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Updated: Jun 20, 2026

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase
Published on: November 23, 2016
The NEXT-A (N-terminal EXtension with Transferase and ARS) reaction
Masumi Taki1, Hiroyuki Kuroiwa, Masahiko Sisido
1The Graduate School of Natural Science and Technology, Okayama University, Okayama 700-0082, Japan.
Abstract:
L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein. A nonnatural amino acid is once charged onto an E. coli tRNA(Phe) by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides alphaA294G mutation on the ARS, alphaT251A, betaG318W, or betaA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.
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