In vivo incorporation of AzPhe into a reporter protein, GFP(TAG) by AzPheRS-mediated amber suppression
(A) GFP fluorescence of bacterial cells cultured in the presence or absence of 1 mM AzPhe. Each label indicates the following: pAcamG, bacteria expressing only fMamtRNACUA; pCNFRS, bacteria expressing AzPheRS derived from Mj and its cognate suppressor tRNA; AzPheRS-1, -2, and -3, bacteria expressing AzPheRSs derived from Sc and fMamtRNACUA. (B) SDS-PAGE analysis of purified recombinant GFP(TAG) proteins produced by amber suppression in the presence or absence of 1 mM AzPhe. (C) MALDI-TOF mass spectra of tryptic peptides digested from GFP(TAG) proteins produced by either Sc TyrRS or AzPheRS-3. The spectrum of AzPheRS-3 sample shows a peak at m/z 2002.68, which matches the expected mass 2002.35 for the AzPhe-containing peptide. This peak was not observed in the spectrum of the Sc TyrRS sample. The amino acid sequence of the AzPhe-containing tryptic peptide is indicated. (D) Enhanced amber suppression activity of AzPheRS-3 with P320A/D321A mutations. P320A and D321A mutations were introduced into the AzPheRS-3 gene, and its amber suppression activity was compared to its parental type AzPheRS-3 by measuring the fluorescence produced through amber suppression of GFP(TAG). *P < 0.01; **P < 0.005.
© 2024 Korean J. Microbiol.
