Vol.1 No.1 2008

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Research paper : Mass preparation and technological development of an antifreeze protein (Y. Nishimiya, et al.)−8−Synthesiology - English edition Vol.1 No.1 (2008) AFP. For example, an AFP-assembled plate (Figure 1C) may be capable of ice-nucleation, enabling efficient freezing of attached water near 0 °C (Figure 1A). Strong inhibition of ice growth by AFP in home freezer (Figure 1D) and cell preservation with AFP near 0 °C (Figure 1E) [3, 4] also suggests the potential for AFP as an environment-friendly cryotechnology. Since large amounts of AFP are required to achieve this goal, we conducted studies to develop a mass preparation method for AFP. In addition, we explored the potential of AFP in sample cryopreservation for industry and medicine. 2 BackgroundOrganisms produce a variety of proteins with various functions including metabolism, transportation, storage, structural formation, and immunoreaction. Many enzymes are utilized commercially and medically. Proteins recovered from extreme environments including volcanoes, hydrothermal vents, deep sea, deserts, polar regions, and certain hazardous materials have industrial potential. AFP is an example of such protein. AFP was discovered in 1969 in the serum of Antarctic cold-adaptive fish [5]. Similar ice crystallization inhibition is demonstrated with biosurfactants [6] and poly vinyl alcohol [7], but their property (determined as thermal hysteresis activity [3]) is markedly weaker than that of AFP.Modern technological developments have dramatically improved instrument sensitivity, reducing the amount of sample needed for experimental determinations. For example, 1 µg of protein is sufficient to analyze amino acid sequence and few milligrams enable determination of three-dimensional (3D) structure by employing nuclear magnetic resonance and X-ray diffraction techniques. Biochemically, 10~20 mg of protein represents a large amount [8], and techniques that yield quantities in grams or kilograms were not considered a priority. However, such quantities are necessary to enable AFP research to advance its utility in different fields such as engineering, medicine, and food technology. Biotechnology has long been concerned with large-scale purification of target proteins [9]. Genetic engineering combined with large-scale culturing of the transformed cells has achieved production of commercial quantities of several proteins such as cold temperature active enzymes used in laundry detergent, starch processing, pharmaceutical proteins, diagnostic antibodies, and bio-ethanol. However, large-scale preparation of genetically engineered proteins is difficult [9]. Recognizing these continuing challenges, we decided at present it would be most prudent to consider other methods such as chemical synthesis and purification to mass-produce AFP from natural sources.3 ScenarioResearchers from many different fields including biology, biochemistry, molecular biology, ice physics, biophysics, structural biology, and computer chemistry have studied AFP, and many papers have been published in reputable journals. Potential applicability of AFP in industrial and medical fields has been suggested [10]. However, this potential has not yet been realized. Production of AFP still involves using needle and syringe to collect the protein from serum of live fish. This arduous method and low product yield are reflected in the high purchase price of AFP (approximately USD 10/mg). Typically, AFP is collected on-site (during Arctic fishing) and transported south. However, we have observed that fish near Japan (~43 ºN) also produce AFP [11]. To investigate this Fig. 2 Yield and antifreeze activity of AFP. A: Results of total purified AFP (yield) per 100 g of different portions of an AFP-containing fish (material). B: Dependence of thermal hysteresis activity (i.e. antifreeze activity) on AFP concentration. A less active AFP isoform (SP-type) exhibits a substantial level of activity by addition of small amount of active AFP isoform [12]. abcd75 mg73 mg90 mg107 mgAB2.00.51.01.50.00.80.20.40.60.0Figure 2 Materials Yield / 100 gMuscleHeadHeartEntrailsMuscleHeadMuscleHeartHeadMuscleThermal Hysteresis (oC)isoform mixturessingle isoform (SP-type)Concentration (mM)

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