| 研究課題/領域番号 |
22K14828
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| 研究種目 |
若手研究
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| 配分区分 | 基金 |
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| 審査区分 |
小区分38030:応用生物化学関連
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| 研究機関 | 名古屋大学 |
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研究代表者 |
ダムナニョヴィッチ ヤスミナ 名古屋大学, 生命農学研究科, 講師 (00754673)
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| 研究期間 (年度) |
2022-04-01 – 2025-03-31
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| 研究課題ステータス |
交付 (2023年度)
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| 配分額 *注記 |
3,640千円 (直接経費: 2,800千円、間接経費: 840千円)
2024年度: 1,040千円 (直接経費: 800千円、間接経費: 240千円)
2023年度: 910千円 (直接経費: 700千円、間接経費: 210千円)
2022年度: 1,690千円 (直接経費: 1,300千円、間接経費: 390千円)
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| キーワード | protein engineering / enzyme engineering / single-molecule display / in vitro translation / activity-based selection / NGS / bioinformatics / in vitro display / cDNA display |
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| 研究開始時の研究の概要 |
This research aims to develop a system for the rapid development of enzymes, as environmentally friendly, safe, and inexpensive bio-tools used to drive the conversions of various compounds into useful products. Enzymes with improved properties have a wide range of biotechnological applications (industry, pharma, diagnostics).
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| 研究実績の概要 |
DAAO engineering for specific detection of D-Ala: We mapped the sites of interest for mutagenesis and conducted DAAO random library screening, as well as established a reliable assay to monitor the selection progress across multiple selection rounds. To increase the likelihood of selecting improved variants, we introduced negative selection steps aimed at eliminating non-specific DAAO variants.
MTG engineering for site-specific antibody-drug conjugation: The selection system for sequence-specific MTG has been established, and its first evaluation completed.
PLD engineering for improved synthesis of designer phospholipids: We discovered ways to improve the efficiency of enzyme display formation, and are currently evaluating the PLD selection system.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
In the AY2023, the project continued for all three set themes. Engineering of DAAO is proceeding as planned. We created several mutant libraries to probe different parts of the enzyme structure for substrate specificity change. We have also introduced a control library where we mutated one residue of the active site and performed selection to verify the enrichment of the active DAAO by NGS.
Engineering of TG continues with selection evaluation using model binary libraries. Engineering of PLD proceeds at the slowest pace, since we encountered problems with PLD display formation efficiency, which we are addressing by changing its sequence, and since recently, by mutagenesis using structural bioinformatics.
The progress of all three themes has been reported at local and international meetings.
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| 今後の研究の推進方策 |
In AY2024, we plan to continue according to the experimental plan. After completing the DAAO selection, we will analyze the genes of selected DAAO variants and design second-generation libraries to fine-tune the specificity of DAAO for D-Ala.
MTG engineering will continue with the preparation and selection of mutant MTG libraries, followed by NGS analysis.
We will use structural bioinformatics to predict mutation sites in PLD, and proceed to evaluate the PLD selection system.
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