title: Protein ligation in living cells using sortase.
abstract: 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.
authors.affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
authors.forename: Karin
authors.initials: K
authors.lastname: Strijbis
authors.forename: Eric
authors.initials: E
authors.lastname: Spooner
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism / Q000502:physiology*; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D003585:Cell Biology; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D049452:Green Fluorescent Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases