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Research paper : Mass preparation and technological development of an antifreeze protein (Y. Nishimiya, et al.)−13−Synthesiology - English edition Vol.1 No.1 (2008) AuthorsYoshiyuki NishimiyaFunctional Protein Research Group, Research Institute of Genome-based Biofactory of AISTSenior researcher (2000-present). Research interests are identification, functional analysis, mass-preparation method, and practical application of antifreeze protein. Main research fields are molecular biology and directed molecular evolution. Ph.D. Graduate School of Engineering, Tohoku University (2000).Yasuhiro MieBiomolecular Engineering Group, Research Institute of Genome-based Biofactory of AISTResearcher (2005-present). Research interests are assembly of proteins on a surface that functions as useful material and tool. Electron transfer between electrode and metalloprotein. Main research fields are electrochemistry and biomedical engineering (biosensor). Ph.D. Graduate School of Engineering, Kumamoto University (2000).Yu HiranoBiomolecular Engineering Group, Research Institute of Genome-based Biofactory of AISTResearcher (2005-present). Research interests are development of scanning electrochemical microscopy for the analysis of cell preservation mechanism by antifreeze protein. Main research fields are electrochemistry and biomedical engineering (biosensor). Education: Ph.D. Graduate School of Engineering, Tohoku University (2005).Hidemasa KondoFunctional Protein Research Group, Research Institute of Genome-based Biofactory of AISTSenior researcher (2005-present). Research interests are elucidation of molecular mechanism of antifreeze protein and industrial enzyme by using X-ray crystallography. Main research fields are protein crystallography and structural biology. Ph.D. Graduate School of Science, Hokkaido University (1997).Ai MiuraFunctional Protein Research Group, Research Institute of Genome-based Biofactory of AISTResearch Assistant (1996-present). Research interests are new exploration of fish antifreeze protein and its technological applications (frozen foods, ice slurry, etc.). Main research fields are preparation and analysis of two- and three-dimensional NMR spectra of a protein. Dietitian (1994).Sakae TsudaFunctional Protein Research Group, Research Institute of Genome-based Biofactory of AISTGroup leader (2001-present) and professor (2003-present). Research interests are low-temperature biology, protein-ice interaction, and development of energy-saving technologies. Main research fields are multidimensional NMR spectroscopy and structural biology. Ph.D. Graduate School of Science, Hokkaido University (1993).Discussion with reviewers1 Quantitative AspectsQuestion (Koichi Mizuno)With regard to the ice-nucleation ability of AFP-assembled aluminum plate, is it possible to numerically quantify the superiority of this plate compared to other materials for freezing water near 0 °C? Answer (Sakae Tsuda)It is very difficult for us to give concrete answer to your question. Suppose that our plate can freeze a 1 mL water droplet at -1 °C, for which -18 °C of Tf was evaluated when the plate was not used, we need to evaluate the amount of energetic barrier which causes ice-nucleation in a water droplet. This energetic barrier, however, depends on many parameters, such as cooling rate, environmental perturbations, and contaminants, which can hardly be evaluated. As for difference in potential energy, approximately 40 J is estimated for 1 mL of ice crystal between -18 and -1 °C. Please note that we avoided complete descriptions of the performance of our developments due to future patentability.2 Cell preservation abilityQuestion (Koichi Mizuno)Did the authors attempt preservation of hepatocells (HepG2) near 0 °C without freezing them? The fact that 90 % of HepG2 cells die in 12 hours using commercial fluid seemed to me that 0 °C is not suitable for cell preservation. I imagine that AFP may be having some biological influence on the cells. Has detailed mechanism of such influence been clarified? Answer (Sakae Tsuda)The method to preserve cells and tissues from heart, liver, and kidney for 1~24 hours near 0 °C without freezing has actually been utilized in the medical fields, such as transplantation T. Inada and S.-S. Lu: Thermal hysteresis caused by non-equilibrium antifreeze activity of poly(vinyl alcohol), Chem. Phys. Lett., 394, 361-365 (2004).M. Okada, K. Miyazaki, eds: Kaitei dai3pan tanpakushitsu jikken noto (jokan)-chushitsu・bunri to kumikae tanpakushitsu no hatsugen-(Protein Experiment Note, 3rd Edition: Extraction, Separation, and Gene Expression of a Protein), 202, Yodo Press (2004) (in Japanese).M. Nomoto:Koso kogaku (The Enzyme Technology), Gakkai Press Center, Tokyo (1993) (in Japanese).R.E. Feeney and Y. Yeh: Antifreeze proteins: Current status and possible food uses, Trends Food Sci Tech., 9, 102-106 (1998).M. Takamichi, Y. Nishimiya, A. Miura and S. Tsuda: Effect of annealing time of an ice crystal on the activity of type III antifreeze protein, FEBS J., 274 (24), 6469-6476 (2007).Y. Nishimiya, R. Sato, M. Takamichi, A. Miura and S. Tsuda: Co-operative effect of the isoforms of type III antifreeze protein expressed in notched-fin eelpout, Zoarces elongatus Kner, FEBS J., 272, 482-492 (2005).Y. Hirano, Y. Nishimiya, S. Matsumoto, M. Matsushita, S. Todo, A. Miura, Y. Komatsu and S. Tsuda: Hypothermic preservation effect on mammalian cells of type III antifreeze protein from notched-fin eelpout, Cryobiology (in press).Y. Nishimiya, S. Ohgiya and S. Tsuda: Artificial multimers of the type III antifreeze protein: effects on thermal hysteresis and ice crystal morphology, J. Biol. Chem., 278 (34), 32307-32312 (2003).Y. Mie, Y. Nishimiya, F. Mizutani and S. Tsuda: Assembly of antifreeze protein reveals the ice nucleation activity, (in preparation).Received original manuscript September 18,2007Revisions received November 19,2007Accepted November 19,2007[7][8][9][10][11][12][13][14][15]

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