Engineering Sortase Variants for Intracellular and Cell Surface Labeling

描述（由申请人提供）：我们建议开发一种基于细菌转肽酶分选酶A（SrtA）的活细胞中位点特异性标记蛋白质的新系统。使用蛋白质工程和定向进化的组合，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（<2KDa）的、明亮荧光的膜可渗透标记物以及仅在与靶蛋白连接时才发荧光的底物。这些标记的减小的尺寸加上它们的强荧光将允许在更低、更生理表达水平下监测蛋白质，同时减少空间干扰的任何可能性。最初的工作将集中在标记分泌蛋白，但该技术将适用于几乎任何蛋白质位于许多细胞区室。公共卫生相关性：我们将开发一种新的酶促方法，用于在活细胞中用小荧光分子标记特定蛋白质。

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.