Polar organisms should overcome the problems of decreased enzyme activity, decreased membrane fluidity, inactivation and improper folding of proteins, formation of intracellular ice crystals, etc. to survive in low-temperature, polar environments. Among others, the formation of ice crystals causes physical damages and dehydration of tissues due to the growth of ice crystals, thus causing serious damage to polar organisms. Polar organisms produce various antifreeze proteins (hereinafter referred to as “AFPs”) to survive at low temperatures. AFPs inhibit the growth of ice crystals in vivo and the recrystallization of ice to protect polar organisms from sub-zero temperatures to survive (Davies, P. L. and Sykes, B. D., Curr. Opin. Struct. Biol. 7, 1997, 828-834; Davies, P. L. et al., Philos Trans R Soc Lond B Biol Sci. 357, 2002, 927-935; D'Amico, S. et al., EMBO Rep. 7, 2006, 385-389).
AFPs are proteins that generally have a flat ice-binding surface and bind to specific surfaces of ice crystals, thus inhibiting the growth of ice crystals and the recrystallization of ice. AFPs create a difference between the melting point and freezing point. This is called thermal hysteresis (TH), which can be measured using a nanoliter osmometer and used as an indicator of AFP activity. Moreover, AFPs do not lower the freezing point in proportion to the concentration, unlike typical antifreeze used in vehicles. That is, AFPs can effectively lower the freezing point even at very low concentrations by direct interaction with ice, thus minimizing damage due to osmotic pressure generated in vivo during freezing (Jia, Z. and Davies P. L., Trends Biochem. Sci. 27, 2002, 101-106).
The unique features of AFPs that prevent the growth of ice crystals and inhibit the recrystallization of ice have been used in various commercial fields. For example, in the agricultural field, AFP expression in plants has been attempted for the purpose of preventing cold-weather damage to plants. Moreover, in the field of fisheries, there has been an attempt to produce a transgenic fish by expressing AFPs in commercially available fish such as Atlantic salmon (Salmo salar) or goldfish (Carassius auratus) so as to enable farming in cold areas. Furthermore, in the medical field, research on the use of AFPs in cryosurgery and as an additive in cryopreservation of blood, stem cells, umbilical cord blood, organs, and germ cells has continued to progress. In addition, in the food field, AFPs are also used in product production for frozen storage of smoother ice scream. In the field of cosmetics, functional cosmetics containing AFPs for preventing frostbite have already been sold. Although AFPs are widely used in various commercial fields as mentioned above, there are still limitations in mass production of recombinant AFPs due to low-level expression of AFPs and folding problems. This is mainly because most AFPs have disulfide bonds and are stabilized by disulfide bonds, which thus makes it difficult to express recombinant proteins and yields improper folding of expressed proteins.
Since AFPs were first discovered in fish living in cold water, various types of new AFPs have been discovered in insects, plants, fungi, microorganisms, etc. New AY30 AFP derived from arctic yeast, Leucosporidium sp., has recently been recovered. The AY30 AFP has no cysteine amino acid residues, and thus during production of recombinant proteins, the level of protein expression is high, and the folding problem due to improperly formed disulfide bonds does not occur, As a result, the AY30 AFP is suitable for mass production of recombinant AFPs.
Therefore, the present inventors have synthesized a recombinant polynucleotide by modifying an AFP gene to be expressed using codon optimization for a yeast expression system and inserted the recombinant polynucleotide into a yeast-derived expression vector so as to mass-produce an antifreeze protein (AFP) derived from arctic yeast by overexpressing AFP in the form of activated protein. As a result, the present inventors have obtained a large amount of AFP and found that the AFP is glycosylated, thus completing the present invention. All references cited in this specification are hereby incorporated by reference in their entirety.