An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis.
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.
Ottesen
Jennifer J
JJ
Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10021, USA.
Bar-Dagan
Maya
M
Giovani
Baldissera
B
Muir
Tom W
TW
D000595:Amino Acid Sequence; D001665:Binding Sites; D002247:Carbon Isotopes / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D008956:Models, Chemical; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010452:Peptide Biosynthesis; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D050737:Proto-Oncogene Proteins c-crk / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D003545:Cysteine