Urchased 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 VariantsThe 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 (inhibitor GFPntOut-F and GFPntM153-R, and GFPntM153-F and GFPntOutR) was performed 
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 subsequent screening of fluorescent colonies. GFPnt-r4M was alsoFigure 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: Nterminal functionalization of active GFP by Met residue specific incorporation methodology. doi:10.1371/journal.pone.0046741.gobtained 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 hexahistidine tags in their C-terminals.Expression and Purification of Met Analogues Incorporated GFP VariantsThe GFP variants in pQE80-L was inhibitor 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?.8 and the target proteins were induced withIn Vivo N-Terminal Functionalization of Protein1 mM IPTG followed by simultaneous addition of either Met or Met analogues (0.5 mM) and allowed the expression for 5 h. The harvested cells were suspended in lysis buffer (50 mM NaPhosphate buffer pH 7.4 containing protease inhibitor) and disrupted by french press. The suspension was subsequently spun down to collect the soluble and insoluble protein fraction for SDSPAGE analysis. The remaining soluble protein fractions were purified by Ni-NTA column chromatography (GE Healthcare Bio-Sciences, Sweden) by standard protocol. Elution fractions were analyzed by SDS-PAGE, and those that were enriched in the desired GFP variant.Urchased 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 VariantsThe 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 performed 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 subsequent screening of fluorescent colonies. GFPnt-r4M was alsoFigure 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: Nterminal functionalization of active GFP by Met residue specific incorporation methodology. doi:10.1371/journal.pone.0046741.gobtained 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 hexahistidine tags in their C-terminals.Expression and Purification of Met Analogues Incorporated GFP VariantsThe 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?.8 and the target proteins were induced withIn Vivo N-Terminal Functionalization of Protein1 mM IPTG followed by simultaneous addition of either Met or Met analogues (0.5 mM) and allowed the expression for 5 h. The harvested cells were suspended in lysis buffer (50 mM NaPhosphate buffer pH 7.4 containing protease inhibitor) and disrupted by french press. The suspension was subsequently spun down to collect the soluble and insoluble protein fraction for SDSPAGE analysis. The remaining soluble protein fractions were purified by Ni-NTA column chromatography (GE Healthcare Bio-Sciences, Sweden) by standard protocol. Elution fractions were analyzed by SDS-PAGE, and those that were enriched in the desired GFP variant.
