Virtual Gating Machines for Protein Purification

描述（由申请人提供）：大多数商业化的大规模蛋白质生产使用柱层析和合成膜来分离和浓缩蛋白质。虽然必要的多个回收步骤可以提高纯度，但产量会迅速下降，成本也会上升。因此，提高这些过程的性能是当务之急。自然界已经用核孔复合物（NPC）解决了这种蛋白质富集问题，核孔复合物是一种大分子机器，可以在所有真核细胞的细胞核和细胞质之间有效地分离蛋白质。由于我们现在已经了解了NPC转运蛋白质的机制，我们的目标是在一个强大的合成平台上模拟NPC的分子机制，具有精致的选择性和高通量。我们的方法是逆向工程之一，其中我们将剖析核传输系统，阐明其关键要素，以便复制它们。我们将使用酵母NPC，最容易理解的系统，作为我们的起点。我们的方法是逆向工程之一，其中我们将剖析核传输系统，阐明其关键要素，以便复制它们。我们将使用酵母NPC，最容易理解的系统，作为我们的起点。具体目标1和具体目标2试图详细说明NPC如何配置为运输者。为此，我们必须将那些对运输至关重要的组件与其他NPC功能（如自组装和NE维护）所需的组件分开，并了解这些组件的功能。接下来，我们将详细研究核运输所需的关键成分的行为，以了解为什么它们使NPC在体内如此有效地发挥作用。在具体目标3中，我们将开发NPC的计算模拟，以探索如何将这些最佳参数转化为人工机器，并在具体目标4中，我们将探索几种途径来构建各种规模的此类机器。

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.