Streamlined expressed protein ligation using split inteins

Miquel Vila-Perelló, Zhihua Liu, Neel H. Shah, John A. Willis, Juliana Idoyaga, Tom W. Muir

Research output: Contribution to journalArticlepeer-review

53 Scopus citations

Abstract

Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

Original languageEnglish (US)
Pages (from-to)286-292
Number of pages7
JournalJournal of the American Chemical Society
Volume135
Issue number1
DOIs
StatePublished - Jan 9 2013

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Fingerprint Dive into the research topics of 'Streamlined expressed protein ligation using split inteins'. Together they form a unique fingerprint.

Cite this