Artificial initiation codons and engineered initiator tRNAs enable N-terminal noncanonical amino acid incorporation in intact cell-free translation systems

Noncanonical amino acid (ncAA) incorporation at the protein N -terminus provides a powerful strategy for installing defined chemical handles while minimizing perturbation of internal protein sequences. However, highly efficient initiation-based ncAA incorporation systems suppress the native methionine pathway by removing methionine or methionyl-tRNA synthetase, limiting their use for proteins containing internal methionine residues. Here, we developed an orthogonal initiation system for selective N -terminal ncAA incorporation into proteins in intact cell-free translation systems. We systematically profiled background initiation from all 64 codons in reconstituted translation systems and identified low-background artificial initiation codons. Engineered initiator tRNAs, termed tRNA ^IniTx , were then designed to decode selected codons and support ncAA-dependent initiation. The optimized CAC/tRNA ^IniTx04 _GUG pair enabled efficient N -terminal incorporation of N -biotinyl-L-phenylalanine without removing methionine or methionyl-tRNA synthetase, reaching over 90% incorporation. The system was further extended to p -azido-L-phenylalanine and to an Escherichia coli extract-based cell-free translation system. Finally, N -terminally biotinylated proteins were directly immobilized on streptavidin biosensors for purification-free biolayer interferometry analysis of computationally designed Brd4 ^BD2 binders. This work establishes a codon-guided orthogonal initiation strategy for N -terminal protein functionalization while preserving the native methionine translation pathway.