High-speed method for selection of synthetic binding proteins and the application for quantitative detection of a target protein at single-molecule resolution
| Project/Area Number |
15H02006
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Nanobioscience
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| Research Institution | Nagoya University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
矢島 潤一郎 東京大学, 大学院総合文化研究科, 准教授 (00453499)
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| Project Period (FY) |
2015-04-01 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥41,990,000 (Direct Cost: ¥32,300,000、Indirect Cost: ¥9,690,000)
Fiscal Year 2018: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000)
Fiscal Year 2017: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000)
Fiscal Year 2016: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000)
Fiscal Year 2015: ¥17,030,000 (Direct Cost: ¥13,100,000、Indirect Cost: ¥3,930,000)
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| Keywords | binding protein / screening / translation / single-molecule / 人工抗体 / 1分子定量 / タンパク質 / １分子定量 / 一分子定量 / 抗体 / １分子 / 血管内皮細胞増殖因子受容体 |
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| Outline of Final Research Achievements |
The purpose of this research was to develop a high-speed method for selection of synthetic binding proteins (SBPs) and quantitative detection of the corresponding target proteins at single-molecule resolution. First, we modified our previous high-speed method of screening functional polypeptides (transcription-translation coupled with association of puromycin linker [TRAP] display; JACS, 2013) for SBPs selection. Then, using the improved TRAP display, we demonstrated successful selection of high-affinity (nM level) SBPs against model target proteins, EGFR1 and HER2. Finally, we applied the modified method to obtain several SBPs against low-copy-number proteins and to detect a target protein immobilized on a glass plate using a fluorescently labeled SBP. The improved version of the TRAP display for SBP selection and single-molecule detection of target proteins with the obtained SBPs could become a basic high-precision technology for protein quantification at single-molecule resolution.
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| Academic Significance and Societal Importance of the Research Achievements |
１細胞におけるタンパク質の分子数は、タンパク質の種類によって大きく異なる。これら様々な数からなるタンパク質群の定量は、生物科学において個々の細胞の個性を理解するための重要な課題である。そこで近年、タンパク質を1分子単位で定量する新手法の開発が進められている。このボトルネックは、標的に対して高い特異性と親和性を持った高性能抗体の作製である。本研究では、簡便に高速で人工抗体を作製する手法を開発した。さらに得られた人工抗体を用いてガラス基板上に固定化した標的を1分子単位で検出することができることを示した。本方法は、タンパク質の１分子定量を可能にする重要な基盤技術となると期待される。
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Report
(5 results)
Research Products
(17 results)
