Development of an Optimized System for Non-covalent Delivery of Proteins into Cel

描述（由申请人提供）：在单分子跟踪中，将标记蛋白引入细胞对于许多情况是必不可少的。现有的方法涉及细胞的扰动、遗传编码系统或使蛋白质被困在内体中的输入机制。PI和他的两名研究人员最近发表的一项发现强调了一种名为Pep-1的肽和两种Arg 9系统如何用于将蛋白质递送到COS-7细胞的胞质溶胶中，而不会捕获到内体中。关键是导入是在4 ° C下进行的，其中内体形成受到抑制。该建议是将这一发现发展成一种实用的、稳健的系统，用于将蛋白质递送到细胞中而不进行内体包埋。该项目的关键步骤包括：（i）在一组不同的细胞系中建立一系列蛋白质的一般性;（ii）系统地制备和测试递送系统的类似物，以获得优化的载体，这些载体在逐步更严格的实验中测试游离功能蛋白质;以及（iii）机制研究，以使递送剂的结构与其功能相关。公共卫生相关性：这项研究旨在开发一种系统，允许标记的蛋白质导入细胞，而不会显著干扰蛋白质或细胞。这将是同类试剂中第一个强大的试剂，并将打开活细胞中蛋白质单分子跟踪的大门。

DESCRIPTION (provided by applicant): Introduction of labeled proteins into cells is essential for many situations in single molecule tracking. The existing methods involve perturbation of the cells, genetically encoded systems, or import mechanisms that leave the protein trapped in endosomes. A recent discovery published by the PI and his the two investigators on this grant, highlights how a peptide called Pep-1, and two Arg9 systems can be used to deliver proteins into the cytosol of COS-7 cells without capture into endosomes. They key is that the import was performed at 4¿C, where endosome formation is suppressed. This proposal is to develop this discovery into a practical, robust system for delivery of proteins into cells without endosomyl entrapment. Key steps in the project include: (i) establishing generality with respect to a range of proteins in a diverse set of cell lines; (ii) systematically preparing and testing analogs of the delivery systems to arrive at optimized vehicles that are tested in progressively more rigorous experiments for free functional protein; and, (iii) mechanistic studies to allow the structures of the delivery agents to be correlated with their functions. PUBLIC HEALTH RELEVANCE: This research is to develop a system that allows labeled proteins to be imported into cells without dramatically perturbing either the protein or the cell. This would be the first robust reagent of its kind, and would open the doors to single molecule tracking of proteins in living cells.