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（由适用提供）：将标记的蛋白质引入细胞中对于单分子跟踪中的许多情况至关重要。现有的方法涉及细胞的扰动，遗传编码的系统或将蛋白质捕获到内体中的进口机制。 PI和他的两名研究人员对这笔赠款发表的最新发现突出了一个称为PEP-1的肽，并且可以使用两个ARG9系统将蛋白质输送到无捕获成内体的COS-7细胞的细胞质中。他们的关键是该导入是在4¿C下进行的，其中抑制了内体形成。该建议是将这一发现开发成一个实用的，可靠的系统，用于将蛋白质递送到无内粒内膜中的细胞中。该项目的关键步骤包括：（i）在潜水细胞系中的一系列蛋白质方面建立普遍性；（ii）系统地准备和测试输送系统的类似物，以达到优化的车辆，这些车辆在更严格的实验中进行了自由功能蛋白的测试；（iii）机械研究允许输送剂的结构与其功能相关。公共卫生相关性：这项研究是开发一种系统，该系统允许将标记的蛋白质进口到细胞中，而不会极大地扰动蛋白质或细胞。这将是同类的第一个强大试剂，并将为活细胞中蛋白质的单分子跟踪打开门。