Cell Free Protein Labeling with Molecular Partners

使用分子伙伴进行无细胞蛋白质标记

• 批准号: 6582647
• 负责人: JERZY OLEJNIK
• 金额: $ 10万
• 依托单位: AMBERGEN, INC
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6582647
• 关键词: affinity labeling; aminoacyl tRNA; biotechnology; chemical synthesis; conformation; enzyme linked immunosorbent assay; fluorescence; hydroxamate; mass spectrometry; protein engineering; protein protein interaction; technology /technique development

DESCRIPTION (provided by applicant): Cell free protein expression labeling provides a means for incorporation of fluorescent markers (fluorotags) and affinity tags (affytags) into proteins which can be used for a variety of biotechnological applications including proteomics and drug discovery. It relies on the attachment of a non-native amino acid to a specific tRNA using non-enzymatic chemical aminoacylation and the introduction of the misaminoacylated tRNA into a cell-free protein expression system. Such labels allow cell-free expressed proteins to be tracked, purified, isolated, conformation changes measured and interaction with other molecules detected. However, this approach is severely limited by restrictions imposed by the protein translation system on the structure of the amino acid side chains that can be incorporated into the protein. The proposed research will develop and evaluate a new approach, which circumvents labeling restrictions imposed by the protein translational apparatus. Molecular partners, small organic molecules, which exhibit highly selective and strong interaction between each other are utilized. By incorporating one of these partners into the protein and by using the other as a linker, versatile labeling and surface attachment of proteins can be achieved. Phase I will focus on the use of 1,3-phenyldiboronic acid (PDBA) and salicylhydroxamic acid (SHA) derivatives as molecular partners. A novel class of affinity ligands, which form highly stable complexes with PDBA will be synthesized based on bis-salicylhydroxamic acid (bis-SHA). As opposed to currently used mono-SHA derivatives, these compounds are expected to have superior properties including complex stability and a functional group that enables easy conjugation with fluorotags, affytags and surfaces. A variety of applications of this new labeling technology will be developed during Phase II including utilization of mass spectrometry to analyze protein-protein interactions.

描述（由申请人提供）： 无细胞蛋白表达标记提供了掺入荧光标记物的手段 将荧光标记（fluorotags）和亲和标记（affytags）引入蛋白质中，其可用于多种生物技术应用，包括蛋白质组学和药物发现。它依赖于使用非酶化学氨酰化将非天然氨基酸连接到特定的tRNA上，并将错氨酰化的tRNA引入无细胞蛋白质表达系统中。这种标记允许无细胞表达的蛋白质被追踪、纯化、分离、测量构象变化以及检测与其他分子的相互作用。然而，这种方法受到蛋白质翻译系统对可以掺入蛋白质的氨基酸侧链结构的限制的严重限制。拟议的研究将开发和评估一种新的方法，该方法规避了蛋白质翻译装置施加的标记限制。分子伴侣，小的有机分子，它们表现出高度选择性和彼此之间的强相互作用。通过将这些配偶体之一并入蛋白质中并通过使用另一个作为接头，可以实现蛋白质的通用标记和表面附着。第一阶段将集中于使用1，3-苯基二硼酸（PDBA）和水杨基异羟肟酸（SHA）衍生物作为分子伴侣。基于双水杨基异羟肟酸（bis-SHA），将合成一类新型的亲和配体，它与PDBA形成高度稳定的配合物。与目前使用的单SHA衍生物相反，预期这些化合物具有上级性质，包括复合物稳定性和能够容易地与荧光标签、亲和标签和表面缀合的官能团。这种新的标记技术的各种应用将在第二阶段开发，包括利用质谱分析蛋白质-蛋白质相互作用。