STRUCTURES AND FUNCTIONS OF PROTEINS FROM ORPHAN GENES

孤儿基因蛋白质的结构和功能

• 批准号: 6347559
• 负责人: Edward Eisenstein
• 金额: $ 15.24万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6347559
• 关键词: Haemophilus; bacterial proteins; circular dichroism; gene expression; matrix assisted laser desorption ionization; molecular cloning; nuclear magnetic resonance spectroscopy; open reading frames; protein folding; protein structure function; ultracentrifugation

DESCRIPTION: Recent develops in automated techniques for DNA sequencing have led to an explosion of information on the complete sequences for the genomes of several organisms. Entire genomic sequences of 11 microorganisms are available now, and soon the genomes of almost three dozen additional organisms will be completed. These revolutionary data have stimulated mosaic research from the basic science, medical, and biotechnology communities that is focused on determining the essential complement of genetic information and functional attributes of an organism that is need to sustain life. A striking observation that has been made as each organism's genome is analyze is that almost one third of the putative open reading frames, although conserved among several organisms, encode for hypothetical proteins of no known function. The physiological function of the protein products represent a major gap in our understanding of the full complement of genetic information that is needed for the viability of any free living organism. The overall goal of this research program is to elucidate the function of roughly 50 proteins from a set of 65 bonafide hypothetical proteins from Haemophilus influenzae, an organism of moderate genetic size, by determining their high-resolution atomic structures. The first step is to develop high throughput methodology for subcloning the open reading frames that have already been screened for expressible polypeptides into high-level expression vectors to optimize the yields of soluble protein products. The second phase is to develop efficient protocols for high yield purification of native proteins of suitable quality and in sufficient quantities to begin large-scale crystallization studies. The final component is to characterize the targeted proteins in terms of their quaternary structure, and also in terms of their solubility and stability in solution. These data will permit protein targets to be identified for structure determination by NMR methods, provide clues for the crystallization of challenging proteins, and yield data on the physical and chemical properties of the hypothetical proteins that will be useful for functional determinations.

DNA测序自动化技术的最新发展 已经导致了关于完整序列的信息爆炸， 几种生物的基因组。全基因组序列11 微生物现在是可用的，很快就有近三个的基因组 十多个新的生物体将被完成。这些革命性的数据 从基础科学、医学和 生物技术社区的重点是确定基本的 生物体的遗传信息和功能属性的补充 这是维持生命所必需的。一个惊人的观察， 每种生物基因组分析结果几乎是假定的三分之一 开放阅读框架，虽然在几种生物中是保守的， 对于功能未知的假设蛋白质。生理功能 的蛋白质产品代表了一个主要的差距，我们的理解， 完整的遗传信息，这是必要的可行性， 任何自由的生物体。该研究项目的总体目标是 从一组65个bonafide中阐明大约50个蛋白质的功能 来自流感嗜血杆菌的假设蛋白质， 基因大小，通过确定它们的高分辨率原子结构。的 第一步是开发高通量方法，用于亚克隆 开放的阅读框架，已经筛选出可表达的 将多肽导入高水平表达载体以优化多肽的产量。 可溶性蛋白质产品。第二阶段是发展高效 用于高产量纯化合适的天然蛋白质的方案 质量和足够的数量来开始开始大规模结晶 问题研究最后一个组成部分是表征目标蛋白质， 就其四级结构而言，以及就其溶解度而言， 和溶液中的稳定性。这些数据将允许蛋白质靶点被 通过NMR方法确定结构，为 挑战性蛋白质的结晶，以及 假设的蛋白质的物理和化学性质， 用于功能测定。