The NEXT-A (N-terminal EXtension with Transferase and ARS) reaction.
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.
Taki
Masumi
M
The Graduate School of Natural Science and Technology, Okayama University, Okayama 700-0082, Japan.
Kuroiwa
Hiroyuki
H
Sisido
Masahiko
M
D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D009154:Mutation; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012360:RNA, Transfer, Phe; D014166:Transferases / Q000378:metabolism*
D000596:Amino Acids; D005456:Fluorescent Dyes; D011506:Proteins; D012360:RNA, Transfer, Phe; D014166:Transferases; D010652:Phenylalanine-tRNA Ligase