A preprint about a method for introducing unnatural amino acids to the N-terminus of proteins and de novo binder screening has been posted on bioRxiv

A preprint has been posted on bioRxiv. This is a collaborative research project with Associate Professor Yasunori Okamoto in The Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences (NINS).

H. Furukawa, Y. Okamoto, N. Terasaka*. Artificial initiation codons and engineered initiator tRNAs enable N-terminal noncanonical amino acid incorporation in intact cell-free translation systems. bioRxiv (2026)

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 tRNAIniTx, were then designed to decode selected codons and support ncAA-dependent initiation. The optimized CAC/tRNAIniTx04GUG 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 Brd4BD2 binders. This work establishes a codon-guided orthogonal initiation strategy for N-terminal protein functionalization while preserving the native methionine translation pathway.