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

开发用于将蛋白质非共价递送至细胞的优化系统

基本信息

批准号：
8548535
负责人：
KEVIN BURGESS
金额：
$ 32.37万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2015-08-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：将标记的蛋白质引入细胞对于单分子追踪的许多情况是必不可少的，现有的方法涉及细胞的扰动、基因编码系统或使蛋白质被困在内体中的导入机制。 PI 和他的两名研究人员在这项资助中发表的论文强调了如何使用一种名为 Pep-1 的肽和两个 Arg9 系统将蛋白质递送到 COS-7 细胞的胞质溶胶中，而不捕获到内体中。关键是导入是在 4¿ C，内体形成受到抑制。该提案旨在将这一发现发展成一种实用的、强大的系统，用于将蛋白质递送到细胞中，而不会被内体截留。该项目的关键步骤包括：（i）建立一系列蛋白质的通用性。在不同的细胞系中；（ii）系统地制备和测试递送系统的类似物，以得到优化的载体，并在逐渐更严格的实验中测试游离功能蛋白；以及（iii）机制研究以允许结构要关联的递送代理与公共健康相关性：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中，而不会显着干扰蛋白质或细胞。这将是同类中第一个强大的试剂，并将打开大门。活细胞中蛋白质的单分子追踪。