concept [8]. In the study, the internal Met residues of the single chain fragment variable (scFv) 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 [13,14]. 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 [15]. 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 (GFP) to be an internal Met-free protein sequence and demonstrated its N- terminal 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 [16]. 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, which yielded an internal Met-free GFP sequence that can be properly folded. Subsequently, bio-orthogonally reactive amino acid analogues were introduced at the N-terminus of the engineered GFP (Figure 1). Then a protein-protein conjugation was demonstrated using the N-terminally modified GFPs. Materials and Methods Materials T4 DNA ligase, restriction endonucleases and PCR reagents, were purchased from New England Biolabs (Tokyo, Japan). The isopropyl-D-thiogalactopyranoside (IPTG) and other chemicals were purchased from Sigma chemicals (St. Louis, MO, USA) unless otherwise indicated. Hpg and Aha were purchased from Chiralix (Nijmegen, Netherlands). Escherichia coli strain M15A methionine auxotroph was kindly donated by Prof. David A. Tirrell (Caltech, USA) and plasmid pQE80-L was obtained from Qiagen (Valencia, CA, USA). Construction of the GFP Variants The pQE-80-GFP constructed in our previous study [17] was used for the template of GFPnt. Mutagenesis of the Met codons in GFPnt were carried out by assembly PCR method [18]. Table S1 shows the primers used in the mutagenesis. The amplification of GFPnt in pQE80-L using two sets of primers (GFPntOut-F and GFPntM153-R, and GFPntM153-F and GFPntOutR) was per- formed with VentR DNA Polymerase (New England Biolabs) to mutate M153 to Thr. The DNA fragments obtained from these PCR steps were purified on 1.5% agarose gels (Qiagen Gel Extraction Kit). Equimolar quantities of the fragments were used as the template for the assembly PCR using the following pairs of primers: GFPntOut-F and GFPntOut-R. Similarly, M233K mutation was introduced into GFPnt containing M153T using two sets of primers (GFPntOut-F and GFPntM233-R, and GFPntM233-F and GFPntOutR) followed by assembly PCR described above, which generated GFPnt-r2M. GFPnt-r3M was obtained by the saturation mutagenesis of the M218 position in GFPnt-r2M using two sets of primers (GFPntOut-F and GFPntM218-R, and GFPntM218-F and GFPntOutR) and subse- quent screening of fluorescent colonies. GFPnt-r4M was also In Vivo N-Terminal Functionalization of Protein Figure 1. Schematic illustration of the approach for the generation of N-terminal functionalized green fluorescent protein (GFP). Step 1: generation of internal methionine (Met) free GFP by semi-rational mutagenesis using similar physicochemical amino acids. Step 2: introduction of folding enhancement mutations to rescue the fluorescence activity of internal Met-free native GFP. Step 3: N- terminal functionalization of active GFP by Met residue specific incorporation methodology. doi:10.1371/journal.pone.0046741.g001 obtained by the saturation mutagenesis of the M78 and M88 positions in GFPnt-r2M using respective sets of primers and subsequent screening of fluorescent colonies. GFPhs-r5M was commercially synthesized by Genescript Corporation (New Jersey, USA). The GFPnt-r2M, GFPnt-r3M, GFPnt-r4M and GFPnt-r5M were cloned into pQE80-L by using BamHI and HindIII restriction sites, and GFPhs-r5M was cloned into the same plasmid by using EcoRI and HindIII sites. All of the constructs possessed hexa- histidine tags in their C-terminals. Expression and Purification of Met Analogues Incorporated GFP Variants The GFP variants in pQE80-L was transformed into E. coli M15A Met auxotroph and expressed in minimal medium according to previously described protocols [14]. Briefly, the limiting concentration of Met (0.035 mM) allowed the cells to attain an OD600 0.6–0.8 and the target proteins were induced with PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46741