Suppression of Formylation Provides an Alternative Approach to Vacant Codon Creation in Bacterial In Vitro Translation

Genetic code reprogramming is a powerful approach to controlled protein modification. A remaining challenge, however, is the generation of vacant codons. We targeted the initiation machinery of E. coli, showing that restriction of the formyl donor or inhibition of the formyl transferase during in vitro translation is sufficient to prevent formylation of the acylated initiating tRNA and thereby create a vacant initiation codon that can be reprogrammed by exogenously charged tRNA. Our approach conveniently generates peptides and proteins tagged N‐terminally with non‐canonical functional groups at up to 99 % reprogramming efficiency, in combination with decoding the AUG elongation codons either with native methionine or with further reprogramming with azide‐ and alkyne‐containing cognates. We further show macrocyclization and intermolecular modifications with these click handles, thus emphasizing the applicability of our method to current challenges in peptide and protein chemistry.