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必不可少的。然而，目前缺乏高质量的蛋白质捕获试剂是生物医学科学几乎所有领域的主要瓶颈。该项目的总体目标是开发一种具有高保真度和可预测性的创新和强大的蛋白质捕获技术。我们的目标是克服目前可用技术的一个主要限制，即在生成蛋白质捕获试剂后，必须通过费力的方法单独测试特异性和表位。我们引入了一个新的概念，“c -箝位”，使捕获试剂（“c -箝位”）能够以高保真度和高亲和力专门捕获蛋白质的c -末端6-8残基。C-clamps的形式是利用最先进的噬菌体展示技术生成的强大重组结合蛋白。几乎每个蛋白质都有一个独特的c端特征，可以被c -夹高效率地识别。表位位置的先验知识允许人们通过数据库搜索识别潜在的交叉反应性来准确预测特异性水平，并实施消除脱靶的策略。这些特性使得C-clamps特别适合作为生成一套全面的蛋白质捕获试剂的核心技术。此外，c -箝位非常适合检测由蛋白质水解产生的蛋白水解“新表位”，这是生物医学上重要过程（如细胞凋亡）的标记。我们的概念验证实验已经成功地证明了c -夹紧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（包括完整膜蛋白、剪接变异体、病毒蛋白和caspase新表位）的高性能捕获试剂，将c夹紧建立为一种通用技术。c -箝位代表了捕获试剂产生的范式转变，c -箝位的建立将对整个分子生物医学科学做出重大贡献。

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.