JP3854995B2 - Cell-permeable carrier peptide - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、細胞透過性キャリアペプチド及び該キャリアペプチドと異種ポリペプチド、ＤＮＡ及び糖からなる群から選ばれるいずれかを連結してなるキャリアペプチドコンジュゲートに関する。また、本発明は、ポリペプチドないしタンパク質、さらにはＤＮＡや糖を細胞内に導入するための細胞透過性キャリアペプチドの使用に関する。
【０００２】
【従来の技術】
近年、合成ペプチドやタンパク質、さらにはＤＮＡや糖を細胞内に導入し、細胞内でのタンパク質相互作用を調節することにより細胞内情報伝達や転写などに関する情報を得ようとする試みがなされている。このアプローチにより、標的タンパクをコードした遺伝子を導入する方法によっては困難なペプチド導入量の調節や、非天然アミノ酸骨格を分子内に有するポリペプチドの細胞内導入が期待できる。
【０００３】
一般にポリペプチド、核酸、糖は親水性が高く、細胞膜を通過することは困難であり、ポリペプチド、核酸、糖の細胞内導入には、マイクロインジェクションの他、脂肪酸などの脂溶性分子とのハイブリッド化による方法やリポソームを用いる方法が従来採られてきた。
【０００４】
これらの方法とは別に、近年、疎水性のシグナルペプチド（Lin Y.Z., et al. (1995) J. Biol. Chem., 270, 14255-14258; Rojas M., et al. (1998) Nat. Biotechnol., 16, 370-375)、或いはHIV-1 Tat タンパク質やショウジョウバエAntennapediaタンパク質由来の塩基性ペプチドを用いてペプチド・タンパク質を導入する試みが知られている（特開平１０−３３１８６号公報、Derossi D. et al. (1998) Trends. Cell Biol., 8, 84-87; Fawell S., et al. (1994) Proc. Acad. Natl. Sci., USA, 91, 664-668; Nagahara H. et al. (1998) Nat. Med., 4, 1449-1452)。
【０００５】
しかしながら、これら従来技術の試みでは、ペプチドないしタンパク質、さらにはＤＮＡや糖を十分に細胞内へ導入することができなかった。
【０００６】
【発明が解決しようとする課題】
本発明の目的は、効率よく細胞内にポリペプチドないしタンパク質、ＤＮＡ、糖を導入できる細胞透過性キャリアペプチド、該キャリアペプチドとポリペプチド、さらにはＤＮＡや糖を結合したキャリアペプチドコンジュゲートを提供することにある。
【０００７】
また、本発明の目的は、ポリペプチド、さらにはＤＮＡや糖を細胞内に導入するための細胞透過性キャリアペプチドの使用を提供することである。
【０００８】
【課題を解決するための手段】
本発明は、以下の（ａ）または（ｂ）のポリペプチドを含む細胞透過性キャリアペプチド。
（ａ）配列番号２〜１４のいずれかで表されるポリペプチド；
（ｂ）ポリペプチド(ａ)において１又は数個のアミノ酸が置換、欠失又は付加したポリペプチドを含み、細胞透過性キャリアペプチド活性を有するポリペプチドに関する。
【０００９】
また、本発明は、前記（ａ）または（ｂ）のポリペプチドを含む細胞透過性キャリアペプチドと異種ポリペプチド、ＤＮＡ及び糖からなる群から選ばれるいずれかを、必要に応じて架橋剤を介して連結してなるキャリアペプチドコンジュゲートに関する。
【００１０】
さらに、本発明は、前記細胞透過性キャリアペプチドの、異種ポリペプチド、ＤＮＡ及び糖からなる群から選ばれるいずれかを細胞内に導入するための使用に関する。
【００１１】
【発明の実施の形態】
本発明の細胞透過性キャリアペプチドとしては、配列番号２〜１４のいずれかで表されるポリペプチドが挙げられ、該配列は、細胞透過性キャリアペプチド活性を保持する限りにおいて１又は数個のアミノ酸が置換、欠失又は付加したポリペプチドを含む。
【００１２】
「アミノ酸の欠失、置換又は付加」の程度及びそれらの位置などは、改変されたポリペプチドが、細胞透過性キャリアペプチド活性を有する限り特に制限されない。本発明において「細胞透過性キャリアペプチド活性」とは、配列番号２〜１４のポリペプチドと結合したタンパク質ないしポリペプチドが、細胞膜を透過し、細胞内に導入されることを意味する。
【００１３】
置換・付加・欠失の具体的手段としては、該キャリヤペプチドをコードするＤＮＡを経由して行う場合には、例えばサイトスペシフィック・ミュータゲネシス〔Methods in Enzymology, 154, 350, 367-382 (1987)；同 100, 468 (1983)；Nucleic Acids Res., 12, 9441 (1984)；続生化学実験講座１「遺伝子研究法II」、日本生化学会編, p105 (1986)〕などの遺伝子工学的手法、リン酸トリエステル法やリン酸アミダイト法などの化学合成手段〔J. Am. Chem. Soc., 89, 4801 (1967)；同 91, 3350 (1969)；Science, 150, 178 (1968)；Tetrahedron Lett., 22, 1859 (1981)；同 24, 245 (1983)〕及びそれらの組合せ方法などが例示できる。より具体的には、ＤＮＡの合成は、ホスホルアミダイト法またはトリエステル法による化学合成によることもでき、市販されている自動オリゴヌクレオチド合成装置上で行うこともできる。二本鎖断片は、相補鎖を合成し、適当な条件下で該鎖を共にアニーリングさせるか、または適当なプライマー配列と共にＤＮＡポリメラーゼを用い相補鎖を付加するかによって、化学合成した一本鎖生成物から得ることもできる。さらに、本発明の細胞透過性キャリアペプチドは、ペプチド合成機を用いて固相合成法により合成することもでき、置換・付加・欠失は、ペプチド合成機を用いる場合には保護アミノ酸の種類を変えることにより容易に行うことができる。又、Ｄ−アミノ酸やサルコシン（N-メチルグリシン）等の特殊なアミノ酸を導入することもできる。
【００１４】
本発明の細胞透過性キャリアペプチドに連結されて細胞内に導入されるポリペプチドは特に限定されず、任意のポリペプチドが挙げられる。該ポリペプチドの分子量は５００程度から１００万程度、好ましくは１０００〜５０万程度が例示され、分泌タンパク、膜結合タンパク、ペプチドホルモンなどの種類も問わない。
【００１５】
細胞内に導入されるポリペプチドの具体例としては、カルボニックアンヒドラーゼ、ミオグロビン、西洋わさびパーオキシダーゼ、β−ガラクトシダーゼ、ロイシンジッパーや亜鉛フィンガーモチーフを有する転写因子、Fas p53などのアポトーシス誘導タンパク質、欠損により代謝異常の疾病を誘発するアデノシンデアミナーゼなどのタンパク質及び酵素阻害剤（例えばカルパインインヒビター）、遺伝情報発現調節因子（例えばＩκＢ）、ペプチドホルモン（インスリン、カルシトニン等）などが例示される。
【００１６】
細胞内に導入されるポリペプチドと本発明の細胞透過性キャリアペプチドは、細胞透過性キャリアペプチドがシステイン残基を有する場合には、ポリペプチドのシステイン残基と−ＳＳ−結合を介して連結してもよく、適当な架橋剤を介して連結してもよい。また、本発明の細胞透過性キャリアペプチドをコードするポリヌクレオチドと導入されるポリペプチドをコードするポリヌクレオチド（遺伝子）を、好ましくは直接に結合し、ベクターに導入し、大腸菌等の宿主細胞内で発現させるなどの常法により、本発明の細胞透過性キャリアペプチドのＣ末端側に導入されるタンパク質ないしポリペプチドが直接連結されたペプチドコンジュゲートを得ることができる。同様に、ＤＮＡや糖についても、適当な架橋剤を介して連結することができる。
【００１７】
架橋剤としては、本発明の細胞透過性キャリアペプチドと導入されるタンパク質ないしポリペプチド、ＤＮＡまたは糖を結合できる少なくとも２価の架橋剤であれば特に限定されないが、例えばN-(6-マレイミドカプロイルオキシ)コハク酸イミドエステル(EMCS)などが挙げられる。
【００１８】
本発明の細胞透過性キャリアペプチドのＣ末端側には、例えばCys、Gly-Cysなどの様式でCys残基をさらに結合するのが好ましい。該Cys残基のＳＨ基は、EMCSのマレイミド基に付加反応させたり、導入されるタンパク質ないしポリペプチドがフリーのＳＨ基を有する場合には−ＳＳ−結合により導入されるタンパク質ないしポリペプチドに、連結することができる。−ＳＳ−結合を介して連結した場合には、細胞内で該−ＳＳ−結合が還元され、非修飾のタンパク質ないしポリペプチドが遊離されるので好ましい。
【００１９】
【発明の効果】
本発明によれば、単独で投与した場合に細胞内に移行し難いポリペプチド、ＤＮＡ、糖を高効率で細胞内に導入することができる。
【００２０】
細胞内に導入されて生物活性を示すポリペプチド、ＤＮＡ、糖などの生理活性物質を細胞内に導入することにより、各種の疾患の治療が期待できる。
【００２１】
【実施例】
以下、実施例についてさらに詳しく説明するが、本発明はこれらの実施例になんら限定されるものではない。
実施例１〜１３
配列番号２〜１４で表されるポリペプチドのＣ末端にGly-Cys-アミドが付加されたポリペプチドを、島津PSSM-8型ペプチド合成機、Rinkアミド樹脂を用いてFmoc固相合成法で合成した。得られたポリペプチドは、各々トリフルオロ酢酸−エタンジチオール（９５：５）で室温１．５時間処理し、脱保護と樹脂からの切り出しを行い、Waters社Symmetry 300 C18（5μm、4.6×150mm)を用い、0.1%TFAを含有するアセトニトリル−水グラジエント（アセトニトリル濃度５％〜５０％）で溶出する逆相ＨＰＬＣにより精製した。さらに、該ポリペプチドのCys残基を1.5当量の5-マレイミドフルオレセインジアセテートで蛍光標識して得られたポリペプチドについて質量分析（MALDI-TOF-MS(Shimadzu MALDI-2))を行った。結果を表１に示す。
実施例１４、１５
配列番号２，３で表されるポリペプチドのＣ末端にGly-Cys-アミドが付加されたポリペプチドを、実施例１〜１３と同様にして固相合成し、逆相ＨＰＬＣで精製した。得られたポリペプチドの質量分析結果を表１に示す。なお、実施例１４で得られたポリペプチドは、フルオレセインで蛍光標識されていない点を除いて、実施例１で得られたポリペプチドと同一である。同様に、実施例１５で得られたポリペプチドは、フルオレセインで蛍光標識されていない点を除いて、実施例２で得られたポリペプチドと同一である。
比較例１
配列番号１で表されるポリペプチドのＣ末端にGly-Cys-アミドが付加されたポリペプチドを実施例１〜１３と同様にして合成した。得られた蛍光ラベルされたポリペプチドの質量分析結果を表１に示す。
比較例２
配列番号１５で表されるポリペプチドのＣ末端にGly-Cys-アミドが付加されたポリペプチドを実施例１〜１３と同様にして合成した。得られた蛍光ラベルされたポリペプチドの質量分析結果を表１に示す。
【００２２】
【表１】
表１において、Human cFos-(139-164)の配列を一部改変したのでanalogとした。また、(Fl)はフルオレセインを示す。
実施例１６
図１で示されるように、配列番号２で表されるHIV-1 Rev-(34-50)のＣ末端と配列番号１６で表されるカルパイン阻害剤のＮ末端をGlyを介して結合し、さらに、カルパイン阻害剤のＣ末端にGly-Cys-amideを結合した合成ポリペプチドを島津PSSM-8型ペプチド合成機、Rinkアミド樹脂を用いてFmoc固相合成法で合成した。得られたポリペプチドは、各々トリフルオロ酢酸−エタンジチオール（９５：５）で室温１．５時間処理し、脱保護と樹脂からの切り出しを行い、Waters社Symmetry 300 C18（5μm、4.6×150mm)を用い、0.1%TFAを含有するアセトニトリル−水グラジエント（アセトニトリル濃度５％〜５０％）で溶出する逆相ＨＰＬＣで精製した。さらに、該ポリペプチドを１．５当量のフルオレセイン-5(6)-カルボキサミドカプロン酸N-ヒドロキシコハク酸イミドエステルと反応させて、蛍光標識したHIV-1 Rev-(34-50)−Gly−カルパイン阻害剤−GlyCys(フルオレセイン)-amideを得た。得られた蛍光標識体の質量分析（MALDI-TOF-MS(Shimadzu MALDI-2))結果を表２に示す。
実施例１７
配列番号１６で表されるカルパイン阻害剤の配列に代えて、配列番号１７で表されるIκＢ(15-41)の配列を利用した他は実施例１６と同様にして、蛍光標識体HIV-1 Rev-(34-50)−Gly−IκＢ(15-41)−GlyCys(フルオレセイン)-amideを得た。得られた蛍光標識体の質量分析（MALDI-TOF-MS(Shimadzu MALDI-2))結果を表２に示す。
実施例１８
HIV-1 Rev-(34-50)の配列に代えてFHV coat-(35-49)の配列を利用した他は実施例１６と同様にして、蛍光標識体FHV coat-(35-49)−Gly−カルパイン阻害剤−GlyCys(フルオレセイン)-amideを得た。得られた蛍光標識体の質量分析（MALDI-TOF-MS(Shimadzu MALDI-2))結果を表２に示す。
実施例１９
配列番号１６で表されるカルパイン阻害剤の配列に代えて、配列番号１７で表されるIκＢ(15-41)の配列を利用した他は実施例１６と同様にして、蛍光標識体FHV coat-(35-49)−Gly−IκＢ(15-41)−GlyCys(フルオレセイン)-amideを得た。得られた蛍光標識体の質量分析（MALDI-TOF-MS(Shimadzu MALDI-2))結果を表２に示す。
【００２４】
【表２】
実施例２０
図２で示されるように、カルボニックアンヒドラーゼ(CA、分子量29000)をＰＢＳ中で１５当量のN-(6-マレイミドカプロイルオキシ)コハク酸イミドエステル(EMCS)及び１５当量のフルオレセイン-5(6)-カルボキサミドカプロン酸N-ヒドロキシコハク酸イミドエステルを室温で２時間反応させて、マレイミド基及びFITC基を平均で１〜２個ずつ結合した蛍光標識カルボニックアンヒドラーゼ誘導体を得た。
【００２６】
得られた蛍光標識カルボニックアンヒドラーゼ誘導体と実施例１４で得られたフルオレセインで標識されていないポリペプチド（HIV-1 Rev-(34-50)-GlyCys-amide）をＰＢＳ中室温で２０時間反応させることにより、該ポリペプチド（HIV-1 Rev-(34-50)-GlyCys-amide）のＣ末端のCysのＳＨ基がFITC標識カルボニックアンヒドラーゼ誘導体のマレイミド基に付加した本発明の細胞透過性キャリアペプチド−異種ポリペプチド・コンジュゲートを得た。なお、得られたペプチドコンジュゲートの精製は、PBS緩衝液及びSephadex G25を用いたゲル濾過により行った。
【００２７】
マクロファージ由来のＲＡＷ２６４．７細胞と、RPMI 1640培地中チェンバースライド上で１６時間培養し、RPMI 1640培地を交換し、上記で得られた蛍光標識ペプチドコンジュゲートを１０μＭの濃度になるように加え、３７℃で３時間インキュベーションした後、ＰＢＳで洗浄し、アセトン：メタノール（１：１）で固定した。固定化されたＲＡＷ２６４．７細胞を蛍光顕微鏡で観察した。その結果、蛍光標識ペプチドコンジュゲートは全ての細胞内に入ったことが確認された。
実施例２１
実施例１４で得られた非蛍光標識ポリペプチド（HIV-1 Rev-(34-50)-GlyCys-amide）に代えて実施例１５で得られた非蛍光標識ポリペプチド（FHV coat-(35-49)-GlyCys-amide)を用いた他は実施例２０と同様にして本発明の細胞透過性キャリアペプチド−異種ポリペプチド・コンジュゲートを得た。
【００２８】
マクロファージ由来のＲＡＷ２６４．７細胞と、RPMI 1640培地中チェンバースライド上で１６時間培養し、RPMI 1640培地を交換し、上記で得られた蛍光標識ペプチドコンジュゲートを１０μＭの濃度になるように加え、３７℃で３時間インキュベーションした後、ＰＢＳで洗浄し、アセトン：メタノール（１：１）で固定した。固定化されたＲＡＷ２６４．７細胞を蛍光顕微鏡で観察した。その結果、蛍光標識ペプチドコンジュゲートは全ての細胞内に入ったことが確認された。
実施例２２
カルボニックアンヒドラーゼに代えてミオグロビン（分子量：１７０００）を用いた他は実施例２０と同様にして、本発明の細胞透過性キャリアペプチド−異種ポリペプチド・コンジュゲートを得た。
【００２９】
マクロファージ由来のＲＡＷ２６４．７細胞と、RPMI 1640培地中チェンバースライド上で１６時間培養し、RPMI 1640培地を交換し、上記で得られた蛍光標識ペプチドコンジュゲートを１０μＭの濃度になるように加え、３７℃で３時間インキュベーションした後、ＰＢＳで洗浄し、アセトン：メタノール（１：１）で固定した。固定化されたＲＡＷ２６４．７細胞を蛍光顕微鏡で観察した。その結果、蛍光標識ペプチドコンジュゲートは全ての細胞内に入ったことが確認された。
実施例２３
カルボニックアンヒドラーゼに代えてミオグロビン（分子量：１７０００）を用いた他は実施例２１と同様にして、本発明の細胞透過性キャリアペプチド−異種ポリペプチド・コンジュゲートを得た。
【００３０】
マクロファージ由来のＲＡＷ２６４．７細胞と、RPMI 1640培地中チェンバースライド上で１６時間培養し、RPMI 1640培地を交換し、上記で得られた蛍光標識ペプチドコンジュゲートを１０μＭの濃度になるように加え、３７℃で３時間インキュベーションした後、ＰＢＳで洗浄し、アセトン：メタノール（１：１）で固定した。固定化されたＲＡＷ２６４．７細胞を蛍光顕微鏡で観察した。その結果、蛍光標識ペプチドコンジュゲートは全ての細胞内に入ったことが確認された。
実施例２４〜３４
マクロファージ由来のＲＡＷ２６４．７細胞と、RPMI 1640培地中チェンバースライド上で１６時間培養し、RPMI 1640培地を交換し、実施例３〜１３で得られたＣ末端にGly-Cys(フルオレセイン)-amide構造を有する各標識ポリペプチドを１０μＭの濃度になるように加え、３７℃で３時間インキュベーションした後、ＰＢＳで洗浄し、アセトン：メタノール（１：１）で固定した。固定化されたＲＡＷ２６４．７細胞を蛍光顕微鏡で観察した。その結果、蛍光標識ポリペプチドは全ての細胞内に入ったことが確認された。
実施例３５〜３８
実施例３〜１３で得られたＣ末端にGly-Cys(フルオレセイン)-amide構造を有する各標識ポリペプチドに代えて、実施例１６〜１９で得られたカルパイン阻害剤またはIκＢ(15-41)を有する各標識ポリペプチドを用いて実施例２４〜３４と同様に試験を行ったところ、実施例１６〜１９の蛍光標識ポリペプチドは全ての細胞内に入ったことが確認された。
比較例３
比較例２で得られた細胞透過性キャリアペプチド活性を有するポリペプチドと、１．５当量の５−マレイミドフルオレセインジアセテートをジメチルホルムアミド中室温で１時間反応させて、フルオレセインがCys残基に結合した蛍光標識ポリペプチド誘導体を得た。
【００３１】
マクロファージ由来のＲＡＷ２６４．７細胞と、RPMI 1640培地中チェンバースライド上で１６時間培養し、RPMI 1640培地を交換し、上記で得られたＣ末端にGly-Cys(フルオレセイン)構造を有する各標識ポリペプチドを１０μＭの濃度になるように加え、３７℃で３時間インキュベーションした後、ＰＢＳで洗浄し、アセトン：メタノール（１：１）で固定した。固定化されたＲＡＷ２６４．７細胞を蛍光顕微鏡で観察した。その結果、比較例３の蛍光標識ポリペプチドは細胞内にほとんど入らないことが確認された。
【００３２】
【配列表】
【図面の簡単な説明】
【図１】細胞透過性キャリアペプチドとカルパインインヒビター又はＩκＢ(15-41)を連結した蛍光標識ペプチドコンジュゲートを示す。
【図２】カルボニックアンヒドラーゼを細胞透過性キャリアペプチドと連結した蛍光標識ペプチドコンジュゲートの製造スキームを示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cell-permeable carrier peptide and a carrier peptide conjugate formed by linking any one selected from the group consisting of a heterologous polypeptide, DNA, and sugar. The present invention also relates to the use of cell-permeable carrier peptides for introducing polypeptides or proteins, as well as DNA and sugars into cells.
[0002]
[Prior art]
In recent years, attempts have been made to obtain information on intracellular signal transduction and transcription by introducing synthetic peptides and proteins, as well as DNA and sugars into cells, and regulating protein interactions within the cells. . By this approach, it is expected that the amount of peptide introduction, which is difficult depending on the method for introducing a gene encoding a target protein, or intracellular introduction of a polypeptide having an unnatural amino acid skeleton in the molecule can be expected.
[0003]
In general, polypeptides, nucleic acids, and sugars are highly hydrophilic and difficult to pass through cell membranes. For intracellular introduction of polypeptides, nucleic acids, and sugars, in addition to microinjection, hybrids with fat-soluble molecules such as fatty acids Conventionally, a method using crystallization or a method using liposomes has been employed.
[0004]
Apart from these methods, recently hydrophobic signal peptides (Lin YZ, et al. (1995) J. Biol. Chem., 270, 14255-14258; Rojas M., et al. (1998) Nat. Biotechnol) , 16, 370-375), or attempts to introduce peptides and proteins using basic peptides derived from HIV-1 Tat protein or Drosophila Antennapedia protein (Japanese Patent Laid-Open No. 10-33186, Derossi D et al. (1998) Trends. Cell Biol., 8, 84-87; Fawell S., et al. (1994) Proc. Acad. Natl. Sci., USA, 91, 664-668; Nagahara H. et al. (1998) Nat. Med., 4, 1449-1452).
[0005]
However, these prior art attempts have not been able to sufficiently introduce peptides or proteins, as well as DNA and sugar, into cells.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a cell-permeable carrier peptide capable of efficiently introducing a polypeptide, protein, DNA, or sugar into a cell, a carrier peptide and polypeptide, and a carrier peptide conjugate in which DNA or sugar is bound. There is.
[0007]
It is also an object of the present invention to provide the use of a cell permeable carrier peptide for introducing a polypeptide, as well as DNA or sugar, into a cell.
[0008]
[Means for Solving the Problems]
The present invention is a cell-permeable carrier peptide comprising the following polypeptide (a) or (b).
(A) the polypeptide represented by any one of SEQ ID NOs: 2 to 14;
(B) A polypeptide having a cell-permeable carrier peptide activity, comprising a polypeptide in which one or several amino acids are substituted, deleted or added in the polypeptide (a).
[0009]
The present invention also provides a cell-permeable carrier peptide containing the polypeptide of (a) or (b) above and any one selected from the group consisting of a heterologous polypeptide, DNA and sugar via a crosslinking agent as necessary. It is related with the carrier peptide conjugate formed by connecting.
[0010]
Furthermore, the present invention relates to the use of the cell-permeable carrier peptide for introducing any one selected from the group consisting of a heterologous polypeptide, DNA and sugar into a cell.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Examples of the cell-permeable carrier peptide of the present invention include the polypeptide represented by any of SEQ ID NOs: 2 to 14, and the sequence contains one or several amino acids as long as it retains the cell-permeable carrier peptide activity. Includes a substituted, deleted or added polypeptide.
[0012]
The degree of “amino acid deletion, substitution or addition” and their positions are not particularly limited as long as the modified polypeptide has cell-permeable carrier peptide activity. In the present invention, “cell-permeable carrier peptide activity” means that a protein or polypeptide bound to the polypeptide of SEQ ID NOs: 2 to 14 permeates the cell membrane and is introduced into the cell.
[0013]
As specific means of substitution, addition, and deletion, when performed via DNA encoding the carrier peptide, for example, cytospecific mutagenesis [Methods in Enzymology, 154, 350, 367-382 (1987) 100, 468 (1983); Nucleic Acids Res., 12, 9441 (1984); Secondary Biochemistry Experiment Course 1 "Gene Research Method II", edited by Japanese Biochemical Society, p105 (1986)] Chemical synthesis means such as the phosphate triester method and phosphate amidite method [J. Am. Chem. Soc., 89, 4801 (1967); 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981); 24, 245 (1983)], and combinations thereof. More specifically, DNA synthesis can be performed by chemical synthesis by the phosphoramidite method or triester method, and can also be performed on a commercially available automatic oligonucleotide synthesizer. Double-stranded fragments are chemically synthesized single strands produced by synthesizing complementary strands and annealing the strands together under appropriate conditions, or adding complementary strands using DNA polymerase with appropriate primer sequences. It can also be obtained from things. Furthermore, the cell-permeable carrier peptide of the present invention can be synthesized by a solid phase synthesis method using a peptide synthesizer, and substitution, addition, and deletion can be performed by selecting the type of protected amino acid when using the peptide synthesizer. It can be easily done by changing. Also, special amino acids such as D-amino acids and sarcosine (N-methylglycine) can be introduced.
[0014]
The polypeptide linked to the cell-permeable carrier peptide of the present invention and introduced into the cell is not particularly limited, and includes any polypeptide. Examples of the molecular weight of the polypeptide include about 500 to about 1,000,000, preferably about 1,000 to 500,000, and any type of secreted protein, membrane-bound protein, peptide hormone, and the like is acceptable.
[0015]
Specific examples of polypeptides introduced into cells include carbonic anhydrase, myoglobin, horseradish peroxidase, β-galactosidase, transcription factors with leucine zippers and zinc finger motifs, apoptosis-inducing proteins such as Fas p53, and defects. Examples thereof include proteins such as adenosine deaminase that induce metabolic disorders and enzyme inhibitors (eg, calpain inhibitors), genetic information expression regulators (eg, IκB), peptide hormones (insulin, calcitonin, etc.), and the like.
[0016]
When the cell-permeable carrier peptide has a cysteine residue, the polypeptide introduced into the cell and the cell-permeable carrier peptide of the present invention are linked to the cysteine residue of the polypeptide via an -SS-bond. Alternatively, they may be linked via an appropriate crosslinking agent. In addition, the polynucleotide encoding the cell-permeable carrier peptide of the present invention and the polynucleotide (gene) encoding the introduced polypeptide are preferably directly linked, introduced into a vector, and transferred into a host cell such as Escherichia coli. By a conventional method such as expression, a peptide conjugate in which a protein or polypeptide introduced into the C-terminal side of the cell-permeable carrier peptide of the present invention is directly linked can be obtained. Similarly, DNA and sugar can also be linked via an appropriate crosslinking agent.
[0017]
The cross-linking agent is not particularly limited as long as it is an at least divalent cross-linking agent capable of binding the cell-permeable carrier peptide of the present invention and the protein or polypeptide to be introduced, DNA, or sugar. For example, N- (6-maleimide cap) (Royloxy) succinimide ester (EMCS).
[0018]
It is preferable that a Cys residue is further bound to the C-terminal side of the cell-permeable carrier peptide of the present invention in a manner such as Cys or Gly-Cys. The SH group of the Cys residue is added to the maleimide group of EMCS, or when the introduced protein or polypeptide has a free SH group, Can be linked. When linked via -SS-bonding, it is preferable because the -SS-bonding is reduced in the cell and unmodified protein or polypeptide is released.
[0019]
【The invention's effect】
According to the present invention, when administered alone, a polypeptide, DNA, or sugar that is difficult to migrate into the cell can be introduced into the cell with high efficiency.
[0020]
Treatment of various diseases can be expected by introducing into the cell a physiologically active substance such as a polypeptide, DNA, or sugar that is introduced into the cell and exhibits biological activity.
[0021]
【Example】
Hereinafter, examples will be described in more detail, but the present invention is not limited to these examples.
Examples 1-13
A polypeptide in which Gly-Cys-amide is added to the C-terminus of the polypeptide represented by SEQ ID NO: 2 to 14 is synthesized by Fmoc solid phase synthesis using Shimadzu PSSM-8 type peptide synthesizer and Rink amide resin. did. The obtained polypeptides were each treated with trifluoroacetic acid-ethanedithiol (95: 5) for 1.5 hours at room temperature, deprotected and cleaved from the resin, and Waters Symmetry 300 C18 (5 μm, 4.6 × 150 mm) Was purified by reverse phase HPLC eluting with an acetonitrile-water gradient containing 0.1% TFA (
Examples 14 and 15
Polypeptides in which Gly-Cys-amide was added to the C-terminus of the polypeptide represented by SEQ ID NOs: 2 and 3 were synthesized in the same manner as in Examples 1 to 13 and purified by reverse-phase HPLC. The results of mass spectrometry of the obtained polypeptide are shown in Table 1. The polypeptide obtained in Example 14 is the same as the polypeptide obtained in Example 1 except that the polypeptide is not fluorescently labeled with fluorescein. Similarly, the polypeptide obtained in Example 15 is identical to the polypeptide obtained in Example 2 except that it is not fluorescently labeled with fluorescein.
Comparative Example 1
A polypeptide in which Gly-Cys-amide was added to the C-terminus of the polypeptide represented by SEQ ID NO: 1 was synthesized in the same manner as in Examples 1-13. The mass spectrometric results of the obtained fluorescently labeled polypeptide are shown in Table 1.
Comparative Example 2
A polypeptide in which Gly-Cys-amide was added to the C-terminus of the polypeptide represented by SEQ ID NO: 15 was synthesized in the same manner as in Examples 1-13. The mass spectrometric results of the obtained fluorescently labeled polypeptide are shown in Table 1.
[0022]
[Table 1]
In Table 1, since the sequence of Human cFos- (139-164) was partially modified, it was named analog. (Fl) indicates fluorescein.
Example 16
As shown in FIG. 1, the C-terminus of HIV-1 Rev- (34-50) represented by SEQ ID NO: 2 and the N-terminus of the calpain inhibitor represented by SEQ ID NO: 16 are linked via Gly, Furthermore, a synthetic polypeptide in which Gly-Cys-amide was bonded to the C-terminus of the calpain inhibitor was synthesized by Fmoc solid phase synthesis using Shimadzu PSSM-8 type peptide synthesizer and Rink amide resin. The obtained polypeptides were each treated with trifluoroacetic acid-ethanedithiol (95: 5) for 1.5 hours at room temperature, deprotected and cleaved from the resin, and Waters Symmetry 300 C18 (5 μm, 4.6 × 150 mm) Was purified by reverse phase HPLC eluting with an acetonitrile-water gradient containing 0.1% TFA (
Example 17
Instead of the calpain inhibitor sequence represented by SEQ ID NO: 16, the fluorescent marker HIV-1 was used in the same manner as in Example 16 except that the sequence of IκB (15-41) represented by SEQ ID NO: 17 was used. Rev- (34-50) -Gly-IκB (15-41) -GlyCys (fluorescein) -amide was obtained. Table 2 shows the results of mass spectrometry (MALDI-TOF-MS (Shimadzu MALDI-2)) of the obtained fluorescent label.
Example 18
The fluorescent label FHV coat- (35-49)-was used in the same manner as in Example 16 except that the sequence of FHV coat- (35-49) was used instead of the sequence of HIV-1 Rev- (34-50). Gly-calpain inhibitor-GlyCys (fluorescein) -amide was obtained. Table 2 shows the results of mass spectrometry (MALDI-TOF-MS (Shimadzu MALDI-2)) of the obtained fluorescent label.
Example 19
In place of the calpain inhibitor sequence represented by SEQ ID NO: 16, the sequence of IκB (15-41) represented by SEQ ID NO: 17 was used in the same manner as in Example 16, and the fluorescent label FHV coat- (35-49) -Gly-IκB (15-41) -GlyCys (fluorescein) -amide was obtained. Table 2 shows the results of mass spectrometry (MALDI-TOF-MS (Shimadzu MALDI-2)) of the obtained fluorescent label.
[0024]
[Table 2]
Example 20
As shown in FIG. 2, carbonic anhydrase (CA, molecular weight 29000) was dissolved in PBS with 15 equivalents of N- (6-maleimidocaproyloxy) succinimide ester (EMCS) and 15 equivalents of fluorescein-5 ( 6) -Carboxamido caproic acid N-hydroxysuccinimide ester was reacted at room temperature for 2 hours to obtain a fluorescently labeled carbonic anhydrase derivative in which one to two maleimide groups and one FITC group were bonded on average.
[0026]
Reaction of the obtained fluorescently labeled carbonic anhydrase derivative and the polypeptide not labeled with fluorescein obtained in Example 14 (HIV-1 Rev- (34-50) -GlyCys-amide) in PBS at room temperature for 20 hours The cell permeation of the present invention in which the SH group of the C-terminal Cys of the polypeptide (HIV-1 Rev- (34-50) -GlyCys-amide) is added to the maleimide group of the FITC-labeled carbonic anhydrase derivative A sex carrier peptide-heterologous polypeptide conjugate was obtained. The obtained peptide conjugate was purified by gel filtration using PBS buffer and Sephadex G25.
[0027]
Macrophage-derived RAW264.7 cells were cultured for 16 hours on a chamber slide in RPMI 1640 medium, the RPMI 1640 medium was replaced, and the fluorescence-labeled peptide conjugate obtained above was added to a concentration of 10 μM. After incubation at 0 ° C. for 3 hours, the plate was washed with PBS and fixed with acetone: methanol (1: 1). Fixed RAW264.7 cells were observed with a fluorescence microscope. As a result, it was confirmed that the fluorescence-labeled peptide conjugate entered all the cells.
Example 21
Instead of the non-fluorescent labeled polypeptide (HIV-1 Rev- (34-50) -GlyCys-amide) obtained in Example 14, the non-fluorescent labeled polypeptide (FHV coat- (35- The cell-permeable carrier peptide-heterologous polypeptide conjugate of the present invention was obtained in the same manner as in Example 20 except that 49) -GlyCys-amide) was used.
[0028]
Macrophage-derived RAW264.7 cells were cultured for 16 hours on a chamber slide in RPMI 1640 medium, the RPMI 1640 medium was replaced, and the fluorescence-labeled peptide conjugate obtained above was added to a concentration of 10 μM. After incubation at 0 ° C. for 3 hours, the plate was washed with PBS and fixed with acetone: methanol (1: 1). Fixed RAW264.7 cells were observed with a fluorescence microscope. As a result, it was confirmed that the fluorescence-labeled peptide conjugate entered all the cells.
Example 22
A cell-permeable carrier peptide-heterologous polypeptide conjugate of the present invention was obtained in the same manner as in Example 20, except that myoglobin (molecular weight: 17000) was used instead of carbonic anhydrase.
[0029]
Macrophage-derived RAW264.7 cells were cultured for 16 hours on a chamber slide in RPMI 1640 medium, the RPMI 1640 medium was replaced, and the fluorescence-labeled peptide conjugate obtained above was added to a concentration of 10 μM. After incubation at 0 ° C. for 3 hours, the plate was washed with PBS and fixed with acetone: methanol (1: 1). Fixed RAW264.7 cells were observed with a fluorescence microscope. As a result, it was confirmed that the fluorescence-labeled peptide conjugate entered all the cells.
Example 23
A cell-permeable carrier peptide-heterologous polypeptide conjugate of the present invention was obtained in the same manner as in Example 21, except that myoglobin (molecular weight: 17000) was used instead of carbonic anhydrase.
[0030]
Macrophage-derived RAW264.7 cells were cultured for 16 hours on a chamber slide in RPMI 1640 medium, the RPMI 1640 medium was replaced, and the fluorescence-labeled peptide conjugate obtained above was added to a concentration of 10 μM. After incubation at 0 ° C. for 3 hours, the plate was washed with PBS and fixed with acetone: methanol (1: 1). Fixed RAW264.7 cells were observed with a fluorescence microscope. As a result, it was confirmed that the fluorescence-labeled peptide conjugate entered all the cells.
Examples 24-34
Macrophage-derived RAW264.7 cells were cultured for 16 hours on a chamber slide in RPMI 1640 medium, and the RPMI 1640 medium was replaced. A Gly-Cys (fluorescein) -amide structure was obtained at the C-terminus of Examples 3-13 Each of the labeled polypeptides having a concentration of 10 μM was added, incubated at 37 ° C. for 3 hours, washed with PBS, and fixed with acetone: methanol (1: 1). Fixed RAW264.7 cells were observed with a fluorescence microscope. As a result, it was confirmed that the fluorescently labeled polypeptide entered all the cells.
Examples 35-38
Instead of each labeled polypeptide having a Gly-Cys (fluorescein) -amide structure at the C-terminus obtained in Examples 3 to 13, the calpain inhibitor or IκB (15-41) obtained in Examples 16 to 19 was used. When the test was conducted in the same manner as in Examples 24 to 34 using each labeled polypeptide having the above, it was confirmed that the fluorescently labeled polypeptides of Examples 16 to 19 entered all the cells.
Comparative Example 3
The polypeptide having the cell-permeable carrier peptide activity obtained in Comparative Example 2 was reacted with 1.5 equivalents of 5-maleimidofluorescein diacetate in dimethylformamide at room temperature for 1 hour, and fluorescein was bound to the Cys residue. A fluorescently labeled polypeptide derivative was obtained.
[0031]
Macrophage-derived RAW264.7 cells were cultured for 16 hours on a chamber slide in RPMI 1640 medium, RPMI 1640 medium was replaced, and each labeled polypeptide having a Gly-Cys (fluorescein) structure at the C-terminus obtained above Was added to a concentration of 10 μM, incubated at 37 ° C. for 3 hours, washed with PBS, and fixed with acetone: methanol (1: 1). Fixed RAW264.7 cells were observed with a fluorescence microscope. As a result, it was confirmed that the fluorescently labeled polypeptide of Comparative Example 3 hardly enters the cells.
[0032]
[Sequence Listing]
[Brief description of the drawings]
FIG. 1 shows a fluorescence-labeled peptide conjugate in which a cell-permeable carrier peptide and a calpain inhibitor or IκB (15-41) are linked.
FIG. 2 shows a production scheme of a fluorescent-labeled peptide conjugate in which carbonic anhydrase is linked to a cell-permeable carrier peptide.
Claims (5)
（ａ）配列番号３で表されるポリペプチド；
（ｂ）配列番号３で表されるポリペプチドにおいて１又は数個のアミノ酸が置換、欠失又は付加したポリペプチドを含み、細胞透過性キャリアペプチド活性を有するポリペプチド。A cell-permeable carrier peptide comprising the following polypeptide (a) or (b):
(A) the polypeptide represented by SEQ ID NO: 3;
(B) A polypeptide having a cell-permeable carrier peptide activity, comprising a polypeptide represented by SEQ ID NO: 3, wherein one or several amino acids are substituted, deleted or added.
（ａ）配列番号３で表されるポリペプチド。The cell-permeable carrier peptide according to claim 1, comprising the following polypeptide (a):
(A) The polypeptide represented by SEQ ID NO: 3.
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| JP4381822B2 (en) * | 2002-03-22 | 2009-12-09 | 株式会社バイファ | Immunoglobulin hydrophilic peptide complex |
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| WO1993012230A1 (en) * | 1991-12-13 | 1993-06-24 | Sri International | Triple-helix formation at (punpyn).(punpyn) tracts |
| US5654398A (en) * | 1993-06-03 | 1997-08-05 | The Regents Of The University Of California | Compositions and methods for inhibiting replication of human immunodeficiency virus-1 |
| US6495526B2 (en) * | 1996-01-23 | 2002-12-17 | Gpc Biotech, Inc. | Inhibitors of cell-cycle progression and uses related thereto |
| CA2298067A1 (en) * | 1997-07-24 | 1999-02-04 | Ulo Langel | Conjugates of transporter peptides and nucleic acid analogs, and their use |
| AU755564B2 (en) * | 1998-06-20 | 2002-12-12 | Washington University | Membrane-permeant peptide complexes for medical imaging, diagnostics, and pharmaceutical therapy |
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| WO2009093692A1 (en) | 2008-01-25 | 2009-07-30 | Toagosei Co., Ltd. | Artificial peptide and use thereof |
| US8710005B2 (en) | 2009-04-10 | 2014-04-29 | Toagosei Co., Ltd. | Neuronal differentiation-inducing peptide and use thereof |
| US8603967B2 (en) | 2009-07-29 | 2013-12-10 | Toagosei Co., Ltd. | Carrier peptide fragment and use thereof |
| US8673845B2 (en) | 2009-07-29 | 2014-03-18 | Toagosei Co., Ltd. | Carrier peptide fragment and use thereof |
| US8673846B2 (en) | 2009-11-02 | 2014-03-18 | Toagosei Co. Ltd. | Cell proliferation-promoting peptide and use thereof |
| US9133437B2 (en) | 2009-11-02 | 2015-09-15 | Toagosei Co. Ltd. | Cell proliferation-promoting peptide and use thereof |
| US8822408B2 (en) | 2010-06-04 | 2014-09-02 | Toagosei Co., Ltd. | Cell growth-promoting peptide and use thereof |
| US9238796B2 (en) | 2010-06-04 | 2016-01-19 | Toagosei Co. Ltd. | Cell growth-promoting peptide and use thereof |
| US9370182B2 (en) | 2012-05-28 | 2016-06-21 | Toagosei Co., Ltd. | Antimicrobial peptide and use thereof |
| US9480727B2 (en) | 2012-10-18 | 2016-11-01 | Toagosei Co. Ltd. | Synthetic peptide for inhibiting expression of type 2 TNF receptor and use thereof |
| EP3971216A1 (en) | 2020-09-14 | 2022-03-23 | Toagosei Co., Ltd. | Carrier peptide fragment and use thereof |
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|---|---|
| JP2001199997A (en) | 2001-07-24 |
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