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