Development and demonstration of novel purification techniques for disease-specific superficial proteins

• Project/Area Number: 16K14680
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: Osaka University
• Principal Investigator: Inoue Tsuyoshi 大阪大学, 工学研究科, 教授 (20263204)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2017: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: プロテオミクス解析 / 質量分析 / 表在性蛋白質 / 抗原探索 / 非天然ビオチン化ラベル / 改変型ストレプトアビジン / CTC / 血管内皮 / プロテオミクス / ラベル化技術 / 蛋白質精製 / 人工ビオチン / 抗体開発 / 診断薬開発 / 抗体医薬 / イメージング / 診断薬

Outline of Final Research Achievements

Regarding the labeling reagent, succeeded in synthesizing a derivative in which a succinimide group (NHS group) was added to the non-natural biotin derivatives.
As a result of experiments and proteomics analyses of vascular endothelium using mouse, it was found that the surface protein were only purified and the endogenous biotinylated proteins could be excluded by using the new derivatives. Proteomic analysis was also performed on diseased mice seeded with A20 lymphoma. Analyses of the vascular endothelial proteins that increased by 1.5 times or more in tumor-bearing mice were performed. There are 147 kinds of proteins listed only by the conventional method, and 28 kinds of proteins commonly listed in the conventional method and the novel method. However, it turned out that there were 53 kinds of proteins that were only found to be enhanced by the novel method. Validation is now under study.

Research Products

• [Journal Article] Crystal structure of secretory abundant heat soluble protein 4 from one of the toughest “water bears” micro-animals Ramazzottius varieornatus (2018)
  • Author(s): Fukuda Yohta、Inoue Tsuyoshi
  • Journal Title: Protein Science Volume: 印刷中 Issue: 5 Pages: 993-999
  • DOI: 10.1002/pro.3393
  • Peer Reviewed / Open Access
• [Journal Article] New molecular packing in a crystal of pseudoazurin from Alcaligenes faecalis: a double-helical arrangement of blue copper. (2017)
  • Author(s): Yohta Fukuda, Eiichi Mizohata and Tsuyoshi Inoue
  • Journal Title: Acta Cryst. F Volume: 73 Issue: 3 Pages: 159-166
  • DOI: 10.1107/s2053230x17002631
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Structural insights into a secretory abundant heat-soluble protein from an anhydrobiotic tardigrade, Ramazzottius?varieornatus (2017)
  • Author(s): Fukuda Yohta、Miura Yoshimasa、Mizohata Eiichi、Inoue Tsuyoshi
  • Journal Title: FEBS letters Volume: 591 Issue: 16 Pages: 2458-2469
  • DOI: 10.1002/1873-3468.12752
  • Peer Reviewed / Open Access
• [Journal Article] Active site geometry of a novel aminopropyltransferase for biosynthesis of hyperthermophile-specific branched-chain polyamine (2017)
  • Author(s): R. Hidese, K. M. Tse, S. Kimura, E. Mizohata, J. Fujita, Y. Horai, N. Umezawa, T. Higuchi, M. Niitsu, T. Oshima, T. Imanaka, T. Inoue, and S. Fujiwara
  • Journal Title: The FEBS Journal Volume: 284 Issue: 21 Pages: 3684-3701
  • DOI: 10.1111/febs.14262
  • Peer Reviewed / Open Access
• [Patent(Industrial Property Rights)] 抗体医薬の開発に資する創薬標的タンパク質の同定方法及び標的タンパク質に対する抗体の製造方法 (2018)
  • Inventor(s): 鈴木常司, 戸谷由之, 番場伸一, 鎌田春彦, 井上豪, 他
  • Industrial Property Rights Holder: 鈴木常司, 戸谷由之, 番場伸一, 鎌田春彦, 井上豪, 他
  • Industrial Property Rights Type: 特許
  • Filing Date: 2018
  • Overseas