Enzyme-catalyzed expressed protein ligation

The enzyme subtiligase can be used to catalyze expressed protein ligation of proteins of interest with peptides lacking an N-terminal cysteine. This enables the analysis of protein modifications in the context of the native primary sequence. Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.