Engineering Sortase Variants for Intracellular and Cell Surface Labeling

描述(由申请人提供)：我们建议开发一种新的基于细菌转肽酶分类酶A(SrtA)的活细胞中定位标记蛋白质的系统。利用蛋白质工程和定向进化的结合，我们将开发能够在哺乳动物细胞内有效发挥作用的srtA变体。我们还将开发非常小(&lt；2 KDa)、明亮的荧光膜可渗透标签以及仅在附着在目标蛋白质上时才会发光的底物。这些标记的尺寸减小，加上它们的强烈荧光，将允许监测较低、更具生理性的表达水平的蛋白质，同时减少任何空间干扰的可能性。最初的工作将集中在标记分泌蛋白质上，但这项技术将适用于位于许多细胞隔间中的几乎任何蛋白质。与公众健康相关：我们将开发一种新的酶促方法，在活细胞中用小荧光分子标记特定的蛋白质。

DESCRIPTION (provided by applicant): We propose to develop a new system for site specific labeling proteins in live cells based on the bacterial transpeptidase sortase A (SrtA). Using a combination of protein engineering and directed evolution, we will develop SrtA variants capable of efficiently functioning within mammalian cells. We will also develop exceptionally small (<2KDa), brightly fluorescent membrane permeable labels as well as substrates which will fluoresce only upon attachment to the target protein. The reduced size of these labels coupled with their strong fluorescence will permit monitoring of proteins at lower, more physiological expression levels while reducing any potential for steric interference. Initial work will focus on labeling secretory proteins, but the technology will be applicable to almost any protein located in a number of cellular compartments. PUBLIC HEALTH RELEVANCE: We will develop a novel enzymatic method for labeling specific proteins with small fluorescent molecules in live cells.