STRUCTURES AND FUNCTIONS OF PROTEINS FROM ORPHAN GENES

孤儿基因蛋白质的结构和功能

• 批准号: 6204320
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6204320
• 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.

描述：DNA 测序自动化技术的最新发展 导致了有关完整序列的信息爆炸 几种生物体的基因组。 11 的完整基因组序列 微生物现在已经可用，很快就会有近三种微生物的基因组 十几个额外的生物体将被完成。这些革命性的数据 刺激了基础科学、医学和 生物技术界致力于确定必要的 生物体遗传信息和功能属性的补充 那是维持生命的需要。一个惊人的观察结果是 分析的每个生物体的基因组几乎有三分之一是假定的 开放阅读框虽然在多种生物体中保守，但编码 对于未知功能的假设蛋白质。生理功能 蛋白质产品的研究代表了我们对蛋白质产品的理解上的一个主要差距 完整补充生存所需的遗传信息 任何自由的生物体。该研究计划的总体目标是 阐明 65 个 bonafide 集合中大约 50 个蛋白质的功能 来自流感嗜血杆菌的假设蛋白质，流感嗜血杆菌是一种中等程度的生物体 通过确定其高分辨率原子结构来确定遗传大小。这 第一步是开发亚克隆的高通量方法 已经筛选出可表达的开放阅读框 将多肽导入高水平表达载体以优化产量 可溶性蛋白质产品。第二阶段是发展高效 用于高产率纯化合适的天然蛋白质的方案 质量和数量足以开始大规模结晶 研究。最后一部分是表征目标蛋白 就其四级结构以及其溶解度而言 和溶液稳定性。这些数据将使蛋白质靶点成为可能 通过核磁共振方法确定结构，提供线索 具有挑战性的蛋白质的结晶，并产生有关 假设蛋白质的物理和化学性质 对于功能测定很有用。