Use of FALI with cDNA display single-domain antibodies for efficient functional screening of therapeutic targets
| Project/Area Number |
16K15206
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
General pharmacology
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| Research Institution | Juntendo University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
根本 直人 埼玉大学, 理工学研究科, 教授 (60509727)
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| Research Collaborator |
HASHIMOTO yoshie
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Keywords | 組換え抗体 / 神経科学 / プロテオーム / cDNAディスプレイ |
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| Outline of Final Research Achievements |
Fluorophore-assisted light inactivation (FALI) enables real-time inactivation of specific proteins targeted in-situ by fluorophore-labeled probes. In combination with a cell-based assay, FALI is a valuable technique for use as a functional proteomic screen to search for druggable targets. One of the major obstacles of this method is the construction of probe libraries containing high-affinity binders that cover cell surface membrane proteins as potential targets. To overcome this barrier, we used synthetic single domain antibodies derived from camel variable domains of heavy chain antibodies (VHH) and alternative non-antibody scaffolds. The efficiency of cell-free cDNA display expression of the molecules was measured. Through the use of thermostable scaffolds, we improved the expression of non-antibody affinity reagents linked to their genotypes. We attempted high-affinity clone selection for membrane proteins from a newly synthesized non-antibody probe library.
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| Academic Significance and Societal Importance of the Research Achievements |
神経-グリア相互作用などの細胞間相互作用は、その局所における時間依存的なシグナルにより制御されていることから、それに関わるタンパク質の解析・探索には、時間分解能の高い方法が必要とされる。FALIと細胞機能アッセイの組合せはこれに適した方法であるが、その効率化には問題があった。本研究は組換え抗体様分子とcDNA displayによる課題解決を目的としたものであり、実現すればこれまでにない特徴を持った細胞機能スクリーニングとして創薬に結びつけることが可能である。
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Report
(4 results)
Research Products
(15 results)
