Renewable Antibodies for Post Translational Modifications and Protease Activatio

用于翻译后修饰和蛋白酶激活的可再生抗体

• 批准号: 8702418
• 负责人: JAMES A WELLS
• 金额: $ 79.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-24 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702418
• 关键词: Antibodies; Antigens; Bacteriophages; Biomedical Research; Biotinylation; Cells; Development; Enzyme Precursors; Epitopes; Event; Genetic Transcription; Goals; Hot Spot; In Vitro; Industrialization; Integral Membrane Protein; Libraries; Membrane Proteins; Methods; Peptide Hydrolases; Phage Display; Phosphorylation; Phosphorylation Site; Post-Translational Protein Processing; Preparation; Production; Proteins; Reagent; Recombinant Antibody; Research; Robotics; Serine; Signal Transduction; Site; Sorting - Cell Movement; Surface; Technology; Threonine; Translations; Tyrosine; Validation; Yeasts; design; extracellular; in vivo; magnetic beads; novel; open source; scaffold

The long-term goal of the TR&D 3 project is to develop high-impact, renewable, and open-source antibody reagents for cellular signaling and biomedical research. Specifically, we plan to focus on signaling events marked by important protein post-translational modifications (PTMs) such as phosphorylation and proteolytic zymogen activation events and their attendant protein conformational changes. While some antibodies do exist for specific phosphorylation sites, these antibodies are often unreliable, rarely renewable, and only available to a fraction of the modified sites. In addition, detecting functional forms of specific proteases has not been routinely possible in cells or in vivo due to the lack of conformationally selective antibodies. Furthermore, the industrialization of high-throughput recombinant antibody (rAb) selection and preparation has been thwarted by the absence of reliable robotic expression platforms. Thus, our immediate goals are to generate high-quality, open-source antibody reagents, by using novel phage display technologies and libraries developed by the UCSF Antibiome Center, to PTMs including: (i) phosphorylation sites, (ii) proteolytic neo-epitopes, and their attendant conformational changes, and (iii) alternative rapid antigen expression technologies such as in vitro transcription and translation (IVTT) that support all three TR&D projects. These goals compliment TR&Ds 1 and 2 by focusing on PTMs within key transmembrane proteins

TR&D 3项目的长期目标是为细胞信号传导和生物医学研究开发高影响力、可再生和开源的抗体试剂。具体来说，我们计划集中在信号事件标志着重要的蛋白质翻译后修饰（PTM），如磷酸化和蛋白水解 酶原激活事件及其伴随的蛋白质构象变化。虽然一些抗体确实存在于特定的磷酸化位点，但这些抗体通常是不可靠的，很少可再生的，并且仅可用于一部分修饰位点。此外，检测特定蛋白酶的功能形式尚未被广泛应用。 由于缺乏构象选择性抗体，在细胞或体内通常可能。此外，由于缺乏可靠的机器人表达平台，高通量重组抗体（rAb）选择和制备的工业化受到阻碍。因此，我们的近期目标是通过使用UCSF抗生素组中心开发的新型噬菌体展示技术和文库来产生高质量的开源抗体试剂，以PTM包括：（i）磷酸化位点，（ii）蛋白水解新表位及其伴随的构象变化，以及（iii）替代的快速抗原表达技术，例如支持所有三个TR&D项目的体外转录和翻译（IVTT）。这些目标通过关注关键跨膜蛋白内的PTM来补充TR&Ds 1和2