Expressed protein ligation: A general method for protein engineering Academic Article uri icon

Overview

MeSH Major

  • Blood Platelets
  • Glycolysis
  • Oxygen Consumption

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.

publication date

  • June 9, 1998

Research

keywords

  • Academic Article

Identity

Digital Object Identifier (DOI)

  • 10.1073/pnas.95.12.6705

Additional Document Info

start page

  • 6705

end page

  • 10

volume

  • 95

number

  • 12