Technical developmet of drug discovery basis using cell-free system and nanodisc

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K13096
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Chemical biology
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: Kuruma Yutetsu 東京工業大学, 地球生命研究所, 特任准教授 (40508420)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: セルフリー 系 / nanodisc / GPCR / mRNA display

Outline of Final Research Achievements

bacteriorhodopsin (bR) was synthesized in cell-free translation system which including Nanodisc. The synthesized bR were integrated into Nanodisc membrane and used as a bait for in vitro peptide selection. a peptide library containing total random amino acid sequence (10aa) were prepared by the cell-free system. After combining bR-nanodisc and peptide library, candidate peptides which specifically bind to bR were isolated together with their mRNA. the collected mRNA were treated by RT-PCR and applied for the second round peptide library. after repeating these cycle for several times, we analyzed the selected sequence by NGS, and the sequence were converted into amino acids seq. Such peptides were ordered and analyzed for its binding activity to bR.

Free Research Field

合成生物学

Academic Significance and Societal Importance of the Research Achievements

モノクローナル抗体を作製する従来の方法は、ハイブリドーマ法やファージディスプレイ法があるが、いずれの方法においても、ターゲット抗原発現細胞や精製抗原タンパク質の入手が必要である。申請者は先端技術としての試験管内合成法と、最新の脂質操作技術を用いて、正しい構造を維持した膜タンパク質の合成に成功している。本方法により、これまでの生体や培養細胞を用いた方法と比較して、抗体入手までの時間が大幅に短縮できることが期待できる。このようなセルフリー系による抗原作製と、連続した抗体作製ができるようになれば、抗体医薬創薬研究の１次選別技術として広く応用できることが期待できる。