Vol.1 No.1 2008

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Research paper : Mass preparation and technological development of an antifreeze protein (Y. Nishimiya, et al.)−12−Synthesiology - English edition Vol.1 No.1 (2008) 6.0 x 1011 type III AFPs were assembled per square cm. Detailed descriptions of this experiment will be presented in a separate paper. Here, we show some of the results. First, a droplet of water placed on the AFP-assembled plate froze at approximately 5 ºC higher temperature than the unassembled plate, consistent with ice-nucleation ability of the surface. Second, unidirectional freezing occurred from the surface of AFP-assembled plate, which led to formation of extremely clear ice. AFP could be assembled not only on flat metal surface but also on curved substances and particles in various sizes.Figure 6D is a photograph of cell preservation fluid containing AFP. Human and animal cells could not maintain their functions in vitro for a prolonged time. In the fields of organ transplant and regenerative medicine, tremendous efforts are spent to achieve long-term preservation of cells and organs in both frozen and unfrozen states. Earlier in this report, we explained the effectiveness of quick-freezing using very low temperature (e.g. LN2) for preservation of frozen substance (Figure 1). Here, we briefly report our results of cell preservation in unfrozen state, which will be useful in 1~21 day period before transplantation of cultured cells. We attempted to preserve approximately 10,000 unfrozen (0 ºC) human hepatoma cells (HepG2) without AFP. Ninety percent of the HepG2 cells died within 12 h using commercially available preservation fluid. In contrast, AFP-containing preservation fluid (Figure 6D) preserved 90 % of the HepG2 cells even after 72 h [13]. This preservative effect of AFP was also identified in cell lines of small intestine, kidney, umbilical cord, blood, cervix, and pleural effusion. Gram quantities of highly purified AFP were sufficient to examine the preservation effect on cells, but were insufficient for tissue and organ examinations. Further study is necessary to overcome this problem.6 Future developmentAmount of protein that may produce superior performance at molecular level can be insufficient for practical use. In other words, quantity has been a hindrance for expansion of basic research into practical technology. In the case of AFP, the ability to generate grams of product enables collaborative advances in different fields such as food, medicine, and engineering. More collaborations are expected, and product utilization of AFP is becoming a reality. We note with interest that the currently reported technique that brings the benefits of lowered energy consumption and CO2 emission is derived from the classical extraction of target protein from natural resources. At the same time, our technique utilizes advanced studies from molecular biology to 3D structural analysis.Applications of AFP in medical fields will require approval from the appropriate government agencies concerning aspects that include toxicity, mutagenicity, and carcinogenicity. These approvals may require time. It will also be necessary to construct AFP preparation facility that satisfies the regulation of Good Manufacturing Practice (GMP). Although much remains to be done, this study is an encouraging start. In addition to applications in food industry, partially purified AFP may be used in the cold storage systems in office buildings. Most air conditioners work by lowering the temperature of a building through circulation of refrigerant. Replacement of refrigerant with ice slurry would save energy while maintaining the same level of air conditioning. Partially purified AFPs would prevent aggregation of ice slurry that often occurs during circulation. Many biological compounds including antifreeze proteins have potential industrial applications. However, sufficient quantities of these compounds are required for research to further investigate their applications. AFP is one example. Bio-ethanol is another. This study was primarily based on the functional analysis of protein from organisms of Hokkaido. In July 2008, the G8 Summit was held at Lake Toya in Hokkaido. We would like to contribute to reducing global warming through our technologies. AcknowledgementsWe thank Dr. Takaaki Inada (Energy Technology Research Institute (ETRI), AIST), Dr. Shuichiro Matsumoto, Prof. Michiaki Matsushita, and Prof. Satoru Todo (General and Digestive Surgery, Graduate School of Medicine, Hokkaido University) for many helpful advices and discussions. We also thank Fumie Shiraishi, Etsuko Hayashi, and Michiko Ito for developments of mass preparation techniques of AFP.ReferencesP.V. Hobbs: Ice Physics, 18-39, Oxford University Press, London (1974).H.Tsuyuki: Shokuhin kakogaku dai 2 han-kako kara hozo made -(Processing of Food: From Processing to Storage), 14-16, Kyoritsu Press, Tokyo (1990) (in Japanese). ] Y. Yeh, and R.E. Feeney: Antifreeze proteins: Structures and mechanisms of function, Chemical Reviews, 92(2), 601-617 (1996).B. Rubinsky, A. Arav, and G.L. Fletcher: Hypothermic protection: A fundamental property of “antifreeze” proteins, Biochem. Biophys. Res. Commun., 180(2), 566-571 (1991).Z. Jia, and P.L. Davies: Antifreeze proteins: an unusual receptor-ligand interaction, Trends Biochem. Sci., 27, 101-106 (2002).D. Kitamoto, H. Yanagishita, A. Endo, M. Nakaiwa, T. Nakane, and T. Akiya: Remarkable antiagglomeration effect of a yeast biosurfactant, diacylmannosylerythritol, on ice-water slurry for cold thermal storage, Biotechnol. Prog., 17, 362-365 (2001).[1][2][3][4][5][6]

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