Publication Type Academic Article
Authors Muir T, Sondhi D, Cole P
Journal Proc Natl Acad Sci U S A
Volume 95
Issue 12
Pagination 6705-10
Date Published 06/09/1998
ISSN 0027-8424
Keywords Protein Engineering, Proteins
Abstract 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.
DOI 10.1073/pnas.95.12.6705
PubMed ID 9618476
PubMed Central ID PMC22605
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