Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
Traffic. 2012 Jun;13(6):780-9. doi: 10.1111/j.1600-0854.2012.01345.x. Epub 2012 Mar 23.
Sortagging is a versatile method for site-specific modification of proteins as applied to a variety of in vitro reactions. Here, we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging, we employ the Ca²⁺-independent sortase A transpeptidase (SrtA) from Streptococcus pyogenes. Substrate proteins were equipped with the C-terminal sortase-recognition motif (LPXTG); we used proteins with an N-terminal (oligo)glycine as nucleophiles. We show that sortase-dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30 min of translation. The versatility of the method is shown by protein ligation of multiple substrates with green fluorescent protein-based nucleophiles in different intracellular compartments.
分拣标签是一种用于各种体外反应的蛋白质定点修饰的多功能方法。在这里，我们探索了将分拣标签方法适应于活细胞的可能性。对于细胞内分拣标签，我们使用来自酿脓链球菌的无钙依赖性分拣标签 A 转肽酶（SrtA）。底物蛋白被装备有 C 末端分拣标签识别基序（LPXTG）；我们使用具有 N 末端（寡）甘氨酸的蛋白作为亲核试剂。我们表明，依赖于分拣标签的蛋白连接可以在酿酒酵母和哺乳动物 HEK293T 细胞中实现，无论是在细胞质中还是在内质网（ER）的腔中。ER 腔分拣标签使反应产物（包括环状多肽）能够分泌。底物和亲核试剂的蛋白连接在翻译后 30 分钟内发生。该方法的多功能性通过使用基于绿色荧光蛋白的亲核试剂在不同的细胞内隔室中对多个底物进行蛋白连接来展示。
