Rational generation of directed protein-capture reagents

描述(由申请人提供)：蛋白质捕获试剂对于描述疾病的分子机制、检测和表征细胞异常以及表征药物的生物效应是不可或缺的。然而，目前高质量蛋白质捕获试剂的缺乏几乎是生物医学科学所有领域的一个主要瓶颈。该项目的总体目标是开发一种创新的、功能强大的蛋白质捕获技术，具有高水平的保真度和可预测性。我们的目标是克服目前现有技术的一个主要限制，即在产生蛋白质捕获试剂后，必须通过费力的方法分别测试特异性和表位。我们引入了一个新的概念，“C-钳制”，它使捕获试剂(“C-钳制”)能够专门针对高保真和高亲和力的蛋白质的C-末端6-8残基。C-夹的形式是利用最先进的噬菌体展示技术产生的强大的重组结合蛋白。事实上，每种蛋白质都有一个独特的C-末端标记，可以通过C-钳高效地识别。表位位置的先验知识使人们能够通过数据库搜索识别潜在的交叉反应，从而准确地预测特异性水平，并实施消除非靶标的策略。这些特性使得C-夹子特别适合作为产生一套全面的蛋白质捕获试剂的核心技术。此外，C-钳制非常适合于检测由蛋白分解产生的蛋白水解性“新表位”，这是生物医学上重要过程(如细胞凋亡)的标志物。我们的概念验证实验已经成功地证明了C形钳位的可行性和巨大的潜力。拟议的研究旨在通过生产高价值靶标的高性能捕获剂，包括整合膜蛋白、剪接变异体、病毒蛋白和caspase新表位，将C-钳制建立为一项通用技术。C-钳制代表了捕获试剂生成的范式转变，C-钳制的建立将对整个分子生物医学科学做出巨大贡献。 公共卫生相关性：检测和测量蛋白质的数量对于了解细胞和组织的正常和患病状态之间的差异至关重要。该项目将建立一种全新的方法，以方便地产生高性能、易于生产和易于向研究界提供的检测试剂。这项创新和强大的技术将填补目前可用的分子工具中的一个重大空白，并将对分子生物医学科学、诊断和药物开发的几乎所有领域产生重大影响。

DESCRIPTION (provided by applicant): Protein-capture reagents are indispensable for delineating the molecular mechanisms of diseases, to detect and characterize cellular abnormalities, and to characterize biological effects of drugs. However, the current paucity of high-quality protein-capture reagents presents a major bottleneck in virtually all areas of biomedical sciences. The overarching goal of this project is to develop an innovative and powerful protein-capture technology with high levels of fidelity and predictability. We aim to overcome a major limitation of currently available technologies where specificity and epitopes must be individually tested by laborious methods after generating protein-capture reagents. We introduce a new concept, "C-clamping", that enables to direct capture reagents ("C-clamps") exclusively to the C-terminal 6-8 residues of proteins with high fidelity and high affinity. C-clamps are in the form of robust recombinant binding proteins generated using state- of-the-art phage-display technologies. Virtually every protein has a unique C-terminal signature that can be recognized with high efficiency by C-clamps. The a priori knowledge of epitope location allows one to accurately predict the level of specificity by identifying potential cross- reactivity through database search and to implement strategies to eliminate off-targets. These attributes make C-clamps particularly suited as the core technology for generating a comprehensive set of protein capture reagents. Furthermore, C-clamping is ideally suited to detect proteolytic "neo-epitopes" generated by proteolysis, markers of biomedically important processes (e.g. apoptosis). Our proof-of-concept experiments have successfully demonstrated the feasibility and enormous potential of C-clamping. Proposed studies aim to establish C-clamping as a general technology by producing high- performance capture reagents for high-value targets including integral membrane proteins, splice variants, viral proteins and caspase neo-epitopes. C-clamping represents a paradigm shift in capture-reagent generation, and the establishment of C-clamping will make large contributions to the entire molecular biomedical sciences. PUBLIC HEALTH RELEVANCE: Detecting and measuring the amounts of proteins are critical for understanding differences between normal and diseased states of cells and tissues. This project will establish a totally new approach to facile generation of detection reagents that are high performance, easy to produce and easy to made available to the research community. This innovative and powerful technology will fill a major void in the currently available molecular tools and will have a major impact on virtually all areas of molecular biomedical sciences, diagnosis and drug development.