Virtual Gating Machines: A new technology for protein purification

虚拟门控机：蛋白质纯化新技术

基本信息

批准号：
7782870
负责人：
MICHAEL P ROUT
金额：
$ 68.13万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-15 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7782870
关键词：
Architecture Biotechnology Cell Nucleus Cells Chromatography Column Chromatography Computer Simulation Crowding Cytoplasm Development Devices Drops Engineering Environment Evolution Figs - dietary Filtration Fractionation Goals Industry Learning Life Macromolecular Complexes Mass Spectrum Analysis Measures Mediating Membrane Methods Mind Nature Nuclear Pore Complex Nuclear Pore Complex Proteins Peptides Performance Precipitation Process Protein Import Proteins Proteomics Recovery Research Role Solutions Sorting - Cell Movement Speed Structure System Techniques Testing Therapeutic Agents Work base cost design falls heuristics improved in vivo instrument large scale production molecular scale nanoscale new technology next generation nucleocytoplasmic transport operation protein complex protein purification protein transport prototype public health relevance research study trafficking virtual

项目摘要

DESCRIPTION (provided by applicant): Proteins hold increasing promise for the development of new classes of analytical and therapeutic agents, but the cost of their enrichment and purification can be prohibitively high. Thus, there is a great need to develop improved processes for the purification of pharmacologically active proteins and peptides. Nature has already solved this kind of protein enrichment problem with the nuclear pore complex (NPC), the macromolecular complex that efficiently fractionates proteins between the cell nucleus and cytoplasm. Inspired by nature's solution, we have designed an artificial device that can sort nanoscale objects such as protein molecules and protein complexes and that can faithfully reproduce key features of trafficking through the NPC, including transport-factor-mediated cargo import. However, its performance still falls short of the NPC. Our goal is now to gain sufficient understanding of the molecular scale engineering principles behind nuclear transport to allow us to design next generation nanosorters capable of purifying any protein that we desire. We will first employ a "reverse engineering" approach, dissecting the architecture and operation of the NPC to determine which parameters and structures of the NPC are essential to its main transport functions, and how these central parameters have been tuned by evolution to make transport so selective and efficient in the crowded cellular environment. We will also develop methods to assess in detail the performance and mechanism of our current machines in comparison with the equivalent parameters of the NPC, to determine how they work on a molecular scale and discover why the NPC still performs better, in particular with respect to selectivity. We will then use the information garnered in these experiments to guide the design of new and improved versions of our machines, with the aim of increasing their selectivity and efficiency. Our ultimate goal is to create robust, fully synthetic, high-speed protein purification devices. We will investigate how the principles of transport by the NPC can be used to purify any protein, offering potential alternatives to chromatography, filtration and precipitation as protein purification techniques to the biotechnology industry. We will also test the use of our nanosorting devices as components of advanced, mass spectrometry-based, proteomic analytical instruments. PUBLIC HEALTH RELEVANCE: Almost all the active products from the biotechnology industry are proteins and peptides, but their purification remains a costly challenge. However, nature has already devised a rapid and efficient way to purify proteins; nuclear pore complexes sort many hundreds of proteins per second between the nucleus and cytoplasm. By discovering how nuclear pore complexes sort proteins, we are learning how to build prototype nanosorting machines, which eventually should be able to purify many kinds of medically relevant proteins and peptides far more efficiently and at far lower cost than currently possible.