Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 56, Issue 18, Pages 5096-5100Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201611841
Keywords
carbamate; crosslinks; noncanonical amino acids; protein engineering; protein modifications
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Funding
- NIH [R01 GM062159]
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The use of genetically encoded noncanonical amino acids (ncAAs) to construct crosslinks within or between proteins has emerged as a useful method to enhance protein stability, investigate protein-protein interactions, and improve the pharmacological properties of proteins. We report ncAAs with aryl carbamate side chains (PheK and FPheK) that can react with proximal nucleophilic residues to form intra-or intermolecular protein crosslinks. We evolved a pyrrolysyltRNA synthetase that incorporates site-specifically PheK and FPheK into proteins in both E. coli and mammalian cells. PheK and FPheK when incorporated into proteins showed good stability during protein expression and purification. FPheK reacted with adjacent Lys, Cys, and Tyr residues in thioredoxin in high yields. In addition, crosslinks could be formed between FPheK and Lys residue of two interacting proteins, including the heavy chain and light chain of an antibody Fab.
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