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Protein Ligation in Living Cells Using Sortase
Author(s) -
Strijbis Karin,
Spooner Eric,
Ploegh Hidde L.
Publication year - 2012
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/j.1600-0854.2012.01345.x
Subject(s) - sortase , sortase a , biology , cytosol , endoplasmic reticulum , biochemistry , secretion , intracellular , microbiology and biotechnology , enzyme , gene , bacterial protein
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 Ca 2+ ‐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 HEK 293 T 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.