Cell Free Protein Labeling with Molecular Partners

描述(由申请人提供)： 细胞游离蛋白表达标记为掺入荧光标记物提供了一种手段 (荧光标记)和亲和标记(亲和标记)到蛋白质中，可用于包括蛋白质组学和药物发现在内的各种生物技术应用。它依赖于通过非酶化学氨酰化将非天然氨基酸连接到特定的tRNA上，并将氨基酰化的tRNA引入无细胞蛋白质表达系统。这样的标记可以跟踪、纯化、分离无细胞表达的蛋白质，测量构象变化，并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结合到蛋白质中的氨基酸侧链结构的限制。这项拟议的研究将开发和评估一种新的方法，该方法绕过了蛋白质翻译装置施加的标记限制。利用了具有高度选择性和强相互作用的分子伴侣，即有机小分子。通过将这些伙伴中的一个结合到蛋白质中并使用另一个作为连接物，可以实现蛋白质的多功能标记和表面附着。第一阶段将重点使用1，3-苯基二硼酸(PDBA)和水杨基异羟肟酸(SHA)衍生物作为分子伙伴。基于双水杨基异羟肟酸(bi-SHA)合成了一类新型的亲和配体，与PDBA形成高度稳定的络合物。与目前使用的单SHA衍生物不同，这些化合物有望具有优异的性能，包括络合物稳定性和易于与氟标记、亲和标记和表面结合的官能团。在第二阶段将开发这种新标记技术的各种应用，包括利用质谱学来分析蛋白质-蛋白质相互作用。

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.