USING ANTIFREEZE PROTEINS AS CRYOPROTECTANTS FOR FREEZING

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们正在寻求使用抗冻蛋白（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过：鱼类抗冻蛋白综述于：Davies et al.，2002; Harding等人，2003; Inglis等人，Duman，2001中综述了来自甲虫Dendroides canadensis的类似蛋白。 这些防冻剂通常是糖蛋白，并且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 ℃的温度范围内最有效地工作。  约-30 ° C（综述于：Harding等人，2003年）。AFP已被证明可以防止北极鱼类，青蛙和一些昆虫在暴露于非常低的温度时受到伤害。 它们可以在低于其体液平衡冰点的零度以下的温度下生存，有些鱼甚至可以在被冻成冰块的情况下生存。然而，抗冻蛋白的常规功能是在缓慢冷冻条件下，而不是在插入式或高压冷冻机中应用的快速冷冻条件。 在缓慢的冷却速率下，任何类型的低温保护剂最终都会在0 º C以下不太远的地方形成六边形冰。然而，在快速冷却速率下，我们预计AFP具有不同的涂抹效果和提高玻璃化-结晶相变点（纯水= -140 ° C），从而防止冷冻过程中冰晶的形成，基本上与其他冷冻保护剂相同，但对细胞的渗透应力较小。 此外，AFP已被证明可以直接将冰与其表面结合，这似乎是其防止大冰晶形成的一般机制。 当应用于细胞外介质时，它们也可以使冰不那么脆，这可以改善冷冻切片术。因此，对于外部使用，挑战将是用其天然糖基化表达和纯化它们。 为此，我们将调整用于克隆和表达这些蛋白质到我们自己的感兴趣的细胞系统中的方案（例如，参见Macouzet等人，1999年）。 对于细胞内使用，它们将被直接克隆并在稳定的、遗传可及的细胞系中表达。

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.