title: Expressed protein ligation: a general method for protein engineering.
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.
authors.affiliation: Laboratory of Synthetic Protein Chemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. muirt@rockvax.rockefeller.edu
authors.forename: T W
authors.initials: TW
authors.lastname: Muir
authors.forename: D
authors.initials: D
authors.lastname: Sondhi
authors.forename: P A
authors.initials: PA
authors.lastname: Cole
mesh_terms: D000818:Animals; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D011506:Proteins; D011994:Recombinant Proteins