This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We are pursuing the idea of using antifreeze proteins (AFPs) for use as both an intracellular and an extracellular cryoprotectant. Some such proteins have already been studied: fish antifreeze proteins are reviewed in: Davies et al., 2002;Harding et al., 2003;Inglis et al., 2006 and analogous proteins from the beetle Dendroides canadensis are reviewed in Duman, 2001. These anti-freeze agents are often glycoproteins and exhibit a lower osmotic activity than pure sugars. In nature these proteins typically work most efficiently at a temperature range of -2 ?approximately -30?C (reviewed in: Harding et al., 2003). AFPs have been shown to prevent arctic fish, frogs, and also some species of insects from damage when exposed to very low temperatures. They can survive at sub-zero temperatures below the equilibrium freezing point of their body fluids, and some fish even survive being frozen into a block of ice. Nevertheless, the regular functions of antifreeze proteins are at slow-freezing conditions, not the rapid freezing conditions applied in a plunge or high-pressure freezer. At slow cooling rates any type of cryo-protectant will eventually allow the formation of hexagonal ice not too far below 0?C. At rapid cooling rates, however, we expect AFPs to have a different effect of smearing out and raising the vitrified-crystalline phase transition point (pure water= -140?C) and thereby preventing ice-crystal formation during the freezing process, essentially the same way other cryo-protectants do, but with less osmotic stress to the cells. Also, AFPs have been shown to bind ice directly with their surface, which seems to be their general mechanism of preventing the formation of large ice crystals. When applied to the extracellular medium they may also render the ice less brittle, which may improve cryo-microtomy. Hence, for external use the challenge will be to express and purify them with their native glycosylation. To this end we will adapt protocols for cloning and expressing these proteins into our own cell systems of interest (e.g., see Macouzet et al., 1999). For intracellular use they will be directly cloned and expressed in a stable, genetically accessible cell line.
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