Expressed protein ligation: A general method for protein engineering

Expressed protein ligation: A general method for protein engineering

Laboratories of ^*Synthetic Protein Chemistry and ^§Bioorganic Chemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021

Communicated by Samuel J. Danishefsky, Memorial Sloan–Kettering Cancer Center, New York, NY (received for review January 27, 1998)

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.

Footnotes

↵ † T.W.M., D.S., and P.A.C. contributed equally to this work.
↵ ‡ To whom reprint requests should be addressed. e-mail: cole{at}rockvax.rockefeller.edu and muirt{at}rockvax.rockefeller.edu.
↵ ‖ Repression of Src largely requires an unphosphorylated activating loop in addition to tail phosphorylation. A major feature of Src repression appears to be that tail phosphorylation results in a conformational change that prevents autophosphorylation of the activating loop (14, 27). Because Csk does not have an activating loop it would have been impossible to predict the results on Csk’s catalytic activity resulting from the conformational change due to tail phosphorylation. However, these results implicitly argue that one or more structural features that lead to catalytic repression in tail-phosphorylated Src is/are absent in tail-phosphorylated Csk.
↵ ** The simple model where the increased catalytic efficiency results from an interaction of the Lck-SH2 domain with the phosphotyrosine tail of Csk^pPEP appears unlikely because the addition of the peptide NH₂-Cys-Glu-Asp-Asn-Glu-phosTyr-Thr-Ala-Arg-Glu-aminocaproate-Lys-ɛ-[fluorescein]-CO₂H (250 μM) had minimal effect on either Csk^pPEP or Csk^PEP catalyzed Lck phosphorylation.
ABBREVIATIONS:

Csk,

C-terminal Src kinase;

CskPEP,

Csk-[CONH]-peptide (the ligation production of Csk and unphosphorylated peptide);

CskpPEP,

Csk-[CONH]-phosphopeptide (the ligation product of Csk and phosphopeptide);

CBD,

chitin binding domain;

SH2,

Src homology 2