人工タンパク質の技術開発

• Project/Area Number: 17012026
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Japanese Foundation For Cancer Research
• Principal Investigator: 芝 清隆 Japanese Foundation For Cancer Research, 癌研究所・蛋白創製研究部, 部長 (40196415)
• Project Period (FY): 2005 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥47,900,000 (Direct Cost: ¥47,900,000); Fiscal Year 2007: ¥15,700,000 (Direct Cost: ¥15,700,000); Fiscal Year 2006: ¥15,700,000 (Direct Cost: ¥15,700,000); Fiscal Year 2005: ¥16,500,000 (Direct Cost: ¥16,500,000)
• Keywords: 人工タンパク質 / モチーフ / アポトーシス / 信号伝達 / 進化 / 進化分子工学 / 合成生物学 / 構造論的 / 構成論的

Research Abstract

「モチーフ・プログラミング」、すなわち、機能性モチーフを組み合わせ的に重合させ、新しい多機能性人工タンパク質を創製する技術開発に取り組んだ。機能性モチーフの組み合わせ的な重合にはMolCraft法と呼ぶ独自の技術を利用した。人工タンパク質は、がん生物学の研究ツール、がんの治療、がんの診断と幅広い分野でがん研究に関わる事が出来る。ここでは特に、発がん過程に深く関わった信号伝達系に焦点を絞り、アポトーシス経路に遍在するBHI〜BH4モチーフの組み合わせ的重合から、アポトーシス経路をモジュレートする活性をもった人工タンパク質を創製した。また、タンパク質製剤を念頭に置いた、BHモチーフと細胞移入モチーフをプログラムした人工タンパク質も作製しており、その細胞指向性を調べた。また、他の信号伝達系としてインテグリン信号伝達系複合体のメンバーであるp130Casがもつ内部のSrkリン酸化モチーフに注目し、このモチーフ群を2種の代表的モチーフに分類し、この2種のモチーフをいろいろな数と順番でもつp130Cas部分人工タンパク質化した改変体を作製し、その細胞内での活性を調べた。その結果、BHI〜BH4モチーフの組み合わせ的重合からアポトーシス経路を正負に制御する人工制御因子が高頻度に生成すること、BHモチーフと細胞移入モチーフをプログラムした人工タンパク質が、癌細胞パネルに対して明確な細胞選択性をもつこと、p130Casのリン酸かモチーフをモデル化した単純改変体でも、p130Cas欠損細胞株の細胞走化性を維持することが明らかとなった。モチーフ・プログラミングによる人工タンパク質創製から、高効率に機能性人工タンパク質が創製できることがわかった。

Research Products

• [Journal Article] A motif-programmed artificial protein induces apoptosis in several cancer cells by disrupting mitochondria (2008)
  • Author(s): Saito H, Minamisawa T, Yamori T, Shiba K
  • Journal Title: Cancer Science 99(2) Pages: 398-406
  • NAID: 10024002827
  • Peer Reviewed
• [Journal Article] The role of peptide motifs in the evolution of a protein network (2007)
  • Author(s): Saito H, Kashida S, Inoue T, Shiba K
  • Journal Title: Nucl Acid Res 35(19) Pages: 6357-6366
  • Peer Reviewed
• [Journal Article] A synthesis approach to understanding repearted peptides conserved in mineralization proteins (2007)
  • Author(s): Shiba K, Minamisawa T
  • Journal Title: Biomacromolecules 8(9) Pages: 2659-2664
  • Peer Reviewed
• [Journal Article] Dispersion of cisplatin-loaded carbon nanohorns with a conjugate comprised of an artificial peptide aptamer and polyethylene glycol (2007)
  • Author(s): Matsumura S, Ajima K, Yudasaka M Iijima S. Shiba K
  • Journal Title: Mol Pharm 4(5) Pages: 723-729
  • Peer Reviewed
• [Journal Article] Motif programming: A microgene-based method for creating synthetic proteins containing multiple functional motifs (2007)
  • Author(s): Saito H, Minamisawa T, Shiba K
  • Journal Title: Nucl Acid Res 35(6)
  • Peer Reviewed
• [Journal Article] Structural properties of an artificial protein that regulates the nucleation of inorganic and organic crystals (2007)
  • Author(s): Kulp JL III, Minamisawa T, Shiba K, Tejani M, Evans JS
  • Journal Title: Langmuir (in press)
• [Journal Article] Motif programming : A microgene-based method for creating synthetic proteins containin multiple functional motifs (2007)
  • Author(s): Saito H, Minamisawa T, Shiba K
  • Journal Title: Nucl Acid Res (in press)
• [Journal Article] Conversion of a monodispersed globular protein into an amyloid -like filament by appending an artificial peptide at the N-terminal (2007)
  • Author(s): Sano K, Sasaki H, Shiba K
  • Journal Title: Protein Eng Des Sel (in press)
• [Journal Article] Realizing a two-dimensional ordered array of ferritin molecules directly on a solid surface utilizing carbonaceous materials-affinity peptides (2007)
  • Author(s): Matsui T, Matsukawa N, Iwahori K, Sano K, Shiba K, Yamashita I
  • Journal Title: Langmuir 23・4 Pages: 1615-1618
• [Journal Article] Selective nanoscale positioning of ferritin and nanoparticles by means of target-specific peptides (2006)
  • Author(s): Yamashita I, Kirimura H, Okuda M, Nishio K, Sano K, Shiba K, Hayashi T, Hara M, Mishima Y
  • Journal Title: Small 2・10 Pages: 1148-1152
• [Journal Article] Mechanism underlying specificity of proteins targeting inorganic materials (2006)
  • Author(s): Hayashi T, Sano K, Shiba K, Kumashiro Y, Iwahori K, Yamashita I, Hara M
  • Journal Title: Nano Lett 6・3 Pages: 515-519
• [Journal Article] Utilization of the pleiotropy of a peptidic aptamer to fabricate heterogeneous nano-dot-containing multilayer nanostructures (2006)
  • Author(s): Sano K, Sasaki H, Shiba K
  • Journal Title: J Am Chem Soc 128(5) Pages: 1717-1722
• [Journal Article] Frame shuffling : a novel method for in vitro protein evolution (2006)
  • Author(s): Kashiwagi K, Isogai Y, Nishiguchi K, Shiba K
  • Journal Title: Protein Eng Des Sel 19(3) Pages: 135-140
• [Journal Article] Mechanism underlying specificity of proteins targeting inorganic materials (2006)
  • Author(s): Hayashi T, Sano K, Shiba K, Kumashiro Y, Iwahori K, Yamashita I, Hara M
  • Journal Title: Nano Lett 6(3) Pages: 515-519
• [Journal Article] AFMとQCM-Dを用いたチタン結合ペプチドのチタン表面への結合の観察 (2005)
  • Author(s): 佐野健一, 上塚 洋, 大西 洋, 芝 清隆
  • Journal Title: 表面科学 26(7) Pages: 428-431
  • NAID: 10016588150
• [Journal Article] Endowing a ferritin-like cage protein with high affinity and selectivity for certain inorganic materials (2005)
  • Author(s): Sano K, Ajima K, Iwahori K, Yudasaka M, Iijima S, Yamashita I, Shiba K
  • Journal Title: Small 1(8-9) Pages: 826-832
• [Journal Article] Carbon nanohorns as anticancer drug carriers (2005)
  • Author(s): Ajima K, Yudasaka M, Murakami T, Maigne A, Shiba K, Iijima S
  • Journal Title: Mol Pharmaceutics 2(6) Pages: 475-480
• [Journal Article] Probing the conformational features of a phage display polypeptide sequence directed against single wall carbon nanohorn surfaces (2005)
  • Author(s): Kulp JL III, Shiba K, Evans JS
  • Journal Title: Langmuir 21(25) Pages: 11907-11914
• [Book] Nanotubes, nanowires, nanocantilevers and nanorods in cancer treatment and diagnosis (Nanomaterials for cancer diagnosis- Nanotechnologies for the life siciences vol 7) (2007)
  • Author(s): Shiba K, (Challa SSR Kumar ed.)
  • Publisher: Wiley-VCH