biostudies-literatureMorgan HEBiotechnology and Biological Sciences Research Council2341-2347https://www.ebi.ac.uk/biostudies/studies/S-EPMC9782347biostudies-literatureUnknown33(12)<i>Staphylococcus aureus</i> sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is rBioconjugate chemistryCombined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling.PMC9782347BB/R005540/1BB/M011151/1Morgan HEWebb MEKaminski TPArnott ZLPTurnbull WBfalseCombined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling.<i>Staphylococcus aureus</i> sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is r2022-01-01T00:00:00Z2022 Dec2025-04-04T07:59:58.215Z2025-04-04T07:59:58.215ZS-EPMC97823473635616710.1021/acs.bioconjchem.2c00411