Expressed protein ligation: a general method for protein engineering.
| Publication Type | Academic Article |
| Authors | Muir T, Sondhi D, Cole P |
| Journal | Proc Natl Acad Sci U S A |
| Volume | 95 |
| Issue | 12 |
| Pagination | 6705-10 |
| Date Published | 06/09/1998 |
| ISSN | 0027-8424 |
| Keywords | Protein Engineering, Proteins |
| 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. |
| DOI | 10.1073/pnas.95.12.6705 |
| PubMed ID | 9618476 |
| PubMed Central ID | PMC22605 |