USING ANTIFREEZE PROTEINS AS CRYOPROTECTANTS FOR FREEZING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们正在追求使用抗冻蛋白(AFP)作为细胞内和细胞外冷冻保护剂的想法。一些这样的蛋白质已经被研究：鱼的抗冻蛋白在Davies等人，2002；Harding等人，2003；Inglis等人，2006年；以及来自甲虫的加拿大杜鹃的类似蛋白质在Duman，2001中被综述。这些抗冻剂通常是糖蛋白，表现出比纯糖更低的渗透活性。在自然界中，这些蛋白质通常在-2约-30℃的温度范围内最有效地工作(见：Harding等人，2003)。AFPS已经被证明可以防止北极鱼、青蛙和一些种类的昆虫在暴露在非常低的温度下时受到损害。它们可以在低于体液平衡冰点的零下温度下生存，一些鱼甚至可以在冰块中存活下来。然而，抗冻蛋白的正常功能是在缓慢冻结的条件下，而不是在快速冻结条件下应用于跳跃式或高压冷冻机。在缓慢的降温速度下，任何类型的低温保护剂最终都会使六角形的冰形成，但在快速降温速度下，我们预计AFP会有不同的效果，涂抹并提高玻璃晶体的相变点(纯水=-140℃)，从而防止在冷冻过程中形成冰晶，基本上与其他低温保护剂相同，但对细胞的渗透压力较小。此外，AFP已被证明直接将冰与其表面结合，这似乎是它们防止大冰晶形成的一般机制。当应用到细胞外介质中时，它们也可以使冰不那么脆，这可能会改进冷冻显微切割。因此，对于外用，挑战将是用它们的天然糖基化来表达和纯化它们。为此，我们将修改克隆这些蛋白质并将其表达到我们自己感兴趣的细胞系统中的方案(例如，参见Macouzet等人，1999)。为了在细胞内使用，它们将被直接克隆并在稳定的、可遗传的细胞系中表达。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.