JP4799007B2 - Microactuator using bundled actin - Google Patents
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- 102000007469 Actins Human genes 0.000 title claims description 47
- 108010085238 Actins Proteins 0.000 title claims description 47
- 239000000758 substrate Substances 0.000 claims description 56
- 108060008487 Myosin Proteins 0.000 claims description 29
- 102000003505 Myosin Human genes 0.000 claims description 29
- 229910052710 silicon Inorganic materials 0.000 claims description 24
- 239000010703 silicon Substances 0.000 claims description 24
- 102000004169 proteins and genes Human genes 0.000 claims description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 18
- 239000011521 glass Substances 0.000 claims description 15
- 238000004519 manufacturing process Methods 0.000 claims description 9
- MLFHJEHSLIIPHL-UHFFFAOYSA-N isoamyl acetate Chemical compound CC(C)CCOC(C)=O MLFHJEHSLIIPHL-UHFFFAOYSA-N 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 6
- 229940117955 isoamyl acetate Drugs 0.000 claims description 4
- 108010023356 Nonmuscle Myosin Type IIA Proteins 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000003100 immobilizing effect Effects 0.000 claims 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 22
- 239000000835 fiber Substances 0.000 description 22
- 239000000243 solution Substances 0.000 description 19
- 238000003556 assay Methods 0.000 description 16
- 230000004899 motility Effects 0.000 description 16
- 239000011575 calcium Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 6
- 239000000020 Nitrocellulose Substances 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000010884 ion-beam technique Methods 0.000 description 5
- 229920001220 nitrocellulos Polymers 0.000 description 5
- 210000002027 skeletal muscle Anatomy 0.000 description 5
- 102000004204 Fascin Human genes 0.000 description 4
- 108090000786 Fascin Proteins 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 238000003672 processing method Methods 0.000 description 3
- WERLCVLDMOINSQ-UHFFFAOYSA-N 3-methylbutyl acetate 5-methylhexanoic acid Chemical group CC(C)CCCC(O)=O.CC(C)CCOC(C)=O WERLCVLDMOINSQ-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 239000004264 Petrolatum Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005530 etching Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229940066842 petrolatum Drugs 0.000 description 2
- 235000019271 petrolatum Nutrition 0.000 description 2
- 238000001020 plasma etching Methods 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 108700038288 rhodamine-phalloidin Proteins 0.000 description 2
- 239000004065 semiconductor Substances 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 229920000832 Cutin Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 108010045128 Myosin Type II Proteins 0.000 description 1
- 102000005640 Myosin Type II Human genes 0.000 description 1
- 206010034203 Pectus Carinatum Diseases 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- -1 for example Proteins 0.000 description 1
- CKHJYUSOUQDYEN-UHFFFAOYSA-N gallium(3+) Chemical compound [Ga+3] CKHJYUSOUQDYEN-UHFFFAOYSA-N 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229910021421 monocrystalline silicon Inorganic materials 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4716—Muscle proteins, e.g. myosin, actin
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- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
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Description
この出願の発明は、表面にミオシンを担持し、その上に束化アクチンを積載した基板に関する。より詳しくは、束化アクチンを用いたマイクロアクチュエータに関する。 The invention of this application relates to a substrate on which myosin is supported on a surface and bundled actin is loaded thereon. More specifically, the present invention relates to a microactuator using bundled actin.
ミオシン−アクチン系のマイクロアクチュエーターでは、ミオシンの固相化が重要である。すなわち、ミオシンおよびその活性部位であるHMMが酵素活性を維持したまま、アクチン繊維と相互作用を可能とするように一定の方向および適切な密度で基板上に整列配置されなければならないのである。しなしながら、基板上に直接的にミオシンを整列配置させても、アクチン繊維との相互作用は生じず、規則的な整列およびミオシン活性の維持が実現できなかった。 In the myosin-actin microactuator, immobilization of myosin is important. That is, myosin and its active site, HMM, must be aligned on the substrate in a certain direction and at an appropriate density so as to allow interaction with actin fibers while maintaining enzyme activity. However, even when myosin was directly aligned on the substrate, no interaction with actin fibers occurred, and regular alignment and maintenance of myosin activity could not be realized.
この出願の発明は束化アクチンの基板上でのin vitro motility assay系とマイクロアクチュエータを提供することを課題とする。 An object of the invention of this application is to provide an in vitro motility assay system and a microactuator on a substrate of bundled actin.
この出願の発明は上記の課題を解決するために下記の手段を提供する。 The invention of this application provides the following means in order to solve the above problems.
＜１＞濃度が０．２重量％のコロジオン溶液で処理した基板表面にミオシンが担持され、その上に束化アクチンが積載されていることを特徴とするタンパク質構成体。
＜２＞基板がガラス製であることを特徴とする前記＜１＞のタンパク質構成体。
＜３＞基板がシリコン製であることを特徴とする前記＜１＞のタンパク質構成体。
＜４＞コロジオン溶液は、溶質がピロキシリン、溶媒が酢酸イソアミルであることを特徴とする前記＜１＞のタンパク質構成体。
＜５＞前記＜１＞から＜４＞のいずれかに記載のタンパク質構成体の基板表面を３次元パターン加工してなる束化アクチンを作動部とするマイクロアクチュエータ。
＜６＞以下の工程：基板表面を濃度が０．２重量％のコロジオン溶液で処理する工程；この基板表面にミオシンを固定する工程；およびミオシン上に束化アクチンを積載する工程を含むことを特徴とするタンパク質構成体の製造方法。
＜７＞基板がガラス製であることを特徴とする前記＜６＞のタンパク質構成体の製造方法。
＜８＞基板がシリコン製であることを特徴とする前記＜６＞のタンパク質構成体の製造方法。
＜９＞コロジオン溶液は、溶質がピロキシリン、溶媒が酢酸イソアミルであることを特徴とする前記＜６＞のタンパク質構成体の製造方法。
<1> A protein construct, wherein myosin is supported on the surface of a substrate treated with a collodion solution having a concentration of 0.2% by weight , and bundled actin is loaded thereon.
<2> The protein construct according to <1>, wherein the substrate is made of glass.
<3> The protein construct according to <1>, wherein the substrate is made of silicon.
<4> The protein composition according to <1>, wherein the collodion solution has a solute of piroxylin and a solvent of isoamyl acetate.
<5> A microactuator using a bundled actin obtained by processing a three-dimensional pattern on the substrate surface of the protein construct according to any one of <1> to <4> as an operating part.
<6> The following steps: a step of treating the substrate surface with a collodion solution having a concentration of 0.2% by weight; a step of fixing myosin on the surface of the substrate; and a step of loading bundled actin on the myosin A method for producing a characteristic protein construct.
<7> The method for producing a protein construct according to <6>, wherein the substrate is made of glass .
<8> The method for producing a protein construct according to < 6 >, wherein the substrate is made of silicon .
<9> The method for producing a protein construct according to the above < 6 >, wherein the collodion solution is composed of piroxylin as a solute and isoamyl acetate as a solvent .
この出願の発明により束化アクチンの基板上でのin vitro motility assay系、および、マイクロアクチュエータが実現される。 According to the invention of this application, an in vitro motility assay system on a substrate of bundled actin and a microactuator are realized.
この出願の発明において、ガラス基板表面の３次元パターンの作成は収束イオンビーム加工法（などの公知技術を適用できる。収束イオンビーム加工法はガリウムイオンを加速してイオンビームとし、細く絞ってサンプル面に照射し、スパッタリングさせることでサンプル面を加工する方法である。この方法は0.1 μmの精度での加工が可能であり、また、走査型イオン顕微鏡による観察を行いながら加工することが可能であるという特徴をもつ。 In the invention of this application, a known technique such as a focused ion beam processing method (such as the focused ion beam processing method can be applied to create a three-dimensional pattern on the surface of the glass substrate. In the focused ion beam processing method, a gallium ion is accelerated into an ion beam, The sample surface is processed by irradiating and sputtering the surface, which can be processed with an accuracy of 0.1 μm and can be processed while observing with a scanning ion microscope. It has the characteristic of being.
さらに、ガラス基板は、ニトロセルロースによる被覆処理を行う。好ましくはミオシン固定化の前に0.2重量%のコロジオン溶液でガラス基板を処理する。コロジオン溶液は、好ましくは溶質をピロキシリン（ニトロセルロース）、溶媒を酢酸イソアミル（酢酸−3−メチルブチル）とした溶液である。コロジオン溶液をガラス基盤上に直接ピペットで滴下して被覆し、直ちに160℃で乾燥させる。 Further, the glass substrate is coated with nitrocellulose. Preferably, the glass substrate is treated with 0.2% by weight collodion solution before immobilization of myosin. The collodion solution is preferably a solution in which the solute is piroxylin (nitrocellulose) and the solvent is isoamyl acetate (3-methylbutyl acetate). The collodion solution is coated directly onto the glass substrate by pipetting and immediately dried at 160 ° C.
ガラス基板上へのミオシンの固定化は、例えば次の通りとすることができる。すなわちガラス基盤はコロジオン溶液処理後、ワセリンもしくは両面テープをスペーサーとしてスライドグラスに被せ、フローチャンバーを作成する。ここに緩衝液中に希釈したミオシンタンパク質を直接作用させることで固定化する。アクチン繊維のmotility assayはこのチャンバー内にウシ血清アルブミン溶液、蛍光標識アクチン繊維の順で緩衝液を還流し蛍光顕微鏡を用いて行うことができる。観察には、好ましくは高倍率・高開口数（×40倍以上、NA=1以上）の対物レンズを用いる。 Immobilization of myosin on the glass substrate can be performed, for example, as follows. That is, after the glass substrate is treated with the collodion solution, the flow chamber is created by covering the slide glass with petrolatum or double-sided tape as a spacer. Here, the myosin protein diluted in the buffer solution is directly acted to be immobilized. Actin fiber motility assay can be carried out using a fluorescence microscope by refluxing the buffer solution in this order in the order of bovine serum albumin solution and fluorescently labeled actin fiber. For observation, an objective lens having a high magnification and a high numerical aperture (× 40 times or more, NA = 1 or more) is preferably used.
いっぽう、シリコン基板表面の３次元パターンの作成は反応性イオンエッチング加工法などの半導体作成において適用される公知技術を用いることができる。反応性イオンエッチング加工は、装置に導入したガスに高周波電力（例えば13.56MHzが一般的である）を印加してプラズマ状態とし、そこで生じた＋イオンを加速して基板に衝突させ、物理化学的反応を介して基板表面に溝を彫るエッチング反応を促進させる方法である。ガスの圧力を数Pa以下にすると、イオンの運動方向が揃うので、基板に対して垂直方向での加工が可能である。エッチング反応を生じさせるには、被切削物質とガスが反応して揮発性物質が生成することが必須である。従って導入ガスは、好ましくは基板材料と反応しやすく、かつ、揮発性物質を生じやすいフッ素、塩素などのハロゲンを含む化合物を用いることができる。 On the other hand, for the creation of a three-dimensional pattern on the surface of a silicon substrate, a known technique applied in semiconductor production such as a reactive ion etching method can be used. In reactive ion etching processing, high-frequency power (for example, 13.56 MHz is common) is applied to the gas introduced into the apparatus to create a plasma state, and the + ions generated there are accelerated and collided with the substrate. This is a method of promoting an etching reaction in which a groove is carved on the substrate surface through the reaction. When the gas pressure is set to a few Pa or less, the movement direction of ions is aligned, so that processing in a direction perpendicular to the substrate is possible. In order to cause an etching reaction, it is essential that a material to be cut and a gas react to generate a volatile material. Therefore, as the introduced gas, a compound containing a halogen such as fluorine or chlorine which can easily react with the substrate material and easily generate a volatile substance can be used.
さらに、シリコン基板は、ミオシン固定化の前に、ニトロセルロースによる被覆処理を行う。好ましくは、シリコン基板を十分量のコロジオン溶液で表面処理する。好ましくは0.02重量%以上、より好ましくは0.2重量％のコロジオン溶液で処理を行うが、コロジオン溶液がシリコン基板表面を処理するのに十分な濃度であればよい。コロジオン溶液は、好ましくは溶質をピロキシリン（ニトロセルロース）、溶媒を酢酸イソアミル（酢酸−3−メチルブチル）とした溶液である。コロジオン溶液をシリコン基板上に直接ピペットで滴下して被覆させ、直ちに160℃で乾燥させる。 Furthermore, the silicon substrate is coated with nitrocellulose before immobilization of myosin. Preferably, the silicon substrate is surface-treated with a sufficient amount of collodion solution. The treatment is preferably carried out with 0.02% by weight or more, more preferably 0.2% by weight collodion solution, but the concentration of the collodion solution may be sufficient to treat the silicon substrate surface. The collodion solution is preferably a solution in which the solute is piroxylin (nitrocellulose) and the solvent is isoamyl acetate (3-methylbutyl acetate). The collodion solution is coated directly onto the silicon substrate by pipetting and immediately dried at 160 ° C.
シリコン基板へのミオシン固定化は、例えば次の通りとすることができる。すなわち、シリコン基板はコロジオン処理後、ワセリンもしくは両面テープをスペーサーにしてカバーガラスを被せ、フローチャンバーを作成し、ここに緩衝液中に希釈したミオシンタンパク質を直接作用させることで固定化する。アクチン繊維のmotility assayはこのチャンバー内にウシ血清アルブミン溶液、蛍光標識化アクチン繊維の順番で緩衝液を還流し、蛍光顕微鏡を用いて行う。観察には、高倍率・高開口数（×40倍以上、NA＝１以上）の対物レンズを用いる。 Immobilization of myosin on the silicon substrate can be performed as follows, for example. That is, after collodion treatment, the silicon substrate is covered with a cover glass using petrolatum or double-sided tape as a spacer, a flow chamber is created, and the myosin protein diluted in the buffer solution is directly immobilized on the silicon substrate. Actin fiber motility assay is carried out using a fluorescence microscope by refluxing a buffer solution in this order in the order of bovine serum albumin solution and fluorescently labeled actin fiber. For observation, an objective lens having a high magnification and a high numerical aperture (× 40 times or more, NA = 1 or more) is used.
この出願の発明のミオシンおよびアクチンとしては、任意のアクチン、ミオシンを用いることができる。好適なものとしては、ミオシンとしては、例えばウサギ背筋より精製した骨格筋ミオシンIIや特開2004-57152号公報で公知の組換えミオシンを用いることができる。ミオシンは全長ミオシンに限らず、HMM、S1等の活性部位のみを用いてもよい。 Arbitrary actin and myosin can be used as myosin and actin of the invention of this application. As myosin, for example, skeletal muscle myosin II purified from rabbit back muscles or recombinant myosin known in JP-A-2004-57152 can be used. Myosin is not limited to full-length myosin, and only active sites such as HMM and S1 may be used.
アクチンタンパク質としては、例えばウサギ背筋あるいはニワトリ胸筋より精製したアクチンを用いることができる。より好ましくはJ.Neurochem:87 676-685 (2003)で公知の収束化されたアクチン繊維を用いる。アクチン繊維の収束化は好ましくはファシン、より好ましくは脳由来のファッシンによって収束化されたものを用いる。精製アクチンはローダミン−ファイロジンを用いてファイバーの安定化と蛍光標識を行うことができる。 As actin protein, for example, actin purified from rabbit back muscle or chicken breast muscle can be used. More preferably, a converged actin fiber known in J. Neurochem: 87 676-685 (2003) is used. Actin fibers are preferably converged using fascin, more preferably converged with brain-derived fascin. Purified actin can be stabilized and fluorescently labeled with rhodamine-phyllodine.
Ca2+活性型ミオシンとCa2+抑制ミオシンを用いてCa2+濃度により動きを制御できるmotility assayを実現することが可能である。この場合、Ca2+活性型ミオシンとCa2+抑制ミオシンを濃度勾配を作ってシリコン基盤に固定化する。カルシウムイオン濃度を高くするとCa2+活性型ミオシンの並んだ側に、カルシウムイオン濃度を低くするとCa2+抑制ミオシンの並んだ側にアクチン繊維が動く。 It is possible to realize a motility assay that can control movement by Ca 2+ concentration using Ca 2+ activated myosin and Ca 2+ -suppressed myosin. In this case, Ca 2+ activated myosin and Ca 2 + -suppressed myosin are immobilized on a silicon substrate by creating a concentration gradient. When the calcium ion concentration is increased, the actin fiber moves to the side where the Ca 2+ activated myosin is arranged, and when the calcium ion concentration is lowered, the actin fiber is moved to the side where the Ca 2+ inhibitory myosin is arranged.
さらに、アクチン繊維をマイクロアクチュエーターとして使用するには上記の方法を用いて、シリコン基板上に構築した溝の中で束化アクチンの繊維の運動を行わせる。その際、束化クチン繊維の進行方向端に、溝の深さと幅に応じた大きさの栓を置く。この栓は束化アクチン繊維に押されて運動しマイクロアクチュエータとして機能する。この例では出力は直線運動であるが、栓を加工し歯車を組み合わせることで、ピニオン−ラックの原理で出力を回転運動にしてもよい。 Further, in order to use actin fibers as a microactuator, the above-described method is used to cause movement of bundled actin fibers in a groove constructed on a silicon substrate. At that time, a plug having a size corresponding to the depth and width of the groove is placed at the end of the bundled cutin fiber in the traveling direction. This stopper moves when pushed by bundled actin fibers and functions as a microactuator. In this example, the output is a linear motion, but the output may be a rotational motion based on the pinion-rack principle by processing a stopper and combining gears.
アクチン繊維をスイッチングデバイスとして使用するには、上記のカルシウム濃度により動きを制御できるmotility assay系で、カルシウム濃度の変化（入力）に伴うアクチン繊維の運動（出力）を蛍光としてCCDカメラを用いて検出することにより行うことができる。 In order to use actin fibers as a switching device, the movement (output) of actin fibers accompanying changes in calcium concentration (input) is detected as fluorescence using a CCD camera in the motility assay system that can control movement by the calcium concentration described above. This can be done.
以下に実施例を用いてこの出願の発明をさらに詳細に説明するが、下記実施例はこの出願の発明の一態様にすぎず、この出願の発明の実施が下記実施例に制限されるものではない。 The invention of this application will be described in more detail below using examples, but the following examples are only one aspect of the invention of this application, and the implementation of the invention of this application is not limited to the following examples. Absent.
〔実施例１〕ガラス基板上３次元パターンでの束化アクチンのmotility assay
ガラス基板上に収束イオンビーム加工法を用いて3次元パターンを作成した。ガラス基盤を0.2重量%のコロジオン溶液で処理し、ニトロセルロースで被覆した。ミオシンHMMはウサギ骨格筋より精製したものを使用した。アクチンはニワトリ骨格筋より精製し、脳由来のファシン存在下でローダミン−ファロイジンを作用させ束化アクチン繊維として使用した。in vitro motility assayは定法に従い、蛍光顕微鏡（Zeiss Axiovert 100、対物レンズ×100、NA＝1.3）により行った。この結果を図１に示す。束化アクチン繊維を3次元パターンに沿って運動させることができた。
〔実施例２〕シリコン基板上でのアクチンのmotility assay
アクチン−ミオシンを用いたin vitro motility assayをシリコン基板上で試みた。シリコン基板は半導体用単結晶シリコンを使用した。シリコン基板は酸化膜の除去処理を行ったものと未処理のものそれぞれについて、0.2％コロジオン溶液で１分間コロジオンを表面に被覆する処理を行ったものと未処理のもの、計４通りを使用した。ミオシンはウサギ骨格筋より調製した。アクチンはニワトリ骨格筋由来より調製し、ローダミン−ファロイジンで蛍光ラベルを施したものを使用した。in vitro motility assayは定法に従い、実施例１と同様に蛍光顕微鏡により行った。この結果、酸化膜の除去処理の如何にかかわらず、コロジオンで被覆された基板ではシリコン基板上でアクチンの動きを観察することができた。例えば、図２に示す通り、時間の経過とともにアクチン繊維が移動している様子が確認できた。
〔実施例３〕シリコン基板上での束化アクチンのmotility assay
上記実施例２の手法でシリコン基板上で脳由来ファシンで束化したアクチン繊維のmotility assayを行った。この結果、図３に示す通り、コロジオンで被覆されたシリコン基板上で束化アクチンの運動を観察することができた。
[Example 1] motility assay of bundled actin in a three-dimensional pattern on a glass substrate
A three-dimensional pattern was created on a glass substrate using focused ion beam processing. The glass substrate was treated with 0.2 wt% collodion solution and coated with nitrocellulose. Myosin HMM was purified from rabbit skeletal muscle. Actin was purified from chicken skeletal muscle and used as bundled actin fibers by the action of rhodamine-phalloidin in the presence of brain-derived fascin. The in vitro motility assay was performed by a fluorescence microscope (Zeiss Axiovert 100, objective lens × 100, NA = 1.3) according to a conventional method. The result is shown in FIG. The bundled actin fibers could be moved along a 3D pattern.
[Example 2] Actin motility assay on a silicon substrate
An in vitro motility assay using actin-myosin was attempted on a silicon substrate. As the silicon substrate, single crystal silicon for semiconductor was used. For the silicon substrates that had been subjected to the removal treatment of the oxide film and the untreated ones, a total of four types were used, one that had a treatment with a 0.2% collodion solution coated with collodion for 1 minute and the other that had not been treated. . Myosin was prepared from rabbit skeletal muscle. Actin was prepared from chicken skeletal muscle and was fluorescently labeled with rhodamine-phalloidin. The in vitro motility assay was performed by a fluorescence microscope in the same manner as in Example 1 according to a conventional method. As a result, it was possible to observe the movement of actin on the silicon substrate in the substrate coated with collodion regardless of the oxide film removal treatment. For example, as shown in FIG. 2, it was confirmed that the actin fibers were moving over time.
[Example 3] Motility assay of bundled actin on a silicon substrate
A motility assay of actin fibers bundled with brain-derived fascin on a silicon substrate by the method of Example 2 was performed. As a result, as shown in FIG. 3, the movement of bundled actin could be observed on the silicon substrate coated with collodion.
Claims (9)
基板表面を濃度が０．２重量％のコロジオン溶液で処理する工程；Treating the substrate surface with a collodion solution having a concentration of 0.2% by weight;
この基板表面にミオシンを固定する工程；およびImmobilizing myosin on the substrate surface; and
ミオシン上に束化アクチンを積載する工程Loading bundled actin on myosin
を含むことを特徴とするタンパク質構成体の製造方法。A method for producing a protein construct, comprising:
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| PCT/JP2006/303091 WO2006088214A1 (en) | 2005-02-15 | 2006-02-15 | Mictoactuator using bundled actin filaments |
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