JP2005534320A - 細胞中へのタンパク質送達のための方法および器具 - Google Patents
細胞中へのタンパク質送達のための方法および器具 Download PDFInfo
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Abstract
Description
図1の略図に示すように、本方法は最も簡単な形態において以下の工程を含む;タンパク質含有混合物を表面上に沈着させ、表面上に十分な密度でおよびタンパク質含有混合物中のタンパク質を細胞に送達するために適当な条件下で細胞を播種する。たとえば、混合物で被覆された表面上で哺乳類細胞を培養する。細胞はタンパク質を取り込み、それは細胞機能または観察可能な発現に影響を及ぼし得る。一種類以上の目的タンパク質を含む溶液または混合物を表面上に置いて付着させ、その後の細胞培養およびトランスフェクションに用いることができる。
図2に図解される通り、本方法の一つの拡張では、目的タンパク質の組を含む溶液または混合物を表面上の定められた別々の位置に配列し、規定の範囲の付着細胞を有する、細胞の直線マイクロアレイまたはクラスターアレイを作製することができる。そのようなマイクロアレイは、機能または表現型スクリーニング、または他の高性能用途といった、プロテオーム研究に用いることができる。
本発明における逆タンパク質送達に用いるタンパク質を作製するために、さまざまな従来の手段を用いることができる。たとえば、タンパク質は天然起源から得ることができ、または、必要に応じて、組み換えDNA法を用いて過剰発現させることができる。タンパク質は従来の方法を用いて精製することができ、または未精製のままでもよい。多数のタンパク質が市販されており、本発明に用いることができる。
本発明の特定の実施形態はキャリヤー試薬に関して記載されている。キャリヤー試薬はさまざまな種を含むことができる。一実施形態では、キャリヤー試薬は生物活性細胞膜透過性試薬,またはタンパク質−導入ドメイン(PTDs)(すなわち、約15ないし約30残基を含む単一のペプチド配列)を含むペプチドである。タンパク質−導入ドメイン(PTDs)はタンパク質分泌を媒介し、正に荷電したアミノ末端、中心の疎水性核、およびシグナルペプチダーゼによって認識されるカルボキシル末端切断部位から構成される。そのような膜導入ペプチドの例は、Trojanペプチド、ヒト免疫不全ウイルス(HIV)−1転写活性化因子(TAT)タンパク質またはその機能ドメインペプチド、および、Drosophiliaホメオティック転写因子Antemlapedia(Antp)および単純ヘルペスウイルスDNA結合タンパク質、VP22、などのような輸送タンパク(translocationprotein)質に由来するタンパク質−導入ドメイン(PTDs)を含む他のペプチドを含む。一部の市販のペプチド、たとえば、ペネトラチン1、Pep−1(Chariot reagent, Active Motif Inc., カリフォルニア州)およびHIV GP41 断片(519−541)を用いることができる。
本発明の特定の実施形態は補助試薬に関して記載される。一実施形態では、補助試薬はDEAE−デキストラン、デキストラン、ポリリジン、およびポリエチルアミンのようなポリマーである。他の一実施形態では、補助試薬はまた、フィブロネクチンおよびゼラチンのような細胞接着促進タンパク質であってよい。補助試薬は糖を基礎とするゼラチン(たとえば、ポリエチレングリコール)または、アクリルアミドのような合成のまたは化学品を基礎とするゼラチンであってよい。別の一実施形態では、補助試薬はArg−Gly−Asp−Ser、Arg−Gly−Asp−Ser−Pro−Ala−Ser−Ser−Lys−Pro、などといったRGDペプチドであってもよい。あるいは、補助試薬はハイドロゲルおよびRGDペプチドの混合物、および前述の分子の任意のものの混合物である。補助試薬の使用は細胞へのタンパク質送達の効率を高める。
本発明で用いられるアレイ用の基材は、プローブスポット(タンパク質含有混合物マイクロスポット)のパターンを付着することができる少なくとも1つの表面を含む。表面は中空でないかまたは多孔質であってよい。また、表面は滑らかで実質的に平面であるか、あるいは、窪みまたは隆起といった不規則性を有する。スポットのパターンが存在する表面は、表面化学的性質を望ましい方法で修飾する1つ以上の異なる層の化合物で修飾されていてよい。たとえば、タンパク質含有混合物の結合を促進することができる一方、タンパク質含有混合物マイクロスポット上に重ねられた細胞にタンパク質がトランスフェクションされるのを可能にする化学分子で表面を被覆できる。たとえば、γ−アミノプロピルシラン(GAPS)またはポリリジンの被覆をガラス表面に塗布することができる。
本発明のためのアレイは複数のマイクロパターン技術を用いて作製することができる。一実施形態では、機械の突起すなわちプローブの先端(「ピン」ともいう)をタンパク質含有混合物の溶液に浸す。先端を、先端に付着したある量の溶液と共に、溶液から取り出す。濡れた先端を基材の表面に接触させ、それによって溶液を先端から表面に移す。
プリント装置はプリントヘッド、プレート、基材取り扱い部、XYまたはXYZ位置調整ステージ、環境制御部、機器制御ソフトウェア、試料追跡ソフトウェア、などを含み得る。そのような装置はたとえば、Cartesian Technologies, Inc.から入手可能な管状ピンプリンターを含む。
高密度アレイには、プローブの行列を有するプリント用プローブアレイを用いて、行列からの各プローブを対応する原料ウェル(たとえばマイクロタイタープレートからのウェル)に適合するように配列することができる。
タンパク質含有混合物の複数のマイクロスポットを含むアレイを有する基材が一旦作製されると、十分な密度でおよびタンパク質の細胞への導入のために適当な条件下で細胞を基材表面上に播種するか、それとも置く。各位置でタンパク質を取り込んだ、すなわち、タンパク質をトランスフェクションされた生きた細胞のクラスターがアレイを覆う。前述の通り、本発明は、哺乳類細胞(たとえば、ヒト、サル、マウス、など)いった真核細胞、細菌細胞、昆虫細胞、植物細胞を含むさまざまな細胞に適用することができる。好ましくは、細胞(血清培地または無血清培地中の)は、逆トランスフェクションが起こる可能性を高めるため、乾燥したタンパク質を含むスポット上に高密度で(たとえば、0.5〜1x105/cm2)播種する。あるいは、細胞の密度は約0.3x104/cm2から約5x105/cm2まで;または、他の実施形態では、約2x105/cm2から約3x105/cm2まで;または、約0.5x104/cm2から約2x105/cm2まで変動し得る。
材料
使用した材料は下記のものを含む;β−ガラクトシダーゼ(グレードVIII、E.Coliから精製)、RGDペプチドgly−arg−gly−asp−ser、およびgly−arg−gly−asp−ser−pro−lys)、デキストラン(M.W.45000Da)、およびDEAE−デキストラン(M.W.40000Da)(Sigma Chemical社(ミズーリ州St. Louis));Chariot(Pep−1)(Active Motif Inc社、カリフォルニア州Carlsbad); およびHIV GP−41 断片 (519−541)(Bachem社(ペンシルバニア州King of Prussia));およびγ−アミノプロピルシラン(GAPS)スライドはCorning Inc社から入手した(品番2550)(ニューヨーク州Corning)。細胞培地はGibco社から入手した。β−ガラクトシダーゼ染色キットはQiagen社から入手した。他の化学薬品はSigma社から入手した。
ストック溶液調製品
10mM PBS緩衝液(pH7.4)に、β−ガラクトシダーゼを溶解して濃度0.25mg/mlとし、4℃にて保存した。輸送ペプチド(すなわち、キャリヤーペプチド)のChariotまたはGP41断片を、60%DMSOに溶解して濃度2mg/mlとし、使用まで−20℃にて保存した。デキストランまたはDEAE−デキストランはddH2Oに溶解して濃度2%とし、4℃にて保存した。RGDペプチドはPBS緩衝液に溶解して濃度1mg/mlとし使用まで−20℃にて保存した。デキストラン、DEAE−デキストランおよびRGDペプチドは、送達効率を高めるための補助試薬として使用した。
β−ガラクトシダーゼストック溶液をPBS緩衝液で希釈し、タンパク質の終濃度0.1mg/mlとした。ペプチドストック溶液をPBS緩衝液で希釈して、終濃度0.4mg/mlとし、自己凝集を避けるため超音波処理に供した。2つの希釈した溶液を、等しい容量を用いて静かに混合した。補助試薬もまた混合物に添加することができる(これらの補助試薬について最適化された濃度は、デキストランおよびDEAE−デキストランについて1mg/ml、RGDペプチドについて0.lmg/mlである)。結果として生じた混合物を室温にて約30分間ないし1時間インキュベートした。
γ−アミノ−プロピルシラン(Corning Inc.社、ニューヨーク州Coming)で被覆したスライド表面上に、それぞれ1つの格子内に5個の複製スポットを有する4つの別々の格子中に20個のスポットを、5μlの混合物溶液を用いて作製し、室温にて1時間乾燥させた。
タンパク質を含むスポットを有するスライドは、一部の場合には、4℃にて窒素中で数日間ないし数週間保存され、タンパク質トランスフェクション効率またはトランスフェクションされた細胞内でのタンパク質の機能性に観察可能な損失は無かった。
10%ウシ胎仔血清添加Iscove改変Dulbecco培地(IMDM)中で培養された、ヒト胚性腎(HEK)293T細胞およびCHO細胞。
組織培養フード内で、約0.5〜2x105個の細胞(CHOまたはHEK)を、分離したタンパク質スポットを含む各格子の上に播種したか、それとも置いた。細胞懸濁液は血清を含んだかまたは無血清であった。トランスフェクションのために、細胞を接着させ5%CO2および湿度95%下で3時間生育させた。
培養後、PBS緩衝液を用いてスライドをごく穏やかに2回洗浄し、次いで4%ホルムアルデヒド/PBS溶液中で3分間固定し、再びPBS緩衝液を用いて2回洗浄した。
取扱説明書の標準的手順に従って、β−ガラクトシダーゼ染色キット(Invitrogen社)からの試薬溶液を固定した細胞に添加し、細胞と30分間37℃にてインキュベートした。
光学顕微鏡を用いて、青色に染色された細胞の割合を計数し、β−ガラクトシダーゼを用いたタンパク質トランスフェクション効率を計算した。染色された細胞は、染色溶液を捨てて70%グリセロールを重層した後に保存することができた。
Claims (10)
- タンパク質をトランスフェクションされた細胞のアレイを表面上に作製する方法であって、タンパク質含有混合物を、不連続な定められた配置で表面上にスポットし;スポットがアレイ条件下で表面に付着したままであるように、タンパク質含有混合物を有する前記表面を乾燥し;細胞を添加して、タンパク質と細胞の両方を有する表面を作製し;さらに前記タンパク質を前記細胞中に輸送する:工程を含む方法。
- タンパク質の細胞への進入を促進する条件下に前記表面を維持し、前記タンパク質を含む細胞のアレイを作製することをさらに含む、請求項1記載の方法。
- タンパク質含有混合物を提供し;前記タンパク質含有混合物を定められた配置で表面上に置き;前記タンパク質含有混合物を前記表面に付着させ;さらに細胞を表面上に播種する:工程を含む方法によって作製され、前記タンパク質含有混合物が:a)目的タンパク質;b)キャリヤー試薬と複合体を形成した目的タンパク質;c)キャリヤー試薬と目的タンパク質との複合体;または(b)および(c)の混合物の溶液であることを特徴とするアレイ。
- 基材表面上にDNAテンプレートを提供し;表面上で前記DNAテンプレートからタンパク質をin situで合成し; 前記タンパク質を前記表面に付着させ;前記合成されたタンパク質をキャリヤー試薬と反応させ;細胞を前記表面に添加し;さらに前記タンパク質を前記細胞中に輸送する:工程を含む表面媒介性タンパク質送達法によって作製された、トランスフェクションされた細胞クラスターアレイ。
- 前記タンパク質合成工程が、無細胞反応として行われることを特徴とする請求項4記載のアレイ。
- 目的タンパク質を初代細胞株に送達する方法であって、細胞の第1の集団を提供し;前記細胞の第1の集団に、前記目的タンパク質のDNA構築物を導入し;前記細胞の第1の集団を表面上に置き;前記細胞の第1の集団をin situで溶解するか、または、前記目的タンパク質を前記細胞の第1の集団に分泌させ;前記目的タンパク質を前記表面上に捕捉し;前記目的タンパク質をキャリヤー試薬と反応させ;細胞の第2の集団を前記目的タンパク質上に置き;さらに、前記目的タンパク質を前記細胞の第2の集団に輸送する;工程を含む方法。
- 前記目的タンパク質が、生物活性ペプチド、タンパク質ドメイン、細胞内タンパク質、酵素、細胞表面タンパク質、毒素タンパク質、抗体、抗体−核酸複合体、タンパク質−核酸複合体、ペプチド−核酸複合体、タンパク質−ナノ粒子複合体、タンパク質−ポリマー複合体、タンパク質−有機化合物またはタンパク質−無機化合物間の複合体、複数タンパク質複合体、または任意のアミノ酸含有物質を含むことを特徴とする、上記請求項のいずれか1つに記載の発明。
- 前記目的タンパク質が、移送ドメインおよび目的タンパク質ドメインを含む融合タンパク質を含むことを特徴とする上記請求項のいずれか1つに記載の発明。
- 前記キャリヤー試薬が、天然または合成の輸送ペプチド、またはリポソームであるか、あるいは、生物活性細胞膜透過性試薬;細胞膜輸送ペプチド;タンパク質導入ドメイン(PTDs)を含むペプチド;シグナル配列;生物活性ペプチド;または細胞表面受容体に特異的に結合しそれを活性化することができるリガンド;を含むことを特徴とする上記請求項のいずれか1つに記載の発明。
- 前記タンパク質含有混合物が補助試薬をさらに含むことを特徴とする上記請求項のいずれか1つに記載の発明。
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US10/208,894 US7105347B2 (en) | 2002-07-30 | 2002-07-30 | Method and device for protein delivery into cells |
PCT/US2003/023879 WO2004012658A2 (en) | 2002-07-30 | 2003-07-30 | Method and device for protein delivery into cells |
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EP (1) | EP1525473B1 (ja) |
JP (1) | JP4479960B2 (ja) |
CA (1) | CA2493745A1 (ja) |
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EP1525473B1 (en) | 2009-03-18 |
CA2493745A1 (en) | 2004-02-12 |
EP1525473A2 (en) | 2005-04-27 |
WO2004012658A3 (en) | 2004-04-08 |
US20040023391A1 (en) | 2004-02-05 |
US8288113B2 (en) | 2012-10-16 |
US20060105371A1 (en) | 2006-05-18 |
DE60326730D1 (de) | 2009-04-30 |
JP4479960B2 (ja) | 2010-06-09 |
US7105347B2 (en) | 2006-09-12 |
WO2004012658A2 (en) | 2004-02-12 |
US20120088695A1 (en) | 2012-04-12 |
US7829290B2 (en) | 2010-11-09 |
US8293484B2 (en) | 2012-10-23 |
US20120083428A1 (en) | 2012-04-05 |
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