Virtual Gating Machines for Protein Purification

用于蛋白质纯化的虚拟门控机

• 批准号: 7282044
• 负责人: MICHAEL P ROUT
• 金额: $ 48.21万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282044
• 关键词: Behavior; Carrier Proteins; Cell Nucleus; Cells; Column Chromatography; Computer Simulation; Cytoplasm; Data; Drops; Engineering; Eukaryotic Cell; Gene Deletion; Goals; In Situ; In Vitro; Indium; Individual; Kinetics; Learning; Macromolecular Complexes; Maintenance; Membrane; Modeling; Molecular; Nature; Nuclear; Nuclear Pore Complex; Performance; Process; Production; Property; Proteins; Recovery; System; Testing; Translating; Yeasts; base; cell killing; design; improved; in vivo; nucleocytoplasmic transport; protein purification; prototype; research study; self assembly; virtual

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.

描述（由申请人提供）：大多数商业化大规模生产蛋白质都使用柱层析和合成膜来分级和浓缩蛋白质。尽管必要的多个回收步骤可以提高纯度，但产量会迅速下降，费用也会增加。因此，提高这些流程的性能是当务之急。大自然已经通过核孔复合体（NPC）解决了这种蛋白质富集问题，核孔复合体是一种大分子机器，可以有效地将蛋白质分离在所有真核细胞的细胞核和细胞质之间。由于我们现在了解了 NPC 运输蛋白质的机制，我们的目标是在强大的合成平台中模拟 NPC 的分子机制，以其精湛的选择性和高通量。我们的方法是逆向工程之一，我们将剖析核运输系统，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，我们将剖析核运输系统，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 旨在详细说明 NPC 如何配置为充当运输者。为此，我们必须将运输所必需的组件与其他 NPC 功能（例如自组装和 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关键组件的行为，以了解它们为何使 NPC 在体内如此有效地发挥作用。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佳参数转化为人造机器，而在特定目标 4 中，我们将探索多种途径来构建各种规模的此类机器。