Functional analysis and application of an effective cell-preservation peptide CPP

2013 Fiscal Year Final Research Report

• Project/Area Number: 23310171
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Resource conservation science
• Research Institution: National Institute of Advanced Industrial Science and Technology
• Principal Investigator: TSUDA SAKAE 独立行政法人産業技術総合研究所, 生物プロセス研究部門, 上級主任研究員 (70211381)
• Co-Investigator(Kenkyū-buntansha): KONDO Hidemasa 産業技術総合研究所, 生物プロセス研究部門, 主任研究員 (80357045) ; NISHIMIYA Yoshiyuki 産業技術総合研究所, ・生物プロセス研究部門, 主任研究員 (00357716) ; SAKASHITA Mami 産業技術総合研究所, 生物プロセス研究部門, 主任研究員 (20357099)
• Project Period (FY): 2011-04-01 – 2014-03-31
• Keywords: 細胞保護物質 / ペプチド / 不凍蛋白質 / 抗凍結作用 / ３次元分子構造 / 大量生産 / Ｘ線回折法 / ＮＭＲ法

Research Abstract

Antifreeze proteins (AFPs) found from many organisms can inhibit ice crystal growth for cold-survival. We preliminarily found that some AFPs also show cell-preservation ability and named them CPP (Cell-Preservation Peptide). During the 3-year research period, we have been tested structure-function relationship of various AFPs to obtain the most promising CPP. In 2013, we finally could develop a method to alive rat insulinoma cells for more than 5 days at +4 degree C by using a flat-fish-derived CPP (PLoS ONE, 2013). In addition, we published the 1st crystal structure of fungal AFP (PNAS, 2012) and structure-function of CPP3 based on the multi-dimensional NMR experiments (JBNMR, 2013). Biochechemical characterizations and many applicational results about AFP and CPP were also published: 23 publications (incl.19 peer-reviewed articles), 40 presentations (incl.14 invited speeches), and 1 patent claim.

Research Products

• [Journal Article] Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity (2014)
  • Author(s): Basu, K., Garnham, C.P., Nishimiya, Y., Tsuda, S., Braslavsky, I., and Davies, P.L.
  • Journal Title: Journal of Visualized Experiments Volume: 83 Pages: e51185
  • DOI: 10.3791/51185
  • Peer Reviewed
• [Journal Article] Identification of a novel LEA protein involved in freezing tolerance in wheat (2014)
  • Author(s): Sakaki, K., Cristov N., Tsuda, S., and Imai, R
  • Journal Title: Plant and Cell Physiology Volume: 55 (1) Pages: 136-147
  • DOI: 10.1093/pcp/pct164
  • Peer Reviewed
• [Journal Article] Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells during Hypothermic Preservation (2013)
  • Author(s): Kamijima, T, Sakashita, M., Miura, A., Nishimiya, Y., and Tsuda, S.
  • Journal Title: PLoS ONE Volume: 8(9) Pages: e73643
  • DOI: 10.1371/journal.pone.0073643
  • Peer Reviewed
• [Journal Article] A simple medium enables bovine embryos to be held for seven days at 4 oC (2013)
  • Author(s): Ideta, A., Aoyagi, Y., Tsuchiya, K., Kamijima, T., Nishimiya, Y., and Tsuda, S
  • Journal Title: Scientific Reports Volume: 3 (1173) Pages: 1-5
  • DOI: 10.1038/srep01173
  • Peer Reviewed
• [Journal Article] A defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongatus Kner (2013)
  • Author(s): Kumeta, H., Ogura, K., Nishimiya, Y., Miura, A., Inagaki, F., and Tsuda, S.
  • Journal Title: J. Biomol. NMR Volume: 55 (2) Pages: 225-230
  • DOI: 10.1007/s10858-012-9703-9
  • Peer Reviewed
• [Journal Article] Microwave-assisted solid-phase synthesis of antifreeze glycopeptides (2013)
  • Author(s): Izumi, R., Matsushita, T., Ohyabu, N., Fujitani, N., Naruchi, K., Shimizu, H., Tsuda, S., Hinou, H., and Nishimura, S.-I
  • Journal Title: Chem. Eur. J. Volume: 19 Pages: 3913-3920
  • DOI: 10.1002/chem.201203731
  • Peer Reviewed
• [Journal Article] Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice (2012)
  • Author(s): Garnham, C.P., Nishimiya, Y., Tsuda, S., and Davies, P.L.
  • Journal Title: FEBS Letters Volume: 586 Pages: 3876-3881
  • DOI: 10.1016/j.febslet.2012.09.017
  • Peer Reviewed
• [Journal Article] 国産魚類由来の不凍タンパク質 (2012)
  • Author(s): 津田栄
  • Journal Title: 日本極限環境生物学会誌(Mini Review) Volume: vol.11 ,No.2 Pages: 64-69
  • URL: http://www.extremophiles.jp/gakkaishi.html
  • Peer Reviewed
• [Journal Article] Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation (2012)
  • Author(s): Kondo, H., Hanada, Y., Hoshino, T., Garnham, C.P., Davies, P.L., and Tsuda, S.
  • Journal Title: Proc. Natl. Acad. Sci Volume: 109 (24) Pages: 9360-9365
  • DOI: 10.1073/pnas.1121607109
  • Peer Reviewed
• [Journal Article] Synthetic study and structural analysis of the antifreeze agent xylomannan from Upis ceramboides (2011)
  • Author(s): Ishiwata, A., Sakurai, A., Nishimiya, Y., Tsuda, S., and Ito, Y.
  • Journal Title: J. Am. Chem. Soc Volume: 133 Pages: 19524-19535
  • DOI: 10.1021/ja208528c
  • Peer Reviewed
• [Presentation] 不凍蛋白質の機能解明に基づく技術創生 (2014)
  • Author(s): 津田 栄
  • Organizer: 第1回北海道大学オープンファシリティシンポジウム
  • Place of Presentation: 札幌市
  • Year and Date: 2014-03-10
  • Invited
• [Presentation] Hypothermic storage for 10 days of bovine embryos using type III antifreeze protein (2014)
  • Author(s): 出田篤司, 津田栄, 西宮佳志, 土屋加那美, 中村雄気, 青柳敬人
  • Organizer: 2014 Meeting of International Embryo Transfer Society (IETS)
  • Place of Presentation: ネバダ州レノ市,米国
  • Year and Date: 2014-01-11
• [Presentation] トウガレイ由来I 型不凍蛋白質の構造機能解析 (2013)
  • Author(s): 石原和成, 花田祐一, 近藤英昌, 三浦愛, 津田栄
  • Organizer: 日本生物物理学会年会
  • Place of Presentation: 京都市
  • Year and Date: 2013-10-29
• [Presentation] 不凍蛋白質(AFP)を利用した牛初期胚の非凍結保存液開発に関する研究 (2013)
  • Author(s): 出田篤司, 津田栄, 西宮佳志, 土屋加那美, 中村雄気, 青柳敬人
  • Organizer: 日本胚移植研究会研究発表大会
  • Place of Presentation: 札幌市
  • Year and Date: 2013-08-26
• [Presentation] Ice-Binding Structure of a bacterial hyperactive antifreeze protein (2013)
  • Author(s): 花田祐一, 近藤英昌, 三浦愛, 津田栄
  • Organizer: 第7回国際構造ゲノム会議-構造生命科学(ICSG2013- SLS)
  • Place of Presentation: 札幌市
  • Year and Date: 2013-07-30
• [Presentation] Antifreeze Protein from Japanese Organisms : Functional Analysis for the General Use (2013)
  • Author(s): 津田栄
  • Organizer: 50th Annual Meeting of the Society for Cryobiology (CRYO2013)
  • Place of Presentation: ワシントンDC,米国
  • Year and Date: 2013-07-29
  • Invited
• [Presentation] NMR Analyses to identify key determinants to regulate ice-binding activity of type III antifreeze protein (2012)
  • Author(s): 津田栄
  • Organizer: 2012生体磁気共鳴国際会議(ICMRBS2012)
  • Place of Presentation: リヨン,仏国
  • Year and Date: 20120000
• [Presentation] Recent technological developments utilizing antifreeze protein (2011)
  • Author(s): 津田栄
  • Organizer: 1st International Ice-Binding Protein Conference and Workshop(IBP2011)
  • Place of Presentation: キングストン,カナダ
  • Year and Date: 2011-08-03
  • Invited
• [Presentation] 不凍タンパク質の分子機能解明に基づく新技術の創成」 (2011)
  • Author(s): 津田栄
  • Organizer: 極限環境生物学会第12回シンポジウム
  • Place of Presentation: つくば
  • Year and Date: 2011-06-13
  • Invited
• [Presentation] 不凍タンパク質の分子機能解析と応用技術研究 (2011)
  • Author(s): 津田栄
  • Organizer: 酵素工学研究会第65回講演会
  • Place of Presentation: 京都
  • Year and Date: 2011-04-22
  • Invited
• [Book] 極限環境生物の産業展開(監修 : 今中忠行) (2012)
  • Author(s): 近藤英昌, 津田栄
  • Total Pages: 121-129
  • Publisher: シーエムシー出版
• [Remarks] 産総研TODAY Research Hotline「細胞治療に役立つカレイ由来不凍タンパク質ー膵島細胞の120時間チルド保存を可能にー」2014年3月6日(プレスリリース・報道・広報誌ほか)
• [Remarks] つくばサイエンスニュース「マウスの膵島細胞,凍結せず5日間の保存に成功ーカレイから抽出の不凍タンパク質使う」2013年9月16日(プレスリリース・報道・広報誌ほか)
• [Remarks] 日経バイオテクON LINE アカデミック版「産総研,魚の不凍たんぱく質で膵島細胞を120 時間非凍結保存,ニチレイが素材提供」2013年10月2日(プレスリリース・報道・広報誌ほか)
• [Remarks] 産総研公式HP「主な研究成果:カレイ由来の不凍タンパク質により細胞保存期間を延長ー120時間の非凍結保存をマウス膵島細胞で実証ー」2013年9月20日(プレスリリース・報道・広報誌ほか)
• [Remarks] SPring-8 大型放射光施設 光のひろば「キノコの不凍タンパク質 不凍機能の解明」2012年(プレスリリース・報道・広報誌ほか)
• [Remarks] 産総研TODAY Research Hotline「キノコの不凍タンパク質 強力な不凍機能をもつタンパク質の立体構造を決定」2012年12月号(プレスリリース・報道・広報誌ほか)
• [Remarks] つくばサイエンスニュース「キノコの不凍タンパク質の分子構造と機能を解明:産業技術総合研究所/北海道大学など」2012年6月3日(プレスリリース・報道・広報誌ほか)
• [Remarks] 産総研公式HP「プレスリリース:キノコの不凍タンパク質の分子構造と不凍機能のメカニズムを解明ー強力な不凍機能をもつタンパク質をキノコが生産ー」2012年5月29日(プレスリリース・報道・広報誌ほか)
• [Remarks] 研究成果を公開しているHP(プレスリリース・報道・広報誌ほか)
  • URL: https://staff.aist.go.jp/s.tsuda/
• [Patent(Industrial Property Rights)] 細胞延命効果を有するペプチドを用いた牛胚の非凍結保存液及び保存方法 (2012)
  • Inventor(s): 西宮佳志, 上島達郎, 津田栄
  • Industrial Property Rights Holder: (独)産業技術総合研究所
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 特願2012-019052
  • Filing Date: 2012-01-31