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

Incorporation and stability of NBD-Cys-tRNAs in RRL and WG extract translation systems. (A) The aa-tRNAs in Fig. 2A were modified with NBD as described in Materials and Methods and added to RRL (gray bars) or WG extract (white bars) translation reactions programmed with AQP4-P(98) cDNA containing a cognate cysteine (UGC) or amber (UAG) codon at residue 44. [¹⁴C]Cys and [¹⁴C]Lys incorporation into protein was measured by liquid scintillation counting of hot acid precipitable counts as described in Materials and Methods. Results show mean ± SEM (n ≥ 3). (B–D) Deaminoacylation of ɛNBD-Lys-tRNA^Lysamb(•), yeast (▲), E. coli (■), or human (▼) NBD-Cys-tRNA^Cys was determined as described in Fig. 2.