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

描述(由申请人提供)：在单分子追踪的许多情况下，将标记的蛋白质引入细胞是必不可少的。现有的方法涉及对细胞、遗传编码系统或将蛋白质困在内体中的输入机制进行扰动。PI和他的两位研究人员最近发表的一项发现强调了如何使用一种名为Pep-1的多肽和两个Arg9系统将蛋白质输送到COS-7细胞的胞浆中，而不是捕获到内小体。它们的关键是进口是在4℃进行的，在那里内体形成被抑制。这项提议是将这一发现发展成一个实用的、强大的系统，用于将蛋白质输送到细胞中，而不需要内体包裹。该项目的关键步骤包括：(I)针对不同细胞系中的一系列蛋白质建立通用性；(Ii)系统地准备和测试递送系统的类似物，以获得优化的载体，并在逐步更严格的实验中测试游离功能蛋白质；以及(Iii)允许递送剂的结构与其功能相关的机制研究。与公共健康相关：这项研究旨在开发一种系统，允许将标记的蛋白质导入细胞，而不会显著干扰蛋白质或细胞。这将是同类中第一个强大的试剂，并将为在活细胞中追踪蛋白质的单分子打开大门。

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.