JP2007218735A - Pretreatment method of specimen and reagent - Google Patents
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本発明は、電気泳動に供する検体の前処理方法、および前処理用試薬組成物に関する。 The present invention relates to a pretreatment method for a specimen to be subjected to electrophoresis, and a pretreatment reagent composition.
電気泳動、とりわけドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）によるタンパク質の検出は、一般的、標準的なタンパク質の分析方法として広く用いられている。ポリアクリルアミドゲル電気泳動は、アクリルアミドを重合させたゲル中で荷電粒子に電圧を付加し、その移動度によってそれぞれの粒子を分離できることを利用している。ポリアクリルアミドゲルは分子篩として働き、ゲル中では小さい粒子ほど動きやすく大きな粒子ほど動きにくい。また、ゲルの濃度を変えることで、移動度を調整できる。しかし、タンパク質はそのアミノ酸配列により、それぞれ独特な立体構造を持ち、同じ分子量でもその移動度が異なる。またそのタンパク質を構成するアミノ酸残基の電気特性（等電点）もそれぞれ異なり、同じ電圧でもかかる力が異なりそれが移動度にも反映する。この問題を避けるために、ＳＤＳ−ＰＡＧＥは負電荷界面活性剤であるドデシル硫酸ナトリウム（ＳＤＳ）で検体を変性させてから行なう。ＳＤＳは水溶液中では非極性基を内側にしてミセルを作る。この非極性基がタンパク質のアミノ酸残基間の疎水結合を壊すので、タンパク質はほぼ直鎖状に引き伸ばされ、また、イオン性残基の電荷はミセル表面の負電荷にマスクされ単位長さ当たりほぼ一定の電荷となる。これにより、タンパク質はその個々の性質を失いその分子量のみによって分離されるようになる。 Protein detection by electrophoresis, especially sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), is widely used as a general and standard protein analysis method. Polyacrylamide gel electrophoresis utilizes the fact that a voltage is applied to charged particles in a gel polymerized with acrylamide, and each particle can be separated by its mobility. The polyacrylamide gel acts as a molecular sieve, and the smaller particles are easier to move and the larger particles are less likely to move in the gel. Moreover, mobility can be adjusted by changing the density | concentration of a gel. However, proteins have unique three-dimensional structures depending on their amino acid sequences, and their mobility differs even with the same molecular weight. In addition, the electrical properties (isoelectric points) of the amino acid residues constituting the protein are also different, and the applied force is different even at the same voltage, which is reflected in the mobility. In order to avoid this problem, SDS-PAGE is performed after denaturing the specimen with sodium dodecyl sulfate (SDS), which is a negatively charged surfactant. SDS forms micelles with nonpolar groups on the inside in aqueous solutions. Since this nonpolar group breaks the hydrophobic bond between the amino acid residues of the protein, the protein is stretched almost linearly, and the charge of the ionic residue is masked by the negative charge on the micelle surface and is almost per unit length. It becomes a constant charge. This causes the protein to lose its individual properties and be separated only by its molecular weight.
近年、より多くの種類のタンパク質を含む検体より個々のタンパク質を分離する方法として、二次元電気泳動が普及した。二次元電気泳動とは、異なる条件での電気的分離法の２種類を組合せて分離する方法で、一次元目に等電点電気泳動を、二次元目にＳＤＳ−ＰＡＧＥを用いる方法が一般的である。この場合、等電点で分別したタンパク質をさらに分子量の違いにより分別するため、個々のタンパク質がスポットとして分離される。 In recent years, two-dimensional electrophoresis has become widespread as a method for separating individual proteins from specimens containing more types of proteins. Two-dimensional electrophoresis is a method of separation by combining two types of electroseparation methods under different conditions, and generally uses isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. It is. In this case, the proteins separated at the isoelectric point are further separated according to the difference in molecular weight, so that the individual proteins are separated as spots.
検体がタンパク質成分のみである場合や、タンパク質以外の成分が極めて少ない検体であれば、一般に電気泳動は問題無く行なえる。しかしながら、タンパク質以外の成分を多く含む検体を電気泳動すると、タンパク質の分離が十分でない場合が多い。特に、微生物や動植物の細胞や組織を破砕して得られた検体の場合、タンパク質以外に核酸や脂質、低分子化合物などの多様な成分を含むため、明瞭なタンパク質の分離を成功させることは困難である。また、検体中の膜タンパク質を電気泳動するには、タンパク質を膜から抽出せねばならない。したがって、このような検体の電気泳動に際しては、予め何らかの処理を行なうことが必要となる。 If the sample is only a protein component or if the sample contains very few components other than proteins, electrophoresis can generally be performed without problems. However, when a sample containing many components other than protein is electrophoresed, protein separation is often insufficient. In particular, in the case of specimens obtained by disrupting cells and tissues of microorganisms and animals and plants, it is difficult to succeed in clearly separating proteins because they contain various components such as nucleic acids, lipids, and low molecular compounds in addition to proteins. It is. In addition, in order to electrophoretize a membrane protein in a specimen, the protein must be extracted from the membrane. Therefore, it is necessary to perform some kind of processing in advance for electrophoresis of such a specimen.
検体の前処理方法としては、タンパク質成分の検体からの抽出、精製が一般的に行なわれる。例えば、カラムクロマトグラフィーによるタンパク質の吸着と溶出、ゲル濾過による分画、フィルターや膜による分離などは、古くから行なわれている手法で、現在も電気泳動の前処理として常用されている。しかし、これらの方法は時間と手間がかかり、器具や装置、消耗品を使用するためコスト面での負担も大きい。また、検体を界面活性剤で処理する簡便な方法も採用されているが、タンパク質成分を可溶化させる点では有効であるものの、夾雑成分の影響を回避することは難しく、電気泳動によるタンパク質の分離が十分でない場合が多い。また、近年広く行われている、細胞や組織中で発現している全てのタンパク質を網羅的に分析するプロテオーム解析では、検体の前処理によって分析結果が変化することは望ましくないので、精製などによらず夾雑成分による影響の回避することが重要である。しかし、従来は、このような目的に使用される検体を用いて十分な分離を達成することができない場合に分離を改善する有効な手段は知られていなかった。 As a sample pretreatment method, extraction and purification of a protein component from a sample are generally performed. For example, protein adsorption and elution by column chromatography, fractionation by gel filtration, separation by filters and membranes, etc. are methods that have been practiced for a long time, and are still commonly used as pretreatments for electrophoresis. However, these methods are time consuming and labor intensive, and are expensive in terms of cost because they use instruments, devices, and consumables. In addition, a simple method of treating a sample with a surfactant is also adopted, but it is effective in solubilizing protein components, but it is difficult to avoid the influence of contaminating components, and protein separation by electrophoresis is difficult. Is often not enough. In proteome analysis, which is widely performed in recent years and comprehensively analyzes all proteins expressed in cells and tissues, it is not desirable that the analysis results change due to sample pretreatment. Regardless, it is important to avoid the effects of contaminating components. However, conventionally, there has been no known effective means for improving separation when sufficient separation cannot be achieved using a sample used for such a purpose.
したがって、タンパク質以外の成分を多く含む検体の電気泳動における、簡便で低コスト、かつ効果が十分な検体の前処理方法、そのための試薬が望まれていた。 Therefore, there has been a demand for a simple, low-cost, and sufficiently effective sample pretreatment method and reagent therefor in electrophoresis of a sample containing many components other than proteins.
本発明は、タンパク質の電気泳動に供する検体の前処理において、従来は困難であった簡便で低コスト、かつ効果が十分な方法および試薬組成物を提供することを目的とする。 An object of the present invention is to provide a simple method, low cost, and sufficient effect and reagent composition that have been difficult in the pretreatment of specimens for protein electrophoresis.
本発明者らは、上記課題を解決するために種々の検討を行なった。より具体的には、検体を前処理用の溶液にて処理し電気泳動に供する簡便で低コストな方法を想定し、前処理溶液の組成を種々検討することにより、電気泳動でのタンパク質の分離に対し劇的な効果のある溶液組成を見出すことに成功し、本発明を完成するに至った。 The present inventors have made various studies in order to solve the above problems. More specifically, assuming a simple and low-cost method in which a specimen is treated with a pretreatment solution and subjected to electrophoresis, protein separation by electrophoresis is studied by variously examining the composition of the pretreatment solution. As a result, the present inventors have succeeded in finding a solution composition having a dramatic effect, and thus completed the present invention.
すなわち、本発明は以下の通りである。
（１）エステラーゼ活性を有するタンパク質および核酸分解酵素活性を有するタンパク質を含む反応液中で検体を処理する工程を包含することを特徴とする、電気泳動に供する検体の前処理方法。
（２）エステラーゼ活性を有するタンパク質がシュードモナス（Pseudomonas）属細菌由来リパーゼであることを特徴とする、（１）記載の電気泳動に供する検体の前処理方法。
（３）アルコールおよび／または界面活性剤を更に含む反応液中で検体を処理することを特徴とする、（１）または（２）記載の電気泳動に供する検体の前処理方法。
（４）エステラーゼ活性を有するタンパク質および核酸分解酵素活性を有するタンパク質を含む、電気泳動に供する検体の前処理用試薬組成物。
（５）エステラーゼ活性を有するタンパク質がシュードモナス（Pseudomonas）属細菌由来リパーゼであることを特徴とする、（４）記載の電気泳動に供する検体の前処理用試薬組成物。
（６）アルコールおよび／または界面活性剤を更に含む、（４）または（５）記載の電気泳動に供する検体の前処理用試薬組成物。
That is, the present invention is as follows.
(1) A method for pretreating a specimen to be subjected to electrophoresis, comprising a step of treating the specimen in a reaction solution containing a protein having esterase activity and a protein having nucleolytic enzyme activity.
(2) The method for pretreating a specimen to be subjected to electrophoresis according to (1), wherein the protein having esterase activity is a lipase derived from a genus Pseudomonas.
(3) The specimen pretreatment method for electrophoresis as described in (1) or (2), wherein the specimen is treated in a reaction solution further containing alcohol and / or a surfactant.
(4) A reagent composition for pretreatment of a specimen to be subjected to electrophoresis, comprising a protein having esterase activity and a protein having nucleolytic enzyme activity.
(5) The reagent composition for pretreatment of a specimen to be subjected to electrophoresis according to (4), wherein the protein having esterase activity is a lipase derived from a genus Pseudomonas.
(6) The reagent composition for pretreatment of a specimen to be subjected to electrophoresis according to (4) or (5), further comprising an alcohol and / or a surfactant.
本発明により電気泳動に供する検体の前処理方法、および前処理用試薬組成物が提供される。 The present invention provides a pretreatment method of a specimen to be subjected to electrophoresis, and a pretreatment reagent composition.
本発明の電気泳動に供する検体の前処理方法は、エステラーゼ活性を有するタンパク質および核酸分解酵素活性を有するタンパク質を含む反応液中で検体を処理する工程を包含することを特徴とする。また、本発明の検体の前処理用試薬組成物は、エステラーゼ活性を有するタンパク質および核酸分解酵素活性を有するタンパク質を含むことを特徴とする。 The sample pretreatment method for subjecting to electrophoresis according to the present invention includes a step of treating a sample in a reaction solution containing a protein having esterase activity and a protein having nucleolytic enzyme activity. The reagent composition for pretreatment of a specimen of the present invention is characterized by comprising a protein having esterase activity and a protein having nucleolytic enzyme activity.
本発明の前処理方法および前処理用試薬組成物に用いるエステラーゼ活性を有するタンパク質としては、微生物や動植物に由来するエステラーゼ活性を有するタンパク質などを制限無く使用できる。 As the protein having esterase activity used in the pretreatment method and the pretreatment reagent composition of the present invention, proteins having esterase activity derived from microorganisms and animals and plants can be used without limitation.
本明細書において「エステラーゼ活性」とは、エステルを加水分解する活性をいう。また、本明細書において「リパーゼ活性」とは、グリセロールエステルを加水分解して脂肪酸を遊離する活性をいう。リパーゼ活性を有するタンパク質の例としては、トリグリセリド反応性リパーゼが挙げられる。また、トリグリセリド反応性リパーゼの例としては、リポプロテインリパーゼが挙げられる。 As used herein, “esterase activity” refers to an activity of hydrolyzing an ester. In the present specification, “lipase activity” refers to an activity of hydrolyzing a glycerol ester to release fatty acids. Examples of proteins having lipase activity include triglyceride reactive lipase. Examples of the triglyceride reactive lipase include lipoprotein lipase.
本発明において、エステラーゼ活性を有するタンパク質としては、産業用酵素として一般に利用されている微生物や動物由来のトリグリセリド反応性リパーゼを使用することが、コスト低減のために好ましい。このような酵素として、例えばシュードモナス（Pseudomonas）属細菌由来リパーゼ、カンジダ（Candida属）酵母由来リパーゼ、またはリゾプス（Rizopus属）カビ由来リパーゼおよびこれらの混合物を用いることが好適である。本発明の実施例には、シュードモナス・アエルギノサ（Pseudomonas aeruginosa）由来リポプロテインリパーゼを使用した。この酵素は、コレステロールやその他のステロール、各種ラクトン類などに対するエステラーゼ活性を有している。本酵素は、既に臨床検査薬用酵素として産業利用されている。例えば、東洋紡績株式会社から市販されているリポプロテインリパーゼとしてである。尚、本酵素活性は１分間に１マイクロモルのGlycerol（１／２マイクロモルのQuinoneimine色素）を生成する酵素量を１単位（Ｕ）として表される（東洋紡績株式会社ウェブサイト参照、http://www.toyobo.co.jp/e/seihin/xr/enzyme/pdf/205LPL-311.pdf）。 In the present invention, as a protein having esterase activity, it is preferable to use a triglyceride-reactive lipase derived from microorganisms or animals generally used as industrial enzymes for cost reduction. As such an enzyme, it is preferable to use, for example, a lipase derived from bacteria belonging to the genus Pseudomonas, a lipase derived from Candida yeast, a lipase derived from Rhizopus fungus, and a mixture thereof. In the examples of the present invention, Pseudomonas aeruginosa-derived lipoprotein lipase was used. This enzyme has esterase activity for cholesterol, other sterols, various lactones and the like. This enzyme has already been industrially used as a clinical test enzyme. For example, as a lipoprotein lipase commercially available from Toyobo Co., Ltd. This enzyme activity is expressed as 1 unit (U) of the amount of enzyme that produces 1 micromole of Glycerol (1/2 micromole of Quinoneimine dye) per minute (see Toyobo Co., Ltd. website, http: //www.toyobo.co.jp/e/seihin/xr/enzyme/pdf/205LPL-311.pdf).
本発明におけるエステラーゼ活性を有するタンパク質の使用量は、前処理反応液中終濃度で０．００１〜１０Ｕ／ｍｌであるのが好ましく、０．０２〜２Ｕ／ｍｌであるのがより好ましい。ここで「Ｕ」は国際単位を表す。上記エステラーゼ活性を有するタンパク質の量が０．００１Ｕ／ｍｌ未満であると、夾雑脂質成分の分解が不十分で電気泳動によるタンパク質の分離能が低くなってしまう可能性があり、また上記エステラーゼ活性を有するタンパク質の量が１０Ｕ／ｍｌを超えると、このタンパク質自体が電気泳動で他のタンパク質の分離に悪影響を及ぼし、タンパク質の検出効率が低下する可能性がある。当業者は、処理の有効性、分離への影響などを考慮して適切に使用量を決定することができる。 The amount of the protein having esterase activity in the present invention is preferably 0.001 to 10 U / ml, more preferably 0.02 to 2 U / ml, in the final concentration in the pretreatment reaction solution. Here, “U” represents an international unit. If the amount of the protein having esterase activity is less than 0.001 U / ml, there is a possibility that contaminating lipid components are not sufficiently decomposed and the separation ability of the protein by electrophoresis is lowered, and the esterase activity is reduced. If the amount of the protein exceeds 10 U / ml, the protein itself may adversely affect the separation of other proteins by electrophoresis, and the protein detection efficiency may decrease. A person skilled in the art can appropriately determine the amount to be used in consideration of the effectiveness of processing, the influence on separation, and the like.
本発明に係る核酸分解酵素活性を有するタンパク質としては、市販のＤＮＡ分解酵素、ＲＮＡ分解酵素などを制限無く使用できるが、両酵素（ＤＮＡ分解酵素とＲＮＡ分解酵素）を組み合わせて使用することが効果的である。例えば、基質となる核酸（ＤＮＡ、ＲＮＡ）の形態（一本鎖、二本鎖）にかかわらずランダムに分解するエキソ型の酵素を好適に用いることができる。このような酵素として、例えばウシ膵臓由来ＤＮａｓｅＩやその他の動物由来ＤＮａｓｅ、ウシ膵臓ＲＮａｓｅＡやその他の動物由来ＲＮａｓｅ、およびこれらの混合物を用いることが好適である。 As the protein having nucleolytic enzyme activity according to the present invention, commercially available DNA degrading enzymes, RNA degrading enzymes and the like can be used without limitation, but it is effective to use both enzymes (DNA degrading enzyme and RNA degrading enzyme) in combination. Is. For example, an exo-type enzyme that can be decomposed randomly regardless of the form (single-stranded or double-stranded) of nucleic acid (DNA, RNA) serving as a substrate can be suitably used. As such an enzyme, for example, bovine pancreas-derived DNase I, other animal-derived DNases, bovine pancreatic RNase A, other animal-derived RNases, and mixtures thereof are preferably used.
本発明における核酸分解酵素活性を有するタンパク質の使用量は、前処理反応液中終濃度で０．００１〜１０Ｕ／ｍｌであるのが好ましく、０．０４〜４Ｕ／ｍｌであるのがより好ましい。ここで、ＤＮａｓｅの一例であるＤＮａｓｅＩの１単位（Ｕ）は、ｐｏｌｙ（ｄＡ−ｄＴ）をテンプレートプライマーとして用いて、３７℃で３０分間に全体として１０ｎｍｏｌのデオキシリボヌクレオチドを酸沈殿画分に取り込ませる酵素量として定義され、ＲＮａｓｅの一例であるＲＮａｓｅＡの１単位（Ｕ）は、Ｋｕｎｉｔｚの方法に従ってＲＮＡを基質として２５℃で測定した場合に、１分間に吸光度Ａ（終末の吸光度）をＢまで減少（ＡからＢへの減少は全減少量に相当する）させるのに必要な酵素活性として定義される。上記核酸分解酵素活性を有するタンパク質の量が０．００１Ｕ／ｍｌ未満であると、夾雑核酸成分の分解が不十分で電気泳動によるタンパク質の分離能が低くなってしまう可能性があり、また上記核酸分解酵素活性を有するタンパク質の量が１０Ｕ／ｍｌを超えると、電気泳動で他のタンパク質の分離に悪影響を及ぼし、タンパク質の検出効率が低下する可能性がある。当業者は、処理の有効性、分離への影響などを考慮して適切に使用量を決定することができる。 The use amount of the protein having nucleolytic enzyme activity in the present invention is preferably 0.001 to 10 U / ml, more preferably 0.04 to 4 U / ml, as a final concentration in the pretreatment reaction solution. Here, 1 unit (U) of DNase I, which is an example of DNase, uses poly (dA-dT) as a template primer to incorporate 10 nmol of deoxyribonucleotides as a whole into an acid precipitation fraction at 37 ° C. for 30 minutes. One unit (U) of RNase A, which is defined as the amount of enzyme and is an example of RNase, decreases absorbance A (end absorbance) to B per minute when measured at 25 ° C. using RNA as a substrate according to the Kunitz method. (A reduction from A to B corresponds to the total reduction) is defined as the enzyme activity required to make it. If the amount of the protein having the nucleolytic enzyme activity is less than 0.001 U / ml, there is a possibility that the contaminating nucleic acid component is not sufficiently decomposed and the protein separation ability by electrophoresis becomes low, and the nucleic acid If the amount of the protein having a degrading enzyme activity exceeds 10 U / ml, the separation of other proteins may be adversely affected by electrophoresis, and the protein detection efficiency may be reduced. A person skilled in the art can appropriately determine the amount to be used in consideration of the effectiveness of processing, the influence on separation, and the like.
１つの実施態様において、本発明の検体の前処理方法は、アルコールおよび／または界面活性剤を更に含む溶液中で検体を処理することを特徴とする。 In one embodiment, the specimen pretreatment method of the present invention is characterized in that the specimen is treated in a solution further containing an alcohol and / or a surfactant.
本発明に係るアルコールとしては、特に制限されないが、コスト面からメタノール、エタノール、イソプロパノールなどの安価なアルコールが好適である。 Although it does not restrict | limit especially as alcohol which concerns on this invention, Cheap alcohol, such as methanol, ethanol, isopropanol, is suitable from a cost surface.
本発明におけるアルコールの使用量は、前処理反応液中終濃度で１〜２０重量％であるのが好ましく、２〜１０重量％であるのがより好ましい。 The amount of alcohol used in the present invention is preferably 1 to 20% by weight, more preferably 2 to 10% by weight, in the final concentration in the pretreatment reaction solution.
本発明に係る界面活性剤としては、ＴｒｉｔｏｎＸ−１００、ＴｒｉｔｏｎＸ−２００、ＴｒｉｔｏｎＸ−４００、Ｔｗｅｅｎ２０、Ｔｗｅｅｎ８０、ＣＨＡＰＳなどの非イオン系または両性イオン系の界面活性剤が好適である。 As the surfactant according to the present invention, nonionic or zwitterionic surfactants such as Triton X-100, Triton X-200, Triton X-400, Tween 20, Tween 80, and CHAPS are suitable.
本発明における界面活性剤の使用量は、前処理反応液中終濃度で０．０１〜１２重量％であるのが好ましく、０．０２〜１．７重量％であるのがより好ましい。 The amount of the surfactant used in the present invention is preferably 0.01 to 12% by weight, more preferably 0.02 to 1.7% by weight in terms of the final concentration in the pretreatment reaction solution.
本明細書において「検体」とは、それに含まれるタンパク質を分析しようとする検体をいう。本発明の前処理方法および前処理用試薬組成物は、電気泳動において十分な分離が達成されない場合に有効に使用される。従って、対象となる検体としては、電気泳動の際に分離に悪影響を及ぼすタンパク質以外の成分を多く含む検体、例えばタンパク質以外に核酸や脂質、低分子化合物などの多様な成分を含む検体などが挙げられる。具体的には、微生物や動植物の細胞あるいは組織を破砕して得られた検体、細胞の膜画分などが挙げられる。例えば、上記のように、細胞や組織中で発現している全てのタンパク質を網羅的に分析するプロテオーム解析では、検体の前処理によって分析結果が変化することは望ましくないので、精製などによらず夾雑成分による影響の回避することができる本発明の前処理方法および前処理試薬組成物は、プロテオーム解析に供する検体の前処理に有用である。 In this specification, the “sample” refers to a sample to be analyzed for proteins contained therein. The pretreatment method and the pretreatment reagent composition of the present invention are effectively used when sufficient separation is not achieved in electrophoresis. Accordingly, the target specimens include specimens containing many components other than proteins that adversely affect separation during electrophoresis, such as specimens containing various components such as nucleic acids, lipids, and low molecular compounds in addition to proteins. It is done. Specific examples include specimens obtained by disrupting cells or tissues of microorganisms or animals and plants, membrane fractions of cells, and the like. For example, in the proteome analysis that comprehensively analyzes all proteins expressed in cells and tissues as described above, it is not desirable that the analysis results change due to sample pretreatment. The pretreatment method and the pretreatment reagent composition of the present invention that can avoid the influence of contaminating components are useful for pretreatment of a specimen to be subjected to proteome analysis.
本発明を活用して電気泳動し得るタンパク質（目的タンパク質）としては、特に限定されない。細胞質や各種オルガネラの可溶性タンパク質、細胞外分泌タンパク質、膜タンパク質、不溶性タンパク質や凝集体、および糖やリン酸などで修飾を受けたタンパク質などが対象として挙げられる。なお、ここで取り上げられるタンパク質は低分子ペプチドも含む広義のものである。 The protein (target protein) that can be electrophoresed using the present invention is not particularly limited. Examples include soluble proteins of cytoplasm and various organelles, extracellular secreted proteins, membrane proteins, insoluble proteins and aggregates, and proteins modified with sugars and phosphates. In addition, the protein taken up here is a broad meaning including a low molecular weight peptide.
本発明の前処理方法における各条件は、用いる溶液の組成や検体の種類、電気泳動の手法や条件によって適宜選択でき、特に限定されるものではない。以下、検体としてタンパク質以外の成分を多く含む酵母（Saccharomyces cerevisiae）や大腸菌（Escherichia coli）の破砕物を用い、前処理試薬組成物として０．００１〜１Ｕ／ｍｌのシュードモナス（Pseudomonas）属細菌由来リパーゼ、０．００１〜１Ｕ／ｍｌのＤＮａｓｅ（ＤＮＡ分解酵素）、０．００１〜１Ｕ／ｍｌのＲＮａｓｅ（ＲＮＡ分解酵素）、０．０１〜０．１％のＴｒｉｔｏｎＸ−１００、１〜５％のメタノールを含む５０ｍＭリン酸カリウム（ｐＨ７．０）緩衝液を用い、前処理後に二次元電気泳動を行なう場合を例に挙げて、具体的に説明する。 Each condition in the pretreatment method of the present invention can be appropriately selected depending on the composition of the solution to be used, the type of specimen, the electrophoresis technique and conditions, and is not particularly limited. Hereinafter, a lipase derived from bacteria belonging to the genus Pseudomonas of 0.001 to 1 U / ml is used as a pretreatment reagent composition using a crushed product of yeast (Saccharomyces cerevisiae) or Escherichia coli containing a large amount of components other than proteins as a specimen. 0.001-1 U / ml DNase (DNA-degrading enzyme), 0.001-1 U / ml RNase (RNA-degrading enzyme), 0.01-0.1% Triton X-100, 1-5% methanol This will be specifically described with reference to an example in which two-dimensional electrophoresis is performed after pretreatment using a 50 mM potassium phosphate (pH 7.0) buffer solution containing.
検体として細胞や組織を用い、細胞内タンパク質や膜タンパク質を分析する場合、予め検体を破砕する必要がある。破砕方法としては、ソニック破砕、ビーズ破砕やフレンチプレス破砕などの物理的な破砕方法、酵素による細胞壁溶解と浸透圧による破砕など、通常の方法を利用することができる。また、タンパク質の分解や構造変化を防ぐために、細胞や組織をプロテアーゼ阻害剤や有機溶媒などを含む溶解バッファーに懸濁することや、更に物理的破砕を加えるなどの方法も有効である。 When using a cell or tissue as a specimen and analyzing intracellular protein or membrane protein, it is necessary to crush the specimen in advance. As the crushing method, a normal crushing method such as sonic crushing, bead crushing or French press crushing, cell wall lysis by an enzyme and crushing by osmotic pressure can be used. In order to prevent protein degradation and structural changes, it is also effective to suspend cells or tissues in a lysis buffer containing a protease inhibitor or an organic solvent, or to add physical disruption.
これらの方法で検体を処理した後、本発明の前処理試薬組成物と混合して反応を行なう。前処理試薬組成物と検体との混合比率は、検体の種類、濃度などにより異なるため一概に言えないが、前処理試薬組成物を検体に対し１／２０〜２倍容量加えることが好ましく、一般的には１／５〜等量加えると好適である。 After the sample is treated by these methods, the reaction is performed by mixing with the pretreatment reagent composition of the present invention. The mixing ratio of the pretreatment reagent composition and the sample varies depending on the type and concentration of the sample and cannot be generally stated. However, it is preferable to add the pretreatment reagent composition to the sample in an amount of 1/20 to 2 times, Specifically, it is preferable to add 1/5 to an equal amount.
前処理の反応条件も、検体の種類、濃度などにより異なるため一概に言えないが、使用する酵素活性を有するタンパク質の作用に適切な条件が選択される。例えば、反応温度に関しては１０℃以上の反応温度で１０〜１００分間反応させるのが好ましい。本発明の実施例では、室温（２０〜３０℃）にて３０分間反応させている。さらに、適切な反応ｐＨとなるように、反応液中にリン酸緩衝液などの緩衝液を含めることが好ましい。 The reaction conditions for pretreatment also vary depending on the type and concentration of the specimen and cannot be generally stated, but conditions appropriate for the action of the protein having the enzyme activity to be used are selected. For example, the reaction temperature is preferably 10 to 100 minutes at a reaction temperature of 10 ° C. or higher. In the Example of this invention, it is made to react for 30 minutes at room temperature (20-30 degreeC). Furthermore, it is preferable to include a buffer solution such as a phosphate buffer in the reaction solution so as to obtain an appropriate reaction pH.
本発明の検体の前処理方法および本発明の検体の前処理試薬組成物は任意の電気泳動に有効であり、例えば、ゲルを用いるタンパク質の分析のための電気泳動に適用することができる。特に、等電点電気泳動、ＳＤＳ−ＰＡＧＥ、およびその組み合わせである二次元電気泳動に適用することが好ましい。例えば、二次元電気泳動の場合、前処理反応後、常法にしたがい検体をまず等電点電気泳動に供し、ついで二次元目のＳＤＳ−ＰＡＧＥを行なうことにより、タンパク質が十分に分離し、明瞭な二次元電気泳動結果を得ることができる。 The sample pretreatment method of the present invention and the sample pretreatment reagent composition of the present invention are effective for arbitrary electrophoresis, and can be applied to electrophoresis for protein analysis using a gel, for example. In particular, it is preferable to apply to two-dimensional electrophoresis which is isoelectric focusing, SDS-PAGE, and a combination thereof. For example, in the case of two-dimensional electrophoresis, after the pretreatment reaction, the sample is first subjected to isoelectric focusing according to a conventional method, and then the second dimension SDS-PAGE is performed, so that the protein is sufficiently separated and clear. 2D electrophoresis results can be obtained.
以下、実施例及び比較例によって更に本発明を詳細に説明するが、本発明はこれらによって何等限定されるものでない。尚、以下の実施例等に於いて、二次元電気泳動の一次元目の等電点電気泳動はｐＩが３．５〜７の範囲となるよう調製したゲルを使い、ディスク電気泳動装置（日本エイドー製）を用いて実施した。また二次元目のＳＤＳ−ＰＡＧＥは、１７％ゲルを使い、恒温式２連スラブ電気泳動装置（日本エイドー製）を用いて実施した。一次元目の等電点電気泳動のゲルと二次元目のＳＤＳ−ＰＡＧＥのゲルとは、アガロースゲル（ナカライテスク製）を用いて接着した。二次元電気泳動試薬やその他の必要な試薬はナカライテスクより購入し、メーカーのプロトコールにしたがい実施した。 EXAMPLES Hereinafter, although an Example and a comparative example demonstrate this invention further in detail, this invention is not limited at all by these. In the following examples, etc., the first-dimensional isoelectric focusing of the two-dimensional electrophoresis uses a gel prepared so that the pI is in the range of 3.5 to 7, and the disk electrophoresis apparatus (Japan) Aido) was used. The second-dimensional SDS-PAGE was carried out using a 17% gel and a constant temperature double slab electrophoresis apparatus (manufactured by Nippon Aido). The first-dimensional isoelectric focusing gel and the second-dimensional SDS-PAGE gel were bonded using an agarose gel (manufactured by Nacalai Tesque). Two-dimensional electrophoresis reagents and other necessary reagents were purchased from Nacalai Tesque and carried out according to the manufacturer's protocol.
（実施例１）酵母ミクロソーム画分（膜画分）の二次元電気泳動
（１）前処理試薬組成物の調製
以下の組成の前処理試薬組成物を調製し、実験に供した。
５０ｍＭリン酸カリウム緩衝液（ｐＨ７．０）
１Ｕ／ｍｌ シュードモナス・アエルギノサ（Pseudomonas aeruginosa）由来リポプロテインリパーゼ（東洋紡製）
１Ｕ／ｍｌ ＤＮａｓｅＩ（シグマ製）
１Ｕ／ｍｌ ＲＮａｓｅＡ（ナカライテスク製）
０．１％ ＴｒｉｔｏｎＸ−１００
５％メタノール
Example 1 Two-Dimensional Electrophoresis of Yeast Microsome Fraction (Membrane Fraction) (1) Preparation of Pretreatment Reagent Composition A pretreatment reagent composition having the following composition was prepared and used for experiments.
50 mM potassium phosphate buffer (pH 7.0)
1U / ml Lipoprotein lipase from Pseudomonas aeruginosa (Toyobo)
1U / ml DNase I (manufactured by Sigma)
1U / ml RNase A (Nacalai Tesque)
0.1% Triton X-100
5% methanol
（２）検体（酵母ミクロソーム画分）の調製
サッカロミセス・セレビシエ（Saccharomyces cerevisiae）Ｓ２８８Ｃ株（独立行政法人・製品評価技術基盤機構より入手）の菌体を細胞壁分解酵素Zymolyase 100T（生化学工業製）で処理後、遠心分離により酵母細胞のスフェロプラストを回収した。等張液に再懸濁し、テフロンホモジナイザーを用いて細胞を破壊した。細胞破砕液を常法に従って、遠心分離によって細胞内オルガネラを分離し、ミクロソーム画分を得、検体として用いた。
(2) Preparation of Specimen (Yeast Microsome Fraction) Saccharomyces cerevisiae S288C strain (obtained from Independent Administrative Institution / Product Evaluation Technology Infrastructure) cell wall degrading enzyme Zymolyase 100T (manufactured by Seikagaku Corporation) After the treatment, spheroplasts of yeast cells were collected by centrifugation. Resuspended in isotonic solution and disrupted cells using a Teflon homogenizer. In the cell disruption solution, intracellular organelles were separated by centrifugation according to a conventional method to obtain a microsomal fraction, which was used as a specimen.
（３）前処理反応
（２）で調製した検体に等量の（１）で調製した前処理試薬組成物を加え、室温（２０〜３０℃）で３０分間静置して前処理反応を実施した。その後、そのサンプルを実験に供した。
(3) Pretreatment reaction Add the same amount of the pretreatment reagent composition prepared in (1) to the specimen prepared in (2), and leave it at room temperature (20-30 ° C) for 30 minutes to carry out the pretreatment reaction. did. The sample was then subjected to the experiment.
前処理後の検体を、通常通りの方法にて二次元電気泳動に供し染色した。結果を図１に示す。タンパク質が十分に分離し、明瞭な二次元電気泳動結果が得られていることが分かる。 The sample after the pretreatment was subjected to two-dimensional electrophoresis by a usual method and stained. The results are shown in FIG. It can be seen that the protein is sufficiently separated and a clear two-dimensional electrophoresis result is obtained.
（実施例２）大腸菌可溶性画分の二次元電気泳動
（１）前処理試薬組成物の調製
実施例１と同じ組成の前処理試薬組成物を調製し、実験に供した。
Example 2 Two-Dimensional Electrophoresis of E. coli Soluble Fraction (1) Preparation of Pretreatment Reagent Composition A pretreatment reagent composition having the same composition as in Example 1 was prepared and used for experiments.
（２）検体（大腸菌可溶性画分）の調製
大腸菌（Escherichia coli）Ｋ１２ ＭＧ１６５５株（国立遺伝学研究所より入手）の菌体を以下の組成のＬｙｓｉｓ Ｂｕｆｆｅｒに懸濁し、超音波処理により細胞を破戒した。菌破砕液を遠心分離し、不溶性画分と未破壊細胞を除去した。得られた上清を検体として用いた。
尿素（ナカライテスク製） ８Ｍ
ＮＰ−４０（シグマ製） ２％
２−メルカプトエタノール（ナカライテスク製） ５％
(2) Preparation of specimen (E. coli soluble fraction) Suspension of cells of Escherichia coli K12 MG1655 strain (obtained from National Institute of Genetics) in Lysis Buffer having the following composition, and the cells are disrupted by sonication did. The lysate was centrifuged to remove insoluble fractions and unbroken cells. The obtained supernatant was used as a specimen.
Urea (manufactured by Nacalai Tesque) 8M
NP-40 (manufactured by Sigma) 2%
2-mercaptoethanol (Nacalai Tesque) 5%
（３）前処理反応
（２）で調製した検体と（１）で調製した実施例１と同じ組成の前処理試薬組成物とを、検体：前処理溶液＝５：１の容量比で混合し、室温（２０〜３０℃）で３０分間静置して前処理反応を実施した。その後、そのサンプルを実験に供した。
(3) Pretreatment reaction The specimen prepared in (2) and the pretreatment reagent composition having the same composition as in Example 1 prepared in (1) were mixed at a volume ratio of specimen: pretreatment solution = 5: 1. The mixture was allowed to stand at room temperature (20 to 30 ° C.) for 30 minutes to carry out a pretreatment reaction. The sample was then subjected to the experiment.
前処理後の検体を、通常通りの方法にて二次元電気泳動に供し染色した。結果を図２に示す。タンパク質が十分に分離し、明瞭な二次元電気泳動結果が得られていることが分かる。 The sample after the pretreatment was subjected to two-dimensional electrophoresis by a usual method and stained. The results are shown in FIG. It can be seen that the protein is sufficiently separated and a clear two-dimensional electrophoresis result is obtained.
（比較例１）酵母ミクロソーム画分（膜画分）の二次元電気泳動
本発明の前処理を実施しない他は、実施例１と同様の操作にて実験を行なった。結果を図３に示す。タンパク質の分離が不十分で、明瞭な二次元電気泳動結果が得られていない。
Comparative Example 1 Two-dimensional Electrophoresis of Yeast Microsome Fraction (Membrane Fraction) Experiments were performed in the same manner as in Example 1 except that the pretreatment of the present invention was not performed. The results are shown in FIG. Protein separation is insufficient and clear two-dimensional electrophoresis results are not obtained.
（比較例２）大腸菌可溶性画分の二次元電気泳動
本発明の前処理を実施しない他は、実施例２と同様の操作にて実験を行なった。結果を図４に示す。タンパク質の分離が不十分で、明瞭な二次元電気泳動結果が得られていない。
(Comparative Example 2) Two-dimensional electrophoresis of soluble fraction of Escherichia coli An experiment was performed in the same manner as in Example 2 except that the pretreatment of the present invention was not performed. The results are shown in FIG. Protein separation is insufficient and clear two-dimensional electrophoresis results are not obtained.
本発明は、電気泳動に供する検体の簡便で低コストな前処理方法及びそのための試薬組成物を提供するものであり、本発明の方法、試薬組成物を用いれば、従来法に於ける、時間と手間がかかりコスト面での負担が大きいという問題を生じさせることなく、電気泳動により高分解能でタンパク質を分離し得るという効果を奏するので、タンパク質の研究、開発、分析等に貢献するところ極めて大なる発明である。 The present invention provides a simple and low-cost pretreatment method for a sample to be subjected to electrophoresis and a reagent composition therefor. By using the method and reagent composition of the present invention, the time in the conventional method is provided. It can be used to separate proteins with high resolution without causing the problem of costly and labor-intensive tasks, and it contributes to protein research, development, and analysis. It is the invention which becomes.
Claims (6)
The reagent composition for pretreatment of a specimen to be subjected to electrophoresis according to claim 4 or 5, further comprising alcohol and / or a surfactant.
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| JPH03262967A (en) * | 1990-03-13 | 1991-11-22 | Fuji Photo Film Co Ltd | Method for determining high specific gravity lipoprotein cholesterol |
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