JP2005060284A - Protein crystallizing agent and its preparation method - Google Patents
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本発明は、蛋白質含有試料から蛋白質の結晶を析出させる目的で使用する蛋白質結晶化剤に関する。
The present invention relates to a protein crystallizing agent used for the purpose of precipitating protein crystals from a protein-containing sample.
近年、蛋白質の構造を網羅的に解析し、それに基づいて生命現象の仕組みを探索しようとするいわゆる構造ゲノム科学と呼ばれる動きが活発化している。 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.
蛋白質の立体構造解析を行うにはその良好な結晶が必要とされ、蒸気拡散法を始めとしてとして様々な結晶化方法が考案されているが、実験操作が煩雑である等依然として課題は多い。これら従来法に変わる新しい結晶化方法や装置の開発が行われている。例えば特許文献1では、沈殿剤や蛋白質をゲル中に含有させ、これらを積層することにより、溶液中で見られる対流を抑えてゲル中で結晶を成長させる方法が提案されている。また、これ以外にも、結晶化方法を簡略化するため、ゲル状物を利用しようとする試みがなされている。 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.
さらには、結晶化条件の探索に際して、様々な沈殿化剤が提案されている。
しかし、ゲル状物を利用する場合、ゲル状物と沈殿剤の組み合わせによっては、ゲルが白濁したり、ゲル化反応が進行しなかったりという問題があった。 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.
ゲルが白濁すれば、結晶化状態の有無を顕微鏡により観察することができず問題となる。 If the gel becomes cloudy, the presence or absence of the crystallization state cannot be observed with a microscope, which is a problem.
上述した課題を解決するため、本発明者が鋭意検討した結果、蛋白質沈殿剤とゲル状物を構成する成分の組み合わせにより、ゲル化反応が完結し、さらには白濁等を生じないゲル状物を形成するのに重要であることを見出し、本発明を完成した。 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.
即ち、本発明は、(1)アクリルアミド、2−アクリルアミド−2−メチルプロパンスルホン酸、メタクリルジメチルアミノエチルメチルクロライド塩の群から選択される少なくとも1種のモノマーを含むゲル状物に塩化ナトリウムが保持されている蛋白質結晶化用ゲル、(2)ジメチルアクリルアミドを含むゲル状物にMPDが保持されている蛋白質結晶化用ゲル、(3)2−アクリルアミド−2−メチルプロパンスルホン酸を含むゲル状物にリン酸Na/Kが保持されている蛋白質結晶化剤、(4)メタクリルジメチルアミノエチルメチルクロライド塩を含むゲル状物に、硫酸アンモニウムが保持されている蛋白質結晶化用ゲル、(5)アクリルアミドを含むゲル状物に、マロン酸ナトリウムが保持されている蛋白質結晶化用ゲル、(6)ポリオキシエチレンモノアクリレートを含むゲル状物にPEG6kが保持されている蛋白質結晶化用ゲル、である。 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.
本発明によれば、ゲル化反応が完結し、さらには白濁等を生じないゲル状物を形成することができ、蛋白質結晶化剤を保持した透明なゲル状物を提供することができる。 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.
本発明は、蛋白質結晶化剤、不飽和単量体を含む溶液をゲル化することにより、該蛋白質結晶化剤がゲル中に均一に分散、溶解されたことを特徴とする蛋白質結晶化用ゲルである。 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.
蛋白質結晶化剤としては、蛋白質溶液の蛋白質の溶解度を下げることができるものであれば特に限定はない。例えば、塩類として、硫酸アンモニウム、塩化ナトリウム、リン酸ナトリウム、リン酸カリウム、リチウムクロライド、マロン酸ナトリウム、クエン酸ナトリウム、硫酸マグネシウム、硫酸リチウム、硝酸ナトリウム、硫酸カドミウム、硫酸ナトリウム等が挙げられる。有機溶媒としては2−メチル−2,4−ペンタンジオール(以下、MPDとする)、エタノール、イソプロパノール、ジオキサン、メタノール、tert−ブタノール、n−プロパノール等が挙げられる。水溶性高分子化合物としては、ポリエチレングリコール(以下、PEGとする)、ポリエチレングリコールモノアルキルエーテル、ポリエチレンイミン等が挙げられる。 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.
これらの沈殿剤は、単独もしくは2種類以上の組み合わせで使用することができる。これらの中で、特に、硫酸アンモニウム、塩化ナトリウム、リン酸カリウムナトリウム、リチウムクロライド、マロン酸ナトリウム、MPD、PEGが好適である。 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.
市販品として、Emerald BioStructures社製「WIZARD II」、Hampton Research社製「Crystal screen」、「Grid Screen」等を用いることができる。
沈殿剤の使用濃度は、塩類では0.1〜5.0mol/Lが好ましく、有機溶媒では1〜80体積%が好ましく、水溶性高分子化合物では1〜50重量%が好ましい。
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.
蛋白質の結晶化は特定のpH領域で行うことが好ましく、pHを維持するために緩衝液を使用しても良い。使用する緩衝液には特に限定はないが、例えば、クエン酸、2−(N−モリホリノ)エタンスルホン酸、N−2−ヒドロキシエチルピペラジン−N−エタンスルホン酸、トリス(ヒドロキシメチル)アミノメタン、N,N−ビス(ヒドロキシエチル)グリシン等を含有するものが挙げられ、単独もしくは2種類以上の混合物を、必要に応じて酸あるいはアルカリなどで中和し所定のpHに調整する。pHは、3.0〜10.0の範囲が好ましく、4.0〜9.0の範囲がより好ましい。
本発明において、ゲル化剤として不飽和単量体が使用される。不飽和単量体は、水性媒体中で重合することによりゲル形成が可能なものであれば特に限定はないが、(メタ)アクリルアミド系モノマーや(メタ)アクリル系モノマーであることが好ましい。
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.
(メタ)アクリルアミド系モノマーとしては、(メタ)アクリルアミド、N,N−ジメチルアクリルアミド、N,N−ジエチルアクリルアミド等のN,N−ジアルキルアミノ(メタ)アクリルアミド、(メタ)アクリルアミドメタンスルホン酸、(メタ)アクリルアミドエタンスルホン酸、2−(メタ)アクリルアミド−2−メチルプロパンスルホン酸等の(メタ)アクリルアミドアルキルスルホン酸、ジメチルアミノプロピル(メタ)アクリルアミド、ジエチルアミノプロピル(メタ)アクリルアミド、ジメチルアミノエチル(メタ)アクリルアミド等のジアルキルアミノアルキル(メタ)アクリルアミド、ジメチルアミノプロピル(メタ)アクリルアミドメチルクロライド塩、ジエチルアミノプロピル(メタ)アクリルアミドメチルエチルクロライド塩、ジメチルアミノエチル(メタ)アクリルアミドメチルクロライド塩等のジアルキルアミノ(メタ)アクリルアミド4級アンモニウム塩が好適に用いられる。 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.
また、(メタ)アクリル系モノマーとしては、2−ヒドロキシエチル(メタ)アクリレート、4−ヒドロキシブチル(メタ)アクリレート、6−ヒドロキシヘキシル(メタ)アクリレート、ジエチレングリコールモノ(メタ)アクリレート、トリエチレングリコールモノ(メタ)アクリレート、ポリエチレングリコールモノ(メタ)アクリレート等の水酸基含有(メタ)アクリレート、ジメチルアミノエチル(メタ)アクリレート、ジエチルアミノエチル(メタ)アクリレート等のジアルキルアミノアルキル(メタ)アクリレート、ジメチルアミノエチル(メタ)アクリレートメチルクロライド塩、ジエチルアミノエチル(メタ)アクリレートのエチルクロライド塩等のジアルキルアミノアルキル(メタ)アクリレートの4級アンモニウム塩が好適に用いられる。 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.
モノマー濃度は、蛋白質結晶化剤溶液100質量%に対し、0.1〜50質量%が好ましく、1〜10質量%がより好ましい。 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.
また、本発明で必要に応じて使用される上記のモノマーと共重合可能な架橋性モノマーとしては、2官能以上のラジカル重合性基を有する単量体ならば特に限定はないが、例えばN,N’−メチレンビス(メタ)アクリルアミド、エチレングリコールジ(メタ)アクリレート、ジエチレングリコールジ(メタ)アクリレート、トリエチレングリコールジ(メタ)アクリレート、ポリエチレングリコールジ(メタ)アクリレート、トリメチロールプロパントリ(メタ)アクリレート、トリメチロールプロパンエチレンオキサイド変性トリ(メタ)アクリレート等が挙げられるが、特にN,N’−メチレンビス(メタ)アクリルアミド、エチレングリコールジ(メタ)アクリレート、ポリエチレングリコールジ(メタ)アクリレートが好適である。架橋性モノマーの添加量は、(A)群あるいは(B)群のモノマーに対し、0.01〜10質量部であり、好ましくは0.1〜5質量部である。 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.
本発明の蛋白質結晶化剤は、特定の沈殿剤と特定の不飽和単量体とを組み合わせることにより、透明ゲルを得ることができる。ゲルが透明であると、生成した蛋白質結晶の観察が極めて容易となり、光学的検出系による自動化もより容易となる。 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.
透明ゲルが得られる不飽和単量体及び蛋白質結晶化剤の組み合わせとしては、(1)アクリルアミド、2−アクリルアミド−2−メチルプロパンスルホン酸、メタクリルジメチルアミノエチルメチルクロライド塩の群から選択される少なくとも1種のモノマーと塩化ナトリウム、
(2)ジメチルアクリルアミドとMPD、
(3)2−アクリルアミド−2−メチルプロパンスルホン酸リとリン酸Na/K、(4)メタクリルジメチルアミノエチルメチルクロライド塩と硫酸アンモニウム、(5)アクリルアミドとマロン酸ナトリウム、
(6)ポリオキシエチレンモノアクリレートとPEG6k、
である。
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.
本発明の蛋白質結晶化剤は、少なくとも沈殿剤、緩衝液、不飽和単量体を含有してなる水溶液を熱重合させるか、もしくは熱および/または光ラジカル重合開始剤存在下に重合させることによりゲル化させ、沈殿剤をゲル中に均一に保持させることにより調製することができる。ラジカル重合開始剤存在下に重合を行うのが好適である。水溶液中において好適に使用されるラジカル重合開始剤としては、例えば、tert−ブチルハイドロパーオキサイド、過酸化水素、過硫酸アンモニウム、過硫酸カリウム等の過酸化物、2,2’−アゾビス(2−アミジノプロパン)2塩酸塩、2,2’−アゾビス(2−アミジノブタン)2塩酸塩、2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]2塩酸塩等のアゾ系重合開始剤が挙げられる。これらのラジカル重合開始剤は、単独もしくは2種類以上の混合物として使用することができる。また、前記の過酸化物に第三級アミン、亜硫酸塩、第1鉄塩等の還元剤を組み合わせたレドックス系重合開始剤、さらには、レドックス系重合開始剤とアゾ系重合開始剤を組み合わせた併用重合開始剤を使用してもよい。 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.
また、特定波長の光を与える光源下で、光ラジカル重合開始剤を用いて重合を行うこともできる。その際、使用される光ラジカル重合開始剤は、特定波長の範囲内の光照射により分解し、ラジカルを発生するものであれば特に限定はないが、好適に利用できるものとして、例えば、アシルホスフィンオキサイド、ベンゾイン、ベンゾインアルキルエーテル、ベンジル、ベンゾフェノン、アントラキノン等の通常光重合に使用される開始剤、その他に、2,2’−アゾビス(2−メチルプロピオンアミジン)塩酸塩、4,4’−アゾビス(4−シアノ吉草酸)ナトリウム塩、2,2’−アゾビス[2−メチル−N−(2−ヒドロキシエチル)プロピオンアミド]等のアゾ系重合開始剤が挙げられる。これらのうちから、利用する光波長に応じて、任意のものを1種類以上を選択し使用することができる。また、前記の特定波長とは、反応液中に含有される単量体自身による光吸収、ラジカル生成に利用される光量子エネルギーの二つの点を考慮すると、波長が200〜650nmの領域の光を用いることが望ましい。波長が200〜650nmの領域の光照射に利用可能な光源の代表例としては、高圧水銀ランプ、低圧水銀ランプ、メタルハライドランプ、蛍光ケミカルランプ、蛍光青色ランプ等が挙げられる。 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.
以下、実施例により本発明を詳細に説明する。緩衝液は以下のものを使用し、常法に従って調製した。 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.
0.1M−クエン酸緩衝液(pH4.0)、
0.1M−クエン酸緩衝液(pH5.0)、
0.1M−MES(pH6.0)、
0.1M−HEPES(pH7.0)
0.1M−Tris(pH8.0)、
0.1M−Bicine(pH9.0)
<実施例1>
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。また、50%アクリルアミド水溶液(三菱レイヨン製)90gにN,N‘−メチレンビスアクリルアミド(以下、MBAAmと略す)5g、脱イオン水5gを加えて溶解し、50%モノマー溶液(これをb液とする)を調製した。更に、水溶性重合開始剤である2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]二塩酸塩(和光純薬工業製、VA−044)の10%水溶液(これをc液とする)を調製した。2mlのサンプルチューブに、a液を840μl、b液を160μl注入して混合し、蛋白質結晶化剤水溶液(1.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更にc液を10μl添加してよく混合した後、55℃の温浴で3時間重合し、透明なハイドロゲルを得た。
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.
前記の手順でハイドロゲルを形成したサンプルチューブに、40mg/mlの
リゾチーム水溶液1mlを加え、20℃で24時間インキュベートしたところ、リゾチーム水溶液中にリゾチーム結晶が析出していた。尚、結晶生成は肉眼および実体顕微鏡で確認した。
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.
<実施例2>
実施例1において、a液の濃度2.4Mに変更した以外は同様の手順で、結晶化剤水溶液(2.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例3>
実施例1において、a液の濃度3.6Mに変更した以外は同様の手順で、結晶化剤水溶液(3.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例4>
実施例1において、a液の濃度4.8Mに変更した以外は同様の手順で、結晶化剤水溶液(4.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例5〜8>
実施例1〜4において、緩衝液を0.1M−クエン酸緩衝液(pH5.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例9〜12>
実施例1〜4において、緩衝液を0.1M−MES緩衝液(pH6.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例13〜16>
実施例1〜4において、緩衝液を0.1M−HEPES緩衝液(pH7.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例17〜20>
実施例1〜4において、緩衝液を0.1M−Tris緩衝液(pH8.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例21〜24>
実施例1〜4において、緩衝液を0.1M−Bicine緩衝液(pH9.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例25>
沈殿剤であるポリエチレングリコール(和光純薬工業製、PEG6000、重量平均分子量:6000)0.55g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5.5%−PEG水溶液(これをd液とする)を調製した。また、ポリエチレングリコールモノアクリレート(日本油脂製ブレンマ−AE90)95gに、ポリエチレングリコールジアクリレート(新中村化学製NKESTERA−200)を溶解したモノマー溶液(これをe液とする)を調製した。更に、水溶性重合開始剤である2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]二塩酸塩(和光純薬工業製、VA−044)の10%水溶液(これをc液とする)を調製した。2mlのサンプルチューブに、a液を920μl、b液を80μlを注入して混合し、蛋白質結晶化剤水溶液(5%−PEG、pH4.0、モノマー濃度8%)を調製し、更にc液を10μl添加してよく混合した後、55℃の温浴で3時間重合し、透明なハイドロゲルを得た。以下、実施例1と同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例26>
実施例25において、d液の濃度を11%に変更した以外は同様の手順で、結晶化剤水溶液(10%−PEG、pH4.0、モノマー濃度8%)を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例27>
実施例25において、d液の濃度を22%に変更した以外は同様の手順で、結晶化剤水溶液(20%−PEG、pH4.0、モノマー濃度8%)を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例28>
実施例25において、d液の濃度を33%に変更した以外は同様の手順で、結晶化剤水溶液(30%−PEG、pH4.0、モノマー濃度8%)を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例29〜32>
実施例25〜28において、緩衝液を0.1M−クエン酸緩衝液(pH5.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例33〜36>
実施例25〜28において、緩衝液を0.1M−MES緩衝液(pH6.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例37〜40>
実施例25〜28において、緩衝液を0.1M−HEPES緩衝液(pH7.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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.
<実施例41〜44>
実施例25〜28において、緩衝液を0.1M−Tris緩衝液(pH8.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
<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45〜48>
実施例25〜28において、緩衝液を0.1M−Bicine緩衝液(pH9.0)に変更した以外は同様の手順で、結晶化剤水溶液を調製し、更に同様の手順で透明なハイドロゲルを得た。また、同様の方法でリゾチーム結晶の生成を確認した。結果を表―1に示す。
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.
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.
また、2−アクリルアミド−2−メチルプロパンスルホン酸47.5gにN,N‘−メチレンビスアクリルアミド(以下、MBAAmと略す)2.5g、脱イオン水50gを加えて溶解し、50%モノマー溶液(これをb液とする)を調製した。更に、水溶性重合開始剤である2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]二塩酸塩(和光純薬工業製、VA−044)の10%水溶液(これをc液とする)を調製した。2mlのサンプルチューブに、a液を840μl、b液を160μl注入して混合し、蛋白質結晶化剤水溶液(1.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更にc液を10μl添加してよく混合した後、55℃の温浴で3時間重合し、透明なハイドロゲルを得た。 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.
<実施例50>
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。
<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.
また、80%メタクリルジメチルアミノエチルメチルクロライド塩水溶液62.5gにN,N‘−メチレンビスアクリルアミド(以下、MBAAmと略す)2.5g、脱イオン水35gを加えて溶解し、50%モノマー溶液(これをb液とする)を調製した。更に、水溶性重合開始剤である2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]二塩酸塩(和光純薬工業製、VA−044)の10%水溶液(これをc液とする)を調製した。2mlのサンプルチューブに、a液を840μl、b液を160μl注入して混合し、蛋白質結晶化剤水溶液(1.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更にc液を10μl添加してよく混合した後、55℃の温浴で3時間重合し、透明なハイドロゲルを得た。 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.
<実施例51>
沈殿剤で2-メチル-2,4-ペンタンジオール2g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20%−2-メチル-2,4-ペンタンジオール水溶液(これをa液とする)を調製した。
<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.
また、ジメチルアクリルアミド47.5gにN,N‘−メチレンビスアクリルアミド(以下、MBAAmと略す)2.5g、脱イオン水50gを加えて溶解し、50%モノマー溶液(これをb液とする)を調製した。更に、水溶性重合開始剤である2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]二塩酸塩(和光純薬工業製、VA−044)の10%水溶液(これをc液とする)を調製した。2mlのサンプルチューブに、a液を840μl、b液を160μl注入して混合し、蛋白質結晶化剤水溶液(1.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更にc液を10μl添加してよく混合した後、55℃の温浴で3時間重合し、透明なハイドロゲルを得た。 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.
<実施例52>
沈殿剤であるリン酸ナトリウム塩1.176g、リン酸カリウム塩0.035g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1.0M−リン酸ナトリウム/カリウム塩水溶液(これをa液とする)を調製した。
<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.
また、2−アクリルアミド−2−メチルプロパンスルホン酸47.5gにN,N‘−メチレンビスアクリルアミド(以下、MBAAmと略す)2.5g、脱イオン水50gを加えて溶解し、50%モノマー溶液(これをb液とする)を調製した。更に、水溶性重合開始剤である2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]二塩酸塩(和光純薬工業製、VA−044)の10%水溶液(これをc液とする)を調製した。2mlのサンプルチューブに、a液を840μl、b液を160μl注入して混合し、蛋白質結晶化剤水溶液(1.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更にc液を10μl添加してよく混合した後、55℃の温浴で3時間重合し、透明なハイドロゲルを得た。 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.
<実施例53>
沈殿剤である硫酸アンモニウム3.17g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2.4M−硫酸アンモニウム水溶液(これをa液とする)を調製した。
<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.
また、80%メタクリルジメチルアミノエチルメチルクロライド塩水溶液62.5gにN,N‘−メチレンビスアクリルアミド(以下、MBAAmと略す)2.5g、脱イオン水35gを加えて溶解し、50%モノマー溶液(これをb液とする)を調製した。更に、水溶性重合開始剤である2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]二塩酸塩(和光純薬工業製、VA−044)の10%水溶液(これをc液とする)を調製した。2mlのサンプルチューブに、a液を840μl、b液を160μl注入して混合し、蛋白質結晶化剤水溶液(1.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更にc液を10μl添加してよく混合した後、55℃の温浴で3時間重合し、透明なハイドロゲルを得た。 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.
<実施例54>
沈殿剤であるマロン酸1.04gを10mlメスフラスコに秤量し、20%−水酸化ナトリウム水溶液4gを加えて中和したのち、0.1M−クエン酸緩衝液(pH4.0)で定容して1.0M−マロン酸ナトリウム水溶液(これをa液とする)を調製した。
<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.
また、50%アクリルアミド水溶液(三菱レイヨン製)90gにN,N‘−メチレンビスアクリルアミド(以下、MBAAmと略す)5g、脱イオン水5gを加えて溶解し、50%モノマー溶液(これをb液とする)を調製した。更に、水溶性重合開始剤である2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]二塩酸塩(和光純薬工業製、VA−044)の10%水溶液(これをc液とする)を調製した。2mlのサンプルチューブに、a液を840μl、b液を160μl注入して混合し、蛋白質結晶化剤水溶液(1.0M−NaCl、pH4.0、モノマー濃度8%)を調製し、更にc液を10μl添加してよく混合した後、55℃の温浴で3時間重合し、透明なハイドロゲルを得た。 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 polyethylene glycol 6000 is held in a gel-like material containing polyoxyethylene monoacrylate.
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JP2003291308A JP4323258B2 (en) | 2003-08-11 | 2003-08-11 | Protein crystallizing agent and method for preparing the same |
US10/567,666 US20070181058A1 (en) | 2003-08-11 | 2004-08-10 | Protein crystallization apparatus, method of protein crystallization, protein crystallizing agent and process for preparing the same |
PCT/JP2004/011725 WO2005014899A1 (en) | 2003-08-11 | 2004-08-10 | Protein crystallization apparatus, method of protein crystallization, protein crystallizing agent and process for preparing the same |
CN 200710092292 CN101029078A (en) | 2003-08-11 | 2004-08-10 | Gel used for protein crystalling |
CNB2004800295182A CN100355949C (en) | 2003-08-11 | 2004-08-10 | Protein crystallization apparatus, method of protein crystallization, protein crystallizing agent and process for preparing the same |
EP04771686A EP1666646A4 (en) | 2003-08-11 | 2004-08-10 | Protein crystallization apparatus, method of protein crystallization, protein crystallizing agent and process for preparing the same |
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