High-performance affinity reagents for peptide epitopes

肽表位的高性能亲和试剂

基本信息

批准号：
7603122
负责人：
SHOHEI KOIDE
金额：
$ 19.9万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-04 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7603122
关键词：
Address Affinity Amino Acids Antibodies Antigens Architecture Binding Biochemical Biological Models Biotechnology Cancer Biology Cells Chemicals Classification Complex Detection Dissociation Engineering Enhancers Epitopes Figs - dietary Generations Goals Gold Guidelines Immunochemistry Immunoprecipitation Lead Libraries Malignant Neoplasms Measures Mitogen-Activated Protein Kinases Modeling Molecular Molecular Analysis Monoclonal Antibodies Peptides Performance Phosphopeptides Phosphorylation Play Post-Translational Protein Processing Production Protein Engineering Proteins Proteome Proteomics Reagent Receptor Protein-Tyrosine Kinases Regulation Role Series Signal Transduction Specificity System Technology Tertiary Protein Structure Time Tissues Western Blotting base design innovation interest meetings monoclonal antibody production novel polyclonal antibody protein function prototype research study src Homology Region 2 Domain success tool tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Cancer results from the disregulation of signal transduction networks. Thus, quantitative detection, functional assessment and isolation of proteins involved in cancer biology are critically important for molecular analysis of cancer. Short peptide motifs within many of this class of proteins and posttranslational modification thereof play central roles in signal transduction regulation through controlling protein function and interactions. However, there are a very small number of affinity reagents (reagents that bind to a target with high affinity and specificity) to this class of high-value epitopes in cancer analysis. The long-term goal of this project is to develop a powerful technology platform for generating high-performance affinity reagents for short peptide epitopes. The primary products will be novel affinity reagents termed "Epitope Clamps". Our strategy is distinct from many others in that we aim to develop distinct types of affinity reagents, with each type specific to a particular class of short peptide epitopes. This contrasts with the conventional antibody-based approaches where a single, general- purpose platform is used for engineering distinct types of targets. Our innovative protein engineering strategy harnesses the inherent specificity present in the so-called interaction domains and dramatically enhances their affinity and specificity by attaching an "enhancer domain". This makes it possible to generate protein libraries predisposed to binding to a particular motif, and thus it dramatically increases the chance of successfully engineering high- performance affinity reagents. Our proof-of-concept experiments have successfully demonstrated this new paradigm in affinity reagent engineering. These proposed studies will establish the full potential of the Epitope Clamp technology, and will generate high-performance affinity reagents to phosphopeptide epitopes in proteins that are critically involved in cancer biology. The technology and tools developed in this project will make a major impact on molecular analysis of cancer, and more broadly on proteomics and biotechnology. Accurately measuring the amounts of proteins of interest in cells and tissues and assessing their functional state are major technological challenges in molecular analysis of cancer. This application aims to establish a powerful technology platform for facile generation of high- performance reagents that tightly bind to a predefined segment within a protein of interest. We aim to develop such "affinity reagents" to diverse protein segments that carry a chemical signature of activated proteins. This technology will fill a major void in the current technology portfolio for molecular analysis of cancer.

描述（由申请人提供）：癌症是由于疏离信号转导网络的疏离而导致的。因此，对癌症生物学涉及的蛋白质的定量检测，功能评估和分离对于癌症的分子分析至关重要。许多此类蛋白质中的短肽基序和翻译后修饰在信号转导调节中通过控制蛋白质功能和相互作用起着中心作用。但是，在癌症分析中，与这类高价值表位的亲和力试剂（与具有高亲和力和特异性的靶标的试剂结合）。该项目的长期目标是开发一个强大的技术平台，用于为短肽表位生成高性能亲和力试剂。主要产品将是称为“表位夹”的新型亲和力试剂。我们的策略与许多其他策略不同，因为我们旨在开发不同类型的亲和力试剂，每种特定于特定类型的短肽表位。这与传统的基于抗体的方法形成鲜明对比，在该方法中，单个通用平台用于工程不同类型的目标。我们的创新蛋白质工程策略利用所谓的交互域中存在的固有特异性，并通过附加“增强子域”来大大增强其亲和力和特异性。这使得产生易于结合特定基序的蛋白质库，因此它极大地增加了成功设计高性能亲和力试剂的机会。我们的概念验证实验成功地证明了这种新的亲和力试剂工程范式。这些拟议的研究将确定表位夹技术的全部潜力，并将产生高性能的亲和力试剂，以对蛋白质中的磷酸肽表位，这些蛋白质与癌症生物学非常重要。该项目开发的技术和工具将对癌症分子分析以及蛋白质组学和生物技术的更广泛影响产生重大影响。准确地测量细胞和组织中感兴趣的蛋白质量并评估其功能状态是癌症分子分析中的主要技术挑战。该应用程序旨在建立一个强大的技术平台，以促进高性能试剂的生成，该试剂与感兴趣的蛋白质内的预定义细分市场紧密结合。我们旨在将这种“亲和力试剂”开发到具有活化蛋白化学特征的各种蛋白质片段。该技术将填补当前技术组合中用于癌症分子分析的主要空隙。