AbstractExpressed protein ligation (EPL) is a protein semisynthesis technique that allows the site‐specific introduction of unnatural amino acids and biophysical probes into proteins. In the present study, we illustrate the utility of the approach through the generation of two semisynthetic proteins bearing spectroscopic probes. Dihydrofolate reductase containing a single 13C probe in an active site loop was generated through the ligation of a synthetic peptide‐α‐thioester to a recombinantly generated fragment containing an N‐terminal Cys. Similarly, c‐Crk‐II was assembled by the sequential ligation of three recombinant polypeptide building blocks, allowing the incorporation of 15N isotopes in the central domain of the protein. These examples showcase the scope of the protein ligation strategy for selective introduction of isotopic labels into proteins, and the protocols described will be of value to those interested in using EPL on other systems. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90:406–414, 2008.This article was originally published online as an acceptedpreprint. The “Published Online” date corresponds to the preprintversion. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com