Unnatural amino acid mutagenesis allows us to introduce unnatural α-amino acids into internal positions of proteins in response to expanded codons such as amber and four-base codons. To improve the unnatural amino acid mutagenesis, the incorporation of unnatural α-amino acids and non-α-amino acids into the N-terminus of proteins has been achieved using expanded initiation codons. Here, we describe the method for the incorporation of fluorescent-labeled non-α-amino acids into the N-terminus of proteins in a cell-free translation system.
