Expressed protein ligation: A general method for protein engineering

Tom W. Muir, Dolan Sondhi, Philip A. Cole

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1016 Scopus citations


A protein semisynthesis method - expressed protein ligation - is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N- terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail- phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.

Original languageEnglish (US)
Pages (from-to)6705-6710
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number12
StatePublished - Jun 9 1998
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General


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