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Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6705-10.

Expressed protein ligation: a general method for protein engineering.

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  • 1Laboratory of Synthetic Protein Chemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. muirt@rockvax.rockefeller.edu

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.

PMID:
9618476
[PubMed - indexed for MEDLINE]
PMCID:
PMC22605
Free PMC Article
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