Fig. 6: In vitro and in vivo incorporation of novel expanded monomer selection.
a, Workflow for in vitro translation via codon skipping. b, EICs and mass spectra of peptide products obtained using Ma-tRNAPyl-ACC charged with monomers 7 and 13–15 by MaFRS1 (7 and 15) or MaFRS2 (13 and 14). The insets show the mass spectra of the major ions used to generate the EIC of the translated peptide initiated with the indicated monomer. The expected (exp) and observed (obs) m/z peaks in the mass spectra are as follows: l-Phe (7) (M + 3H): exp: 420.51906, obs: 420.52249; α-SH 13 (M + 2H): exp: 638.75554, obs: 638.75614; 2-BMA (14) (M + 2H): exp: 644.76442, obs: 644.76498; N-formyl-l-Phe (N-fPhe) 15 (M + 2H): exp: 644.27242, obs: 644.27167. c, Workflow for the in vivo incorporation of monomers 1, 2, 20 and 21 at position 200 of sfGFP. d, Intact protein mass spectra of sfGFP variants purified from DH10B cells co-expressing MaPylRS (top) or MaFRSA (bottom) in the presence of 1 mM BocK (1), α-OH-BocK (2), m-trifluoromethylphenylalanine (20) or α-OH-m-trifluoromethylphenylalanine (21). e, Fidelity (%) of sfGFP containing the indicated residue at position 200 when expressed in E. coli DH10B (i–vii) or DH10B ∆aspC ∆tyrB (viii and ix) using either terrific broth (i–v, viii and ix) or a defined media lacking glutamate (vi and vii). The fidelity was determined as described in Methods and Supplementary Fig. 39.
