Artificial Initiation Codons and Engineered Initiator tRNAs Enable N-Terminal Noncanonical Amino Acid Incorporation in Cell-Free Translation Systems While Retaining Native Methionine Incorporation.

Noncanonical amino acid (ncAA) incorporation at the protein N-terminus enables defined protein functionalization. However, initiation-based ncAA incorporation systems generally suppress the native methionine pathway, limiting their use for proteins containing internal methionine residues. Here, we developed an orthogonal initiation system for N-terminal ncAA incorporation in cell-free translation systems while retaining native methionine incorporation. We profiled background initiation from all 64 codons in cell-free translation systems and identified low-background artificial initiation codons. Engineered initiator tRNAs were designed to introduce ncAA at the protein N-terminus using artificial initiation codons. This system enabled efficient incorporation of N-biotinyl-l-phenylalanine into protein, reaching over 90% incorporation. This system was further extended to p-azido-l-phenylalanine and to an Escherichia coli extract-based cell-free translation system. Finally, N-biotinylated proteins synthesized in this system were utilized for purification-free biolayer interferometry analysis. This work establishes an orthogonal initiation strategy for N-terminal protein functionalization while preserving the native methionine incorporation.