Engineered proteases for proteomics

用于蛋白质组学的工程蛋白酶

• 批准号: 7670566
• 负责人: Biao Ruan
• 金额: $ 20万
• 依托单位: POTOMAC AFFINITY PROTEINS, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7670566
• 关键词: Affinity; Bacillus (bacterium); Biotinylation; Cleaved cell; Complex; DNA Restriction Enzymes; Detection; Dimensions; Disease; Early Diagnosis; Engineering; Escherichia coli K12; Frequencies; Funding; Gel; Genomics; Lead; Methodology; Methods; Nature; Peptide Hydrolases; Performance; Phage Display; Phase; Proteins; Proteome; Proteomics; Recombinant Proteins; Resolution; Sampling; Slice; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staining method; Stains; Subtilisins; System; Technology; Testing; Work; commercial application; directed evolution; innovation; novel; prototype; public health relevance; technological innovation; therapy development; tool

DESCRIPTION (provided by applicant): Proteomics is a rapidly expanding field but current methodologies remain inadequate for achieving its full potential. The most basic enzymological tool for characterizing proteins is the protease. Proteases are already an essential part of proteomic analysis but more sophisticated tools are needed to identify low abundance proteins in highly complex samples. We have engineered a prototype "restriction" protease which is active in denaturing conditions and which cuts specifically at a well-defined cognate sequence motif. Our basic innovation would be the ability to parse a proteome into sequence-edited slices and to detect these edited portions with high resolution and high sensitivity. In Phase I we propose to test the prototype restriction protease for its suitability in proteomic analysis and to implement a novel directed evolution methodology for the selection of proteases that cut new sequence motifs. The long range objective is to develop a sophisticated set of protease tools to facilitate proteomic analysis in the way restriction endonucleases have facilitated genomic analysis. PUBLIC HEALTH RELEVANCE: The complex and dynamic nature of proteomes make them rich with useful information but difficult to characterize. Our long-range objective is to develop a sophisticated set of protease tools to facilitate proteomic analysis in the way restriction endonucleases have facilitated genomic analysis. Better proteomic tools will lead to earlier detection of disease states, better treatments, better predictability of the effects of various treatments, and the development of individualized therapies.

描述（由申请人提供）：蛋白质组学是一个快速发展的领域，但目前的方法仍然不足以实现其全部潜力。表征蛋白质的最基本的酶学工具是蛋白酶。蛋白酶已经是蛋白质组学分析的重要组成部分，但需要更复杂的工具来识别高度复杂样品中的低丰度蛋白质。我们已经设计了一种原型“限制”蛋白酶，它在变性条件下具有活性，并且在明确定义的同源序列基序处特异性切割。我们的基本创新是能够将蛋白质组解析为序列编辑的切片，并以高分辨率和高灵敏度检测这些编辑的部分。在第一阶段，我们建议测试的原型限制性蛋白酶在蛋白质组学分析的适用性，并实施一种新的定向进化方法选择切割新的序列基序的蛋白酶。长期目标是开发一套复杂的蛋白酶工具，以促进蛋白质组学分析的方式限制性核酸内切酶促进基因组分析。公共卫生相关性：蛋白质组的复杂性和动态性使其具有丰富的有用信息，但难以表征。我们的长期目标是开发一套复杂的蛋白酶工具，以促进蛋白质组学分析的方式限制性核酸内切酶促进基因组分析。更好的蛋白质组学工具将导致疾病状态的早期检测，更好的治疗，更好地预测各种治疗的效果，以及个性化治疗的发展。