Engineering Sortase Variants for Intracellular and Cell Surface Labeling

用于细胞内和细胞表面标记的工程分选酶变体

• 批准号: 8136676
• 负责人: MATTHEW LEVY
• 金额: $ 30.91万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136676
• 关键词: Affect; Amines; Bacterial Proteins; Cell surface; Cells; Chemicals; Cleaved cell; Coupled; Cytoplasm; Endoplasmic Reticulum; Engineering; Environment; Enzymes; Fluorescence; Generations; Goals; Health; Image; In Vitro; Label; Libraries; Life; Ligation; Mammalian Cell; Membrane; Methodology; Methods; Monitor; Pentas; Peptides; Peptidyltransferase; Physiological; Protein Engineering; Proteins; Reaction; Relative (related person); Research; Scheme; Site; Specificity; Substrate Specificity; System; Techniques; Technology; Tertiary Protein Structure; Variant; Work; base; calreticulin; directed evolution; enhanced green fluorescent protein; fluorophore; in vivo; interest; novel; protein expression; protein transport; research study; secretory protein; small molecule; sortase; thioester

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.

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。通过结合蛋白质工程和定向进化，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。我们还将开发特别小（<2KDa）、明亮的荧光膜渗透标记以及仅在附着到目标蛋白时才会发出荧光的底物。这些标记的尺寸减小，加上它们的强荧光，将允许以更低、更生理的表达水平监测蛋白质，同时减少空间干扰的可能性。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多个细胞区室中的几乎任何蛋白质。公共健康相关性：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。