In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

Concurrent incorporation of two modified amino acids at the amber and opal stop codons. (A) Full-length AQP4-P containing L44UAG, L44UGA, or L44UAG plus H68UGA codons (indicated beneath the gel) was translated in WG extract in the presence of a Tran35S Label and an aptamer. ɛNBD-[³H]Lys-tRNA^Lysamb, yeast MBB-[¹⁴C]Cys-tRNA^Cysopl, and yeast NBD-[¹⁴C]Cys-tRNA^Cysopl were added to the translation reactions as indicated above each lane. Products were analyzed by SDS-PAGE and phosphorimaging. Polypeptides that terminate at residue 44, or readthrough residue 44 but terminate at residue 68, are marked by “–” and “*,” respectively. Full-length polypeptides are marked by “**.” Concurrent readthrough efficiency of both stop codons was 16%, compared with WT protein (cf. lanes 1 and 12). Panels shown are taken from the same gel and exposure time. (B) AQP4-P(183) mRNA containing indicated stop codons (shown at bottom) was translated in WG extract in the presence of aa-tRNAs (shown at top). Ribosome–nascent chain complexes were pelleted, RNase treated, and analyzed by SDS-PAGE. The gel was scanned for NBD fluorescence as described in Materials and Methods.