In particular, an approach based on the methionine residue substitution method allows the efficient production of proteins with an N-terminal specific Paclitaxel functional group in vivo, which would pave the way to generate proteins with novel functions. The Met residue substitution method introduces unnatural Met analogues into a protein by reassigning the Met codon globally in a protein sequence. The simple procedure using the Met auxotroph enables the production of a range of proteins with functional groups on a large scale. Bio-orthogonally reactive groups, such as L-homopropargylglycine and L-azidohomoalanine, have been incorporated into the Met positions of proteins in vivo by adding the Met surrogates instead of Met because the wild-type Met-tRNA synthetase recognizes the unnatural amino acids. In addition, engineering of the substrate specificity of Met-tRNA synthetase can expand the scope of this methodology. Bacterial proteins are synthesized from Met and the removal process of the start Met can be suppressed by selecting the second residue next to the Met carefully. Therefore, Met analogues can be incorporated into the N-termini of proteins using the Met residue substitution method. However, the presence of the internal Met codons in the target sequences limits the successful application of the Met residue substitution method for N-terminal specific functionalization due to the reassignment of unnatural Met surrogates to internal Met codons as well as to the first Met codon. This problem can be overcome by engineering the protein sequence to be devoid of internal Met residues. Although this approach sometimes needs time-consuming protein engineering work to find internal Met-free variants having original functions of proteins, to our knowledge, this approach is the only one that makes the N-terminal specific modification of a protein possible. Our previous report showed that a protein sequence could be engineered to be an internal Met-free using a consensus-based concept. In the study, the internal Met residues of the single chain fragment variable antibody sequence were replaced successfully with other conserved amino acids without affecting the activity of the protein. This allowed subsequent N-terminal specific functionalization of the scFv using the Met residue substitution method. The stability of scFv probably contributed to the success of the approach because stability of a protein is known to be related to the resistance to mutations. However, it is easily expected that the Met removal based on consensus sequences may not always work, because most proteins are marginally stable and thus cannot withstand multiple changes in their sequences. In particular, hydrophobic residues such as Met are frequently located in the highly packed hydrophobic core, which makes it harder to generate functional Met-free protein sequences. We here engineered a green fluorescent protein to be an internal Met-free protein sequence and demonstrated its Nterminal functionalization using the in vivo Met residue substitution method. It was previously reported that mutations of the three Met residues in the core hydrophobic regions of GFP based on consensus approach induced complete misfolding of the protein. In the present study, a GFP devoid of internal Met residues was generated by semi-rational mutagenesis and its folding efficiency was improved by introducing mutations for GFP folding enhancement.