k is held in a gel-like material containing oxyethylene monoacrylate.

  According to the present invention, it is possible to form a gel-like product that completes the gelation reaction and does not cause white turbidity, and can provide a transparent gel-like product that retains the protein crystallization agent.

  The present invention relates to a protein crystallization gel, wherein the protein crystallization agent is uniformly dispersed and dissolved in the gel by gelling a solution containing the protein crystallization agent and the unsaturated monomer. It is.

    The protein crystallizing agent is not particularly limited as long as it can reduce the solubility of the protein in the protein solution. Examples of the salts include ammonium sulfate, sodium chloride, sodium phosphate, potassium phosphate, lithium chloride, sodium malonate, sodium citrate, magnesium sulfate, lithium sulfate, sodium nitrate, cadmium sulfate, sodium sulfate and the like. Examples of the organic solvent include 2-methyl-2,4-pentanediol (hereinafter referred to as MPD), ethanol, isopropanol, dioxane, methanol, tert-butanol, and n-propanol. Examples of the water-soluble polymer compound include polyethylene glycol (hereinafter referred to as PEG), polyethylene glycol monoalkyl ether, and polyethyleneimine.

  These precipitants can be used alone or in combination of two or more. Of these, ammonium sulfate, sodium chloride, sodium potassium phosphate, lithium chloride, sodium malonate, MPD, and PEG are particularly preferable.

As commercial products, “WIZARD II” manufactured by Emerald BioStructures, “Crystal screen” manufactured by Hampton Research, “Grid Screen”, and the like can be used.
The concentration of the precipitant used is preferably 0.1 to 5.0 mol / L for salts, 1 to 80% by volume for organic solvents, and 1 to 50% by weight for water-soluble polymer compounds.

Protein crystallization is preferably performed in a specific pH range, and a buffer may be used to maintain the pH. The buffer used is not particularly limited, but examples thereof include citric acid, 2- (N-morpholino) ethanesulfonic acid, N-2-hydroxyethylpiperazine-N-ethanesulfonic acid, tris (hydroxymethyl) aminomethane, Examples thereof include N, N-bis (hydroxyethyl) glycine and the like, and a single or a mixture of two or more kinds is neutralized with an acid or an 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.
In the present invention, an unsaturated monomer is used as a gelling agent. The unsaturated monomer is not particularly limited as long as it can form a gel by polymerization in an aqueous medium, but is preferably a (meth) acrylamide monomer or a (meth) acrylic monomer.

Examples of (meth) acrylamide monomers include (meth) acrylamide, N, N-dimethylacrylamide, N, N-dialkylamino (meth) acrylamide such as N, N-diethylacrylamide, (meth) acrylamide methanesulfonic acid, (meta ) Acrylamide ethanesulfonic acid, (meth) acrylamide alkyl sulfonic acid such as 2- (meth) acrylamide-2-methylpropanesulfonic acid, dimethylaminopropyl (meth) acrylamide, diethylaminopropyl (meth) acrylamide, dimethylaminoethyl (meth) Dialkylaminoalkyl (meth) acrylamide such as acrylamide, dimethylaminopropyl (meth) acrylamide methyl chloride salt, diethylaminopropyl (meth) acrylamide methyl ethyl Chloride salt, dimethylaminoethyl (meth) dialkylamino (meth) acrylamide quaternary ammonium salt such as acrylamide methyl chloride salt is preferably used.

  In addition, (meth) acrylic monomers include 2-hydroxyethyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate, 6-hydroxyhexyl (meth) acrylate, diethylene glycol mono (meth) acrylate, triethylene glycol mono ( Hydroxyl group-containing (meth) acrylates such as (meth) acrylate and polyethylene glycol mono (meth) acrylate, dimethylaminoethyl (meth) acrylate, dialkylaminoalkyl (meth) acrylates such as diethylaminoethyl (meth) acrylate, dimethylaminoethyl (meth) Quaternary ammonium salts of dialkylaminoalkyl (meth) acrylates such as acrylate methyl chloride salts and ethyl chloride salts of diethylaminoethyl (meth) acrylate It is preferably used.

  The monomer concentration is preferably 0.1 to 50% by mass and more preferably 1 to 10% by mass with respect to 100% by mass of the protein crystallization agent solution.

  In addition, the crosslinkable monomer copolymerizable with the above-described monomer used as necessary in the present invention is not particularly limited as long as it is a monomer having a bifunctional or higher radical polymerizable group. 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 (meth) acrylate, Examples include trimethylolpropane ethylene oxide-modified tri (meth) acrylate, and N, N′-methylenebis (meth) acrylamide, ethylene glycol di (meth) acrylate, and polyethylene glycol di (meth) acrylate are particularly preferable.The addition amount of a crosslinkable monomer is 0.01-10 mass parts with respect to the monomer of (A) group or (B) group, Preferably it is 0.1-5 mass parts.

  The protein crystallizing agent of the present invention can obtain a transparent gel by combining a specific precipitant and a specific unsaturated monomer. When the gel is transparent, the produced protein crystals can be observed very easily, and automation by an optical detection system is also facilitated.

The combination of unsaturated monomer and protein crystallizing agent from which a transparent gel can be obtained is (1) at least selected from the group consisting of acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and methacryldimethylaminoethylmethyl chloride salt One monomer and sodium chloride,
(2) Dimethylacrylamide and MPD,
(3) 2-acrylamido-2-methylpropanesulfonic acid and Na / K phosphate, (4) methacryldimethylaminoethyl methyl chloride salt and ammonium sulfate, (5) acrylamide and sodium malonate,
(6) Polyoxyethylene monoacrylate and PEG6k,
It is.

  The protein crystallizing agent of the present invention is obtained by thermally polymerizing an aqueous solution containing at least a precipitant, a buffer solution and an unsaturated monomer, or by polymerizing in the presence of heat and / or a radical photopolymerization initiator. It can be prepared by gelling and keeping the precipitant uniform in the gel. It is preferable to carry out the polymerization in the presence of a radical polymerization initiator. 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 alone 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 redox polymerization initiator and an azo polymerization initiator are combined. A combined polymerization initiator may be used.

  Moreover, it can also superpose | polymerize using the radical photopolymerization initiator under the light source which gives the 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. Initiators usually used for photopolymerization such as oxide, benzoin, benzoin alkyl ether, benzyl, benzophenone, anthraquinone, etc. In addition, 2,2′-azobis (2-methylpropionamidine) hydrochloride, 4,4′-azobis Examples thereof include azo polymerization initiators such as (4-cyanovaleric acid) sodium salt and 2,2′-azobis [2-methyl-N- (2-hydroxyethyl) propionamide]. Among these, one or more arbitrary ones can be selected and used according to the light wavelength to be used. In addition, the specific wavelength refers to light in the region of 200 to 650 nm, considering two points of light absorption by the monomer itself contained in the reaction solution and photon energy used for radical generation. It is desirable to use it. 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.

  Hereinafter, the present invention will be described in detail by way of examples. The following buffers were used and prepared according to a conventional method.

0.1 M citrate buffer (pH 4.0),
0.1 M citrate buffer (pH 5.0),
0.1M-MES (pH 6.0),
0.1M-HEPES (pH 7.0)
0.1M-Tris (pH 8.0),
0.1M-Bicine (pH 9.0)
<Example 1>
0.48 g of sodium chloride as a precipitant is weighed into a 10 ml volumetric flask, and the volume is adjusted with 0.1 M citrate buffer (pH 4.0), and a 1.2 M NaCl aqueous solution (this is referred to as solution a). Prepared. Further, 90 g of 50% acrylamide aqueous solution (manufactured by Mitsubishi Rayon) was dissolved by adding 5 g of N, N′-methylenebisacrylamide (hereinafter abbreviated as MBAAm) and 5 g of deionized water, and dissolved in a 50% monomer solution (this was designated as b solution). Prepared). Furthermore, a 10% aqueous solution of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride (VA-044, manufactured by Wako Pure Chemical Industries, Ltd.), which is a water-soluble polymerization initiator, c liquid) was prepared. Inject 840 μl of solution a and 160 μl of solution b into a 2 ml sample tube and mix to prepare an aqueous protein crystallization agent solution (1.0 M NaCl, pH 4.0, monomer concentration 8%). After adding 10 μl and mixing well, polymerization was carried out in a hot bath at 55 ° C. for 3 hours to obtain a transparent hydrogel.

When 1 ml of 40 mg / ml lysozyme aqueous solution was added to the sample tube formed with the hydrogel by the above procedure and incubated at 20 ° C. for 24 hours, lysozyme crystals were precipitated in the lysozyme aqueous solution. Crystal formation was confirmed with the naked eye and a stereomicroscope.

<Example 2>
In Example 1, a crystallization agent aqueous solution (2.0 M NaCl, pH 4.0, monomer concentration 8%) was prepared in the same procedure except that the concentration of solution a was changed to 2.4 M, and the same procedure was performed. A transparent hydrogel was obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Example 3>
In Example 1, a crystallization agent aqueous solution (3.0 M NaCl, pH 4.0, monomer concentration 8%) was prepared in the same procedure except that the concentration of solution a was changed to 3.6 M, and the same procedure was performed. A transparent hydrogel was obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Example 4>
In Example 1, a crystallization agent aqueous solution (4.0 M-NaCl, pH 4.0, monomer concentration 8%) was prepared in the same procedure except that the concentration of solution a was changed to 4.8 M, and the same procedure was performed. A transparent hydrogel was obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Examples 5 to 8>
In Examples 1 to 4, a crystallization agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M citrate buffer (pH 5.0). Further, a transparent hydrogel was prepared in the same procedure. Got. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Examples 9 to 12>
In Examples 1 to 4, a crystallization agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M-MES buffer (pH 6.0), and a transparent hydrogel was further prepared in the same procedure. Obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Examples 13 to 16>
In Examples 1 to 4, a crystallization agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M HEPES buffer (pH 7.0), and a transparent hydrogel was further prepared in the same procedure. Obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Examples 17 to 20>
In Examples 1 to 4, a crystallization agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M Tris buffer (pH 8.0), and a transparent hydrogel was further prepared in the same procedure. Obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Examples 21 to 24>
In Examples 1 to 4, a crystallization agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M-Bicine buffer (pH 9.0), and a transparent hydrogel was further prepared in the same procedure. Obtained. In addition, the production of lysozyme crystals was confirmed by the same method. The results are shown in Table-1.

<Example 25>
0.55 g of polyethylene glycol (manufactured by Wako Pure Chemical Industries, PEG6000, weight average molecular weight: 6000) as a precipitant is weighed into a 10 ml volumetric flask and fixed with 0.1 M citrate buffer (pH 4.0). A 5.5% -PEG aqueous solution (this was designated as d solution) was prepared. Moreover, the monomer solution (it is set as e liquid) which melt | dissolved polyethyleneglycol diacrylate (Shin-Nakamura Chemical NKESTERA-200) in 95 g of polyethyleneglycol monoacrylate (Nippon Yushi's Bremma-AE90) was prepared. Furthermore, a 10% aqueous solution of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride (VA-044, manufactured by Wako Pure Chemical Industries, Ltd.), which is a water-soluble polymerization initiator, c liquid) was prepared. 920 μl of solution a and 80 μl of solution b are injected into a 2 ml sample tube and mixed to prepare an aqueous protein crystallization agent solution (5% -PEG, pH 4.0, monomer concentration 8%). After adding 10 μl and mixing well, polymerization was carried out in a hot bath at 55 ° C. for 3 hours to obtain a transparent hydrogel. Hereinafter, the production of lysozyme crystals was confirmed by the same method as in Example 1. The results are shown in Table-1.

<Example 26>
In Example 25, a crystallization agent aqueous solution (10% -PEG, pH 4.0, monomer concentration 8%) was prepared in the same procedure except that the concentration of solution d was changed to 11%. A transparent hydrogel was obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Example 27>
In Example 25, an aqueous crystallization agent solution (20% -PEG, pH 4.0, monomer concentration 8%) was prepared in the same procedure except that the concentration of solution d was changed to 22%. A transparent hydrogel was obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Example 28>
In Example 25, an aqueous crystallization agent solution (30% -PEG, pH 4.0, monomer concentration 8%) was prepared in the same procedure except that the concentration of solution d was changed to 33%. A transparent hydrogel was obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Examples 29 to 32>
In Examples 25-28, a crystallizing agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M citrate buffer (pH 5.0), and a transparent hydrogel was further prepared in the same procedure. Got. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Examples 33 to 36>
In Examples 25-28, a crystallization agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M-MES buffer (pH 6.0), and a transparent hydrogel was further prepared in the same procedure. Obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Examples 37 to 40>
In Examples 25-28, a crystallizing agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M HEPES buffer (pH 7.0), and a transparent hydrogel was further prepared in the same procedure. Obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Examples 41 to 44>
In Examples 25 to 28, a crystallizing agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M Tris buffer (pH 8.0), and a transparent hydrogel was further prepared in the same procedure. Obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

＜実施例４９＞
沈殿剤である塩化ナトリウム０．４８ｇを１０ｍｌメスフラスコに秤量し、 ０．１Ｍ−クエン酸緩衝液（ｐＨ４．０）で定容して１．２Ｍ−ＮａＣｌ水溶液（これをa液とする）を調製した。 <Examples 45 to 48>
In Examples 25 to 28, a crystallizing agent aqueous solution was prepared in the same procedure except that the buffer was changed to 0.1 M-Bicine buffer (pH 9.0), and a transparent hydrogel was further prepared in the same procedure. Obtained. Moreover, the production | generation of the lysozyme crystal | crystallization was confirmed by the same method. The results are shown in Table-1.

<Example 49>
0.48 g of sodium chloride as a precipitant is weighed into a 10 ml volumetric flask, and the volume is adjusted with 0.1 M citrate buffer (pH 4.0), and a 1.2 M NaCl aqueous solution (this is referred to as solution a). Prepared.

Also, 2.5 g of N, N′-methylenebisacrylamide (hereinafter abbreviated as MBAAm) and 50 g of deionized water were added to 47.5 g of 2-acrylamido-2-methylpropanesulfonic acid and dissolved to obtain a 50% monomer solution ( This was designated as solution b). Furthermore, a 10% aqueous solution of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride (VA-044, manufactured by Wako Pure Chemical Industries, Ltd.), which is a water-soluble polymerization initiator, c liquid) was prepared. Inject 840 μl of solution a and 160 μl of solution b into a 2 ml sample tube and mix to prepare an aqueous protein crystallization agent solution (1.0 M NaCl, pH 4.0, monomer concentration 8%). After adding 10 μl and mixing well, polymerization was carried out in a hot bath at 55 ° C. for 3 hours to obtain a transparent hydrogel.

<Example 50>
0.48 g of sodium chloride as a precipitant is weighed into a 10 ml volumetric flask, and the volume is adjusted with 0.1 M citrate buffer (pH 4.0), and a 1.2 M NaCl aqueous solution (this is referred to as solution a). Prepared.

Further, 62.5 g of 80% methacryldimethylaminoethyl methyl chloride salt aqueous solution was dissolved by adding 2.5 g of N, N′-methylenebisacrylamide (hereinafter abbreviated as MBAAm) and 35 g of deionized water to obtain a 50% monomer solution ( This was designated as solution b). Furthermore, a 10% aqueous solution of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride (VA-044, manufactured by Wako Pure Chemical Industries, Ltd.), which is a water-soluble polymerization initiator, c liquid) was prepared. Inject 840 μl of solution a and 160 μl of solution b into a 2 ml sample tube and mix to prepare an aqueous protein crystallization agent solution (1.0 M NaCl, pH 4.0, monomer concentration 8%). After adding 10 μl and mixing well, polymerization was carried out in a hot bath at 55 ° C. for 3 hours to obtain a transparent hydrogel.

<Example 51>
Weigh 2 g of 2-methyl-2,4-pentanediol with a precipitating agent into a 10 ml volumetric flask and make a constant volume with 0.1 M citrate buffer (pH 4.0) to give 20% -2-methyl-2,4. -A pentanediol aqueous solution (this is referred to as solution a) was prepared.

Further, 2.5 g of N, N′-methylenebisacrylamide (hereinafter abbreviated as MBAAm) and 50 g of deionized water are dissolved in 47.5 g of dimethylacrylamide and dissolved to obtain a 50% monomer solution (hereinafter referred to as “b solution”). Prepared. Furthermore, a 10% aqueous solution of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride (VA-044, manufactured by Wako Pure Chemical Industries, Ltd.), which is a water-soluble polymerization initiator, c liquid) was prepared. Inject 840 μl of solution a and 160 μl of solution b into a 2 ml sample tube and mix to prepare an aqueous protein crystallization agent solution (1.0 M NaCl, pH 4.0, monomer concentration 8%). After adding 10 μl and mixing well, polymerization was carried out in a hot bath at 55 ° C. for 3 hours to obtain a transparent hydrogel.

<Example 52>
1.176 g of sodium phosphate as a precipitant and 0.035 g of potassium phosphate are weighed into a 10 ml volumetric flask and fixed with 0.1 M citrate buffer (pH 4.0) to give 1.0 M phosphorus. A sodium / potassium acid salt aqueous solution (this is referred to as solution a) was prepared.

Also, 2.5 g of N, N′-methylenebisacrylamide (hereinafter abbreviated as MBAAm) and 50 g of deionized water were added to 47.5 g of 2-acrylamido-2-methylpropanesulfonic acid and dissolved to obtain a 50% monomer solution ( This was designated as solution b). Furthermore, a 10% aqueous solution of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride (VA-044, manufactured by Wako Pure Chemical Industries, Ltd.), which is a water-soluble polymerization initiator, c liquid) was prepared. Inject 840 μl of solution a and 160 μl of solution b into a 2 ml sample tube and mix to prepare an aqueous protein crystallization agent solution (1.0 M NaCl, pH 4.0, monomer concentration 8%). After adding 10 μl and mixing well, polymerization was carried out in a hot bath at 55 ° C. for 3 hours to obtain a transparent hydrogel.

<Example 53>
Weigh out 3.17 g of ammonium sulfate as a precipitating agent into a 10 ml volumetric flask and make a volume with 0.1 M citrate buffer (pH 4.0) to prepare a 2.4 M ammonium sulfate aqueous solution (this is a solution). did.

Further, 62.5 g of 80% methacryldimethylaminoethyl methyl chloride salt aqueous solution was dissolved by adding 2.5 g of N, N′-methylenebisacrylamide (hereinafter abbreviated as MBAAm) and 35 g of deionized water to obtain a 50% monomer solution ( This was designated as solution b). Furthermore, a 10% aqueous solution of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride (VA-044, manufactured by Wako Pure Chemical Industries, Ltd.), which is a water-soluble polymerization initiator, c liquid) was prepared. Inject 840 μl of solution a and 160 μl of solution b into a 2 ml sample tube and mix to prepare an aqueous protein crystallization agent solution (1.0 M NaCl, pH 4.0, monomer concentration 8%). After adding 10 μl and mixing well, polymerization was carried out in a hot bath at 55 ° C. for 3 hours to obtain a transparent hydrogel.

<Example 54>
1.04 g of malonic acid, a precipitant, is weighed into a 10 ml volumetric flask, neutralized by adding 4 g of 20% aqueous sodium hydroxide solution, and then fixed with 0.1 M citrate buffer (pH 4.0). 1.0M sodium malonate aqueous solution (this is referred to as solution a) was prepared.

Further, 90 g of 50% acrylamide aqueous solution (manufactured by Mitsubishi Rayon) was dissolved by adding 5 g of N, N′-methylenebisacrylamide (hereinafter abbreviated as MBAAm) and 5 g of deionized water, and dissolved in a 50% monomer solution (this was designated as b solution). Prepared). Furthermore, a 10% aqueous solution of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride (VA-044, manufactured by Wako Pure Chemical Industries, Ltd.), which is a water-soluble polymerization initiator, c liquid) was prepared. Inject 840 μl of solution a and 160 μl of solution b into a 2 ml sample tube and mix to prepare an aqueous protein crystallization agent solution (1.0 M NaCl, pH 4.0, monomer concentration 8%). After adding 10 μl and mixing well, polymerization was carried out in a hot bath at 55 ° C. for 3 hours to obtain a transparent hydrogel.

Claims (6)

  A protein crystallization gel in which sodium chloride is held in a gel-like material containing at least one monomer selected from the group of acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and methacryldimethylaminoethyl methyl chloride salt.   A protein crystallization gel in which 2-methyl-2,4-pentanediol is held in a gel-like material containing dimethylacrylamide.   A protein crystallizing agent in which sodium phosphate / potassium salt is held in a gel-like material containing 2-acrylamido-2-methylpropanesulfonic acid.     A protein crystallization gel in which ammonium sulfate is held in a gel-like material containing methacryldimethylaminoethyl methyl chloride salt.       A protein crystallization gel in which sodium malonate is retained in a gel containing acrylamide. A protein crystallization gel in which polyethylene glycol 6000 is held in a gel-like material containing polyoxyethylene monoacrylate.