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的新系统。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运作的SrtA变体。我们还将开发极小的（<2KDa），明亮的荧光膜渗透标记以及仅在附着于目标蛋白时才会发出荧光的底物。这些标记的缩小尺寸与它们的强荧光相结合，将允许在更低、更生理的表达水平上监测蛋白质，同时减少任何潜在的空间干扰。最初的工作将集中于标记分泌蛋白，但该技术将适用于位于许多细胞区室中的几乎任何蛋白质。公共卫生相关性：我们将开发一种新的酶法，用小荧光分子标记活细胞中的特定蛋白质。

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.