Virtual Gating Machines for Protein Purification

描述(申请人提供)：大多数商业规模的蛋白质生产使用柱层析和合成膜来分离和浓缩蛋白质。尽管必要的多次回收步骤提高了纯度，但产量迅速下降，成本上升。因此，改善这些进程的绩效是一项高度优先的任务。自然界已经用核孔复合体(NPC)解决了这种蛋白质浓缩的问题，NPC是一种大分子机器，可以有效地将蛋白质在所有真核细胞的细胞核和细胞质之间分离。既然我们现在已经了解了NPC运输蛋白质的机制，我们的目标是在一个强大的合成平台上模拟NPC的分子结构，具有精致的选择性和高通量。我们的方法是逆向工程，在这种方法中，我们将剖析核运输系统，以阐明其关键要素，以便复制它们。我们将使用酵母NPC，这是我们最了解的系统，作为我们的起点。我们的方法是逆向工程，在这种方法中，我们将剖析核运输系统，以阐明其关键要素，以便复制它们。我们将使用酵母NPC，这是我们最了解的系统，作为我们的起点。具体目标1和具体目标2试图详细说明鼻咽癌是如何被配置为运输者的。为此，我们必须将运输所必需的那些部件与其他NPC功能所需的部件分开，例如自组装和NE维护，并了解这些部件是如何发挥作用的。接下来，我们将详细研究核运输所需的关键组件的行为，以了解它们为什么使NPC在体内如此高效地发挥作用。在特定目标3中，我们将开发NPC的计算模拟，以探索如何将这些最优参数转换为人工机器，并且在特定目标4中，我们将探索在不同规模上建造此类机器的几种方法。

DESCRIPTION (provided by applicant): Most commercial large-scale production of proteins uses column chromatography and synthetic membranes to fractionate and concentrate proteins. Though the requisite multiple recovery steps increase purity, yields drop quickly and expenses rise. Improving the performance of these processes is therefore a high priority. Nature has already solved this kind of protein enrichment problem with the nuclear pore complex (NPC), the macromolecular machine that efficiently segregates proteins between the nucleus and cytoplasm of all eukaryotic cells. Since we now have an understanding of the mechanism by which the NPC transports proteins, our goal is to mimic the molecular machinery of the NPC, with its exquisite selectivity and high throughput, in a robust synthetic platform. Our approach is one of reverse engineering, in which we will dissect the nuclear transport system to elucidate its key elements in order to duplicate them. We will use the yeast NPC, the best understood system, as our starting point. Our approach is one of reverse engineering, in which we will dissect the nuclear transport system to elucidate its key elements in order to duplicate them. We will use the yeast NPC, the best understood system, as our starting point. Specific Aim 1 and Specific Aim 2 seek to detail how the NPC is configured to function as a transporter. For this, we must separate those components that are essential for transport from those needed for other NPC functions, such as self-assembly and NE maintenance, and learn how those components function. Following this, we will study in detail the behavior of the key components needed for nuclear transport, to understand why they make the NPC function so efficiently in vivo. In Specific Aim 3 we will develop a computational simulation of the NPC to explore how to translate these optimal parameters into an artificial machine, and in Specific Aim 4 we will explore several avenues to build such machines at various scales.