EXPRESSED PROTEIN LIGATION - A GENERAL-METHOD FOR PROTEIN ENGINEERING

A protein semisynthesis method-expressed protein ligation-is described that involves the chemoselective addition of a peptide to a recombina nt protein. This method was used to ligate a phosphotyrosine peptide t o the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk), By intercepting a thioester generated in the recombinant protei n with an N-terminal cysteine containing synthetic peptide, near quant itative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intram olecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiolo gically relevant substrate compared with the non-tail-phosphorylated c ontrol. This work illustrates that expressed protein ligation is a sim ple and powerful new method in protein engineering to introduce sequen ces of unnatural amino acids, posttranslational modifications, and bio physical probes into proteins of any size.