Development of a system for stable expression of antibodies bearing non-natural amino acids at defined sites

• Project/Area Number: 25670023
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Biological pharmacy
• Research Institution: Osaka University
• Principal Investigator: HINO Nobumasa 大阪大学, 薬学研究科(研究院), 助教 (90469916)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2013: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: 拡張遺伝暗号 / 非天然型アミノ酸 / tRNA / 抗体薬物複合体 / 非天然アミノ酸 / 部位特異的修飾

Outline of Final Research Achievements

For stable production of antibodies with non-natural amino acids at specific positions, it is necessary to establish cell lines that constitutively and efficiently express exogenous amber suppressor tRNAs. To improve expression efficiency of the exogenous tRNAs, I attempted to develop a system that enables to express them from the nucleolus where cellular tRNAs are transcribed. I constructed tRNA expression vectors containing 5S rDNA sequences or genomic sequences surrounding cellular tRNA genes, which are expected to localize themselves to nucleolus. These vectors successfully increased the expression of the amber mutant of GFP by 1.5- to 2-fold as compared with the vector that contains tRNA genes alone.

Research Products

• [Journal Article] Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter (2014)
  • Author(s): Okada, Y., Funahashi, N., Tanaka, T., Nishiyama, Y., Yuan, L., Shirakura, K., Turjman, A. S., Kano, Y., Naruse, H., Suzuki, A., Sakai, M., Zhixia, J., Kitajima, K., Ishimoto, K., Hino, N., Kondoh, M., Mukai, Y., Nakagawa, S., Garcia-Cardena, G., Aird, W. C., Doi, T.
  • Journal Title: Arterioscler Thromb. Vasc. Biol. Volume: 34 Issue: 7 Pages: 1531-1538
  • DOI: 10.1161/atvbaha.114.303818
  • Peer Reviewed
• [Presentation] タンパク質間の相互作用部位の解析を可能にする細胞内光クロスリンク技術の開発 (2014)
  • Author(s): 一瓢奨
  • Organizer: 第13回次世代を担う若手ファーマ・バイオフォーラム2014
  • Place of Presentation: 富山
  • Year and Date: 2014-09-20 – 2014-09-21
• [Presentation] 非天然型アミノ酸含有タンパク質の安定生産系の構築 (2014)
  • Author(s): 喜多絢海、一瓢奨、樋野展正、土井健史
  • Organizer: 日本ケミカルバイオロジー学会 第９回年会
  • Place of Presentation: 大阪
  • Year and Date: 2014-06-11 – 2014-06-13
• [Presentation] プローブ転移能を有する光クロスリンカーを用いた細胞内タンパク質間相互作用解析法の開発 (2014)
  • Author(s): 一瓢奨、喜多絢海、張功幸、高島成二、樋野展正、土井健史
  • Organizer: 日本ケミカルバイオロジー学会 第９回年会
  • Place of Presentation: 大阪
  • Year and Date: 2014-06-11 – 2014-06-13
• [Remarks]
  • URL: http://www.phs.osaka-u.ac.jp/homepage/b018/index.html