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1 pp. β- MRI PRE FMRI in vitro FP Chalfie Tsien Design, synthesis and biological application of chemical probes for imaging protein functions Shin Mizukami and Kazuya Kikuchi Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka, Japan FP Fura- Ca Tsien FMRI

Kazuya Kikuchi Division of Advanced Science and Biotechnology, Graduate School

2 ab FlAsH, ReAsH Griffin et al. HaloTag Los et al. SNAP-tag O - Keppler et al. CLIP-tag SNAP-tag SNAP-tag Gautier et al. Chen et al. Q-tag Lin et al. His-tag Ni -NTA Guignetetal. D -tag Zn Ojida et al. a b kda GFP Da HaloTag kda A β- A β- K β- β-

3 A β- TEM- a TEM- Ser β- acylation Glu deacylation Glu Asn E N TEM k b a TEM-b TEM E N TEM β- a CA E N TEM b c SDS-PAGE CA b E N TEM c WT TEM-

4 E N TEM CA a CA - E N TEM E N TEM CA b CBB E N TEM kda CA TEM- CA c E N TEM CA HaloTag SNAP-tag CA CA b CA fluorogenic β- a CCD E N TEM b SDS-PAGE CCD E N TEM c E N TEM CCD d HEK T E N TEM MBP CCD

5 CCD a CCD CCD β- - N,N - Dabcyl Dabcyl FRET fluorescence resonance energy transfer CCD Dabcyl FRET a β- Dabcyl CA CCD E N TEM SDS-PAGE CCD CCD b c FRET maltose binding protein MBP MBP C E N TEM CCD HEK d MBP E N TEM CCD CCD E N TEM epidermal growth factor receptor EGFR EGFR N E N TEM HEK T µm CCD PBS E N TEM-EGFR CCD EGFR MRI magnetic resonance imaging MRI FMRI MRI MRI MRI MRI MRI Meade MRI Xenopus laevis H MRI

E N TEM CCD CCD E N TEM epidermal growth factor receptor EGFR EGFR N E N TEM HEK T µm CCD PBS E N

6 E N TEM-EGFR EGFR HEK T FMRI HMRI F MRI MRI a FMRI b

7 MRI HMRI F MRI F H NMR F FMRI F MRI HMRI FMRI FMRI FMRI MRI x y z NMR MRI NMR NMR T T T MRI T MRI T paramagnetic relaxation enhancement PRE PRE MRI a DOTA-DEVD-Tfb Gd-DOTA-DEVD-Tfb F NMR b c- Gd-DOTA- DEVD-Tfb b FNMR c FMRI

8 NMR T T Gd f PRE Gd NMR T a Gd NMR F T PRE MRI Gd F TMRI F MRI - FMRI -- DEVD C DEVD Gd F Gd- DOTA-DEVD-Tfb b Gd-DOTA-DEVD-Tfb FNMR Gd DOTA-DEVD-Tfb a T Gd-DOTA-DEVD-Tfb T Gd-DOTA- DEVD-Tfb - FNMR btms - FMRI Gd-DOTA-DEVD-Tfb FMRI - c PRE FMRIin vitro - MRI Gd-DOTA- DEVD-AFC b MRI FMRI FMRI in vivo MRI Chudakov, D.M., Lukyanov, S., & Lukyanov, K.A. Trends Biotechnol.,,. Grynkiewicz, G., Poenie, M., & Tsien, R.Y.J. Biol. Chem.,,. Chen, I. & Ting, A.Y.Curr. Opin. Biotechnol.,,. Griffin, B.A., Adams, S.R., & Tsien, R.Y.Science,,. Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohane, R. F.,Urh,M.,Simpson,D.,Mendez,J.,Zimmerman,K.,Otto, P., Vidugiris, G., Zhu, J., Darzins, A., Klaubert, D.H., Bulleit, R.F., Wood, K.V.ACS Chem. Biol.,,. Keppler, A., Gendreizig, S., Pick, H., Vogel, H., & Johnsson, K.Nat. Biotechnol.,,. Gautier, A., Juillerat, A., Heinis, C., Correa, Jr, I.R, Kindermann, M., Beaufils, F., & Johnsson, K.Chem. Biol.,,. Chen, I., Howarth, M., Lin, W., & Ting, A.Y.Nat. Methods,,. Lin, C.W. & Ting, A.Y.,J. Am. Chem. Soc.,,.

, Poenie, M., & Tsien, R.Y.J. Biol. Chem.,,. Chen, I. & Ting, A.Y.Curr. Opin. Biotechnol.,,. Griffin, B.A., Adams, S.R., & Tsien, R.Y.Science,,. Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.

9 Guignet, E.G., Hovius, R., & Vogel, H.Nat. Biotechnol.,,. Ojida, A., Honda, K., Shinmi, D., Kiyonaka, S., Mori, Y., & Hamachi, I.J. Am. Chem. Soc.,,. Matagne, A., Lammote-Blasseur, J., & Frere, J.M.Biochem. J.,,. Guillaume, G., Vanhove, M., Lammote-Blasseur, J., Ledent, P., Jamin,M.,Joris,B.,&Frere,J.M.J. Biol. Chem.,,. Adachi, H., Ohta, T., & Matsuzawa, H.J. Biol. Chem.,,. Mizukami, S., Watanabe, S., Hori, Y., & Kikuchi, K.J. Am. Chem. Soc.,,. Louie, A.Y., Hüber, M.M., Ahrens, E.T., Rothbächer, U., Moats, R., Jacobs, R.E., Fraser, S.E., & Meade, T.J. Nat. Biotechnol.,,. Ahrens, E.T., Flores, R., Xu, H., & Morel, P.Nat. Biotechnol.,,. Helm, L.Prog. Nucl. Magn. Reson. Spectrosc.,,. Mizukami, S., Takikawa, R., Sugihara, F., Hori, Y., Tochio, H., Wälchli, M., Shirakawa, M., & Kikuchi, K.J. Am. Chem. Soc.,,. Mizukami, S., Takikawa, R., Sugihara, F., Shirakawa, M., & Kikuchi, K.Angew. Chem. Int. Ed.,,.

, Hori, Y., & Kikuchi, K.J. Am. Chem. Soc.,,. Louie, A.Y., Hüber, M.M., Ahrens, E.T., Rothbächer, U., Moats, R., Jacobs, R.E., Fraser, S.E., & Meade, T.J. Nat. Biotechnol.,,. Ahrens, E.T., Flores, R.

37-4.indd

37-4.indd ISSN 0916-3328 東京大学アイソトープ総合センター VOL. 37 NO. 4 2007. 3. 26 アイソトープ総合センターの将来展望 40 18 18 20 3 800 1/3 1300 14 18 20 3 19 2 生きた細胞内の分子過程を時間空間計測する蛍光プローブの開発 1. はじめに Fura-2 Fura-2 198 1, 2 3, 4 2. 生体脂質分子の機能とその分析