@inbook{a8bc3ed39ae84f669215eead5bc5700b,
title = "Analyzing protein posttranslational modifications using enzyme-catalyzed expressed protein ligation",
abstract = "Expressed protein ligation (EPL) allows for the attachment of a synthetic peptide into the N- or C-terminus of a recombinant protein fragment to generate a site-specifically modified protein with substantial yields for biochemical and biophysical studies. In this method, multiple posttranslational modifications (PTMs) can be incorporated into a synthetic peptide containing an N-terminal Cysteine, which selectively reacts with a protein C-terminal thioester to afford an amide bond formation. However, the requirement of a Cysteine at the ligation site can limit EPL's potential applications. Here, we describe a method called enzyme-catalyzed EPL, which uses subtiligase to ligate protein thioesters with Cysteine-free peptides. The procedure includes generating protein C-terminal thioester and peptide, performing the enzymatic EPL reaction, and purifying the protein ligation product. We exemplify this method by generating phospholipid phosphatase PTEN with site-specific phosphorylations installed onto its C-terminal tail for biochemical assays.",
keywords = "Enzymology, Peptide synthesis, Protein posttranslational modifications, Protein semisynthesis, Subtiligase",
author = "Niyi Adelakun and Jordan Parrish and Nam Chu",
note = "Publisher Copyright: {\textcopyright} 2023 Elsevier Inc.",
year = "2023",
month = jan,
doi = "10.1016/bs.mie.2022.12.004",
language = "English",
isbn = "9780443185922",
series = "Methods in Enzymology",
publisher = "Academic Press Inc.",
pages = "319--350",
editor = "Shukla, {Arun K.}",
booktitle = "Integrated Methods in Protein Biochemistry",
}