Combining Sense and Nonsense Codon Reassignment for Site-Selective Protein Modification with Unnatural Amino Acids

Incorporation of unnatural amino acids (uAAs) via codon reassignment is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. However, the site-selective incorporation of multiple uAAs into polypeptides is hampered by the limited number of reassignable nonsense codons. This challenge is addressed in the current work by developing Escherichia coli in vitro translation system depleted of specific endogenous tRNAs. The translational activity in this system is dependent on the addition of synthetic tRNAs for the chosen sense codon. This allows site-selective uAA incorporation via addition of tRNAs pre- or cotranslationally charged with uAA. We demonstrate the utility of this system by incorporating the BODIPY fluorophore into the unique AGG codon of the calmodulin(CaM) open reading frame using in vitro precharged BODIPY-tRNACysCCU. The deacylated tRNACysCCU is a poor substrate for Cysteinyl-tRNA synthetase, which ensures low background incorporation of Cys i...