2021 Fiscal Year Final Research Report

Application of hyperactive antifreeze from a cold-tolerant insect

Research Project

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Project/Area Number	19K22989
Research Category	Grant-in-Aid for Challenging Research (Exploratory)
Allocation Type	Multi-year Fund
Review Section	Medium-sized Section 90:Biomedical engineering and related fields
Research Institution	National Institute of Advanced Industrial Science and Technology
Principal Investigator	TSUDA SAKAE 国立研究開発法人産業技術総合研究所, 生命工学領域, 上級主任研究員 (70211381)
Co-Investigator(Kenkyū-buntansha)	大山恭史国立研究開発法人産業技術総合研究所, 生命工学領域, 主任研究員 (80356675) 近藤英昌国立研究開発法人産業技術総合研究所, 生命工学領域, 主任研究員 (80357045)
Project Period (FY)	2019-06-28 – 2022-03-31
Keywords	不凍タンパク質 / ナノ氷結晶 / 過冷却 / 分子構造 / 熱ヒステリシス
Outline of Final Research Achievements	This research clarified that a stag beetle (Dorcus hopei binodulosus) synthesizes a hyperactive antifreeze protein (DhbAFP), which unexpectedly shares a significant identity with an AFP identified from a phylogenetically distant beetle discovered in 1990s. It also appeared that Dhb larvae are not frozen during minus 5 deg C storage for 24 hrs. The native DhbAFP sample appeared to be a mixture of 6 isoforms, each of which consists of tandem repeats of a 12-residue consensus sequence. A special instrument to purify this beetle AFP by employing ice-probe was also prepared, which simplified the AFP preparation procedure. A supercooled cell storage solution containing DhbAFP appeared to improve the survival rate (%) of rat insulinoma cells, which suggests that AFP binds to embryo ice crystals generated in supercooled water to make them "nano" ice crystals, thereby minimizing physical damages onto the preserved cells. These results were published in world-wide.
Free Research Field	生物物理学、低温生物学
Academic Significance and Societal Importance of the Research Achievements	水を冷却すると過冷却と呼ばれる状態を経て氷になる。過冷却水の中は無数の単結晶氷が発生した状態と考えられ、本研究はそれらをナノサイズ（ナノ氷結晶）に留める高性能不凍タンパク質（DhbAFP）をオオクワガタの体液から抽出することに成功した。DhbAFPのアミノ酸配列、遺伝子配列、生化学的性質、氷結晶結合能、3次元分子構造などが明らかになり、その取得技術も本研究の中で確立された。DhbAFPを含む保存液を使ってマイナス5℃で細胞を非凍結保存したときに20日間保存後で53％という極めて高い生存率が得られたことはナノ氷結晶の有効性を示していると考えられた。