Rational generation of directed protein-capture reagents

蛋白质捕获试剂对于描绘蛋白质降解的分子机制是不可或缺的。 疾病，检测和表征细胞异常，以及表征药物的生物效应。然而，目前缺乏高质量的蛋白质捕获试剂在几乎所有的生物医学科学领域提出了一个主要的瓶颈。该项目的总体目标是开发一种具有高度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主要限制，其中特异性和表位必须在生成蛋白捕获试剂后通过费力的方法单独测试。我们引入了一个新的概念，“C-夹”，其能够以高保真度和高亲和力将捕获试剂（“C-夹”）专门导向蛋白质的C-末端（6-8）残基。C-夹是使用最先进的噬菌体展示技术产生的稳健重组结合蛋白的形式。事实上，每种蛋白质都有一个独特的C-末端特征，可以被C-夹高效识别。表位的先验知识 定位允许人们通过数据库搜索识别潜在的交叉反应性来准确地预测特异性水平，并实施消除脱靶的策略。这些属性使得C-夹特别适合作为核心技术，用于生成一套全面的蛋白质捕获试剂。此外，C-钳位非常适合于检测由蛋白水解产生的蛋白水解“新表位”，生物医学上重要过程（例如凋亡）的标志物。我们的概念验证实验成功地证明了C箝位的可行性和巨大潜力。 拟议的研究旨在通过生产针对高价值靶标的高性能捕获试剂，将C-钳位技术确立为一项通用技术，这些靶标包括整合膜蛋白、剪接变体、病毒蛋白和半胱天冬酶新表位。C-clamping代表了捕获试剂产生的范式转变，C-clamping的建立将对整个分子生物医学科学做出巨大贡献。

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.