An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis

Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10021, USA.
Biopolymers (Impact Factor: 2.39). 01/2008; 90(3):406-14. DOI: 10.1002/bip.20810
Source: PubMed


Expressed 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 (13)C probe in an active site loop was generated through the ligation of a synthetic peptide-alpha-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 (15)N 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.

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    • "Sequential ligation of three recombinant polypeptides. The incorporation of isotopes in the central domain of the protein(Blaschke et al., 2000; Ottesen et al., 2008). "
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