Journal
BIOPOLYMERS
Volume 90, Issue 3, Pages 406-414Publisher
JOHN WILEY & SONS INC
DOI: 10.1002/bip.20810
Keywords
protein; peptide; ligation
Categories
Funding
- NIBIB NIH HHS [EB001991] Funding Source: Medline
- NIGMS NIH HHS [GM068036, GM072015] Funding Source: Medline
- NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING [R01EB001991] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P01GM068036, P20GM072015] Funding Source: NIH RePORTER
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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 C-13 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 N-15 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. (c) 2007 Wiley Periodicals, Inc.
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