| Project/Area Number |
23685038
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| Research Category |
Grant-in-Aid for Young Scientists (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Chemistry related to living body
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| Research Institution | Nagaoka University of Technology |
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Principal Investigator |
TSUKIJI Shinya 長岡技術科学大学, 産学融合トップランナーセンター, 特任准教授 (40359659)
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| Research Collaborator |
HAMACHI Itaru 京都大学, 大学院工学研究科合成生物化学 専攻, 教授 (90202259)
TAMURA Tomonori 京都大学, 大学院工学研究科合成生物化学 専攻, 大学院生
ISHIDA Manabu 長岡技術科学大学, 産学融合トップランナー養成センター, 博士研究員
KIKUCHI Tamaki 長岡技術科学大学, 生物系, 大学院生
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| Project Period (FY) |
2011-11-18 – 2014-03-31
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| Project Status |
Completed (Fiscal Year 2013)
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| Budget Amount *help |
¥15,340,000 (Direct Cost: ¥11,800,000、Indirect Cost: ¥3,540,000)
Fiscal Year 2013: ¥7,670,000 (Direct Cost: ¥5,900,000、Indirect Cost: ¥1,770,000)
Fiscal Year 2012: ¥7,670,000 (Direct Cost: ¥5,900,000、Indirect Cost: ¥1,770,000)
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| Keywords | 蛋白質ラベリング / アフィニティラベリング / 蛍光イメージング / 部位特異的変異法 / 化学修飾 / アフィニティラベル化 / 蛋白質 / リガンド / トシル化学 / 細胞 / FKBP12 / 光クロスリンク / 蛋白質-蛋白質相互作用 |
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| Research Abstract |
Ligand-directed tosyl (LDT) chemistry is a technique that allows selective chemical labeling of specific (native) proteins in cells and in vivo. However, the LDT chemistry is still at the beginning stage, and thus its full potential has yet to be exploited. In this work, we first attempted to apply the LDT chemistry as a novel tool for live-cell fluorescence imaging of endogenous proteins. Using a newly-designed LDT reagent, we were able to selectively label endogenous FKBP12 with an organic fluorescent dye, Oregon Green (OG), in living mammalian cells. It was also possible to visualize the rapamycin-mediated complexation of the OG-labeled FKBP12 and DsRed-fused FRB inside of the cells by FRET imaging. Furthermore, in this work, we demonstrated that the chemical labeling rate and efficiency could be significantly improved by combining the LDT chemistry with the site-directed mutagenesis technique.
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