Obustness were higher than GFPnt. This suggests that the functionalized unnatural 79983-71-4 proteins produced were active and stable enough for further study such as bio-conjugation through the introduced functional groups. The results also support the possibility that the introduced mutations for GFP folding enhancement provided the GFP sequence with sufficient folding robustness to withstand the cumulative effects of internal Met-free mutations.Protein-protein Conjugation Using the Functionalized ProteinsFigure 3. Effect of Met analogue incorporation on the productivity of active protein. (A) Protein expression profile of GFPhs-r5M incorporated with Met analogue in E. coli M15A. GFPhs-r5M was expressed without Met (GFPhs-r5M-Neg) or with Met (GFPhs-r5MPos) or with Met analogues such as Hpg (GFPhs-r5M-Hpg) and Aha (GFPhs-r5M-Aha) in minimal media containing 19 amino acids. The expected size is indicated by an arrow (S, soluble fraction; I, insoluble fraction; M, molecular weight marker). (B) Whole cell fluorescence of GFPhs-r5M expressed in minimal medium without Met (GFPhs-r5MNeg) or with Met (GFPhs-r5M-Pos) or with Met analogues such as Hpg (GFPhs-r5M-Hpg) and Aha (GFPhs-r5M-Aha). The relative fluorescence (in arbitrary units) is the fluorescence of whole cells compared with the fluorescence of cells expressing GFPhs-r5M-Pos. doi:10.1371/journal.pone.0046741.gAzides and alkynes are highly energetic functional groups with a particularly narrow distribution of reactivity. In addition, the copper (I) catalyzed cycloaddition reaction of azide and alkyne yields 1,4-disubstituted 1,2,3-triazole linked conjugates under very mild conditions such as room temperature and in an aqueous buffer. Moreover, the reaction is highly regiospecific, chemoselective and tolerant to a wide range of functional groups [1,30,31]. These outstanding features of click chemistry have been extended to various bio-conjugation applications. Protein-protein bio-conjugation reaction based on the click chemistry described above was performed to evaluate the possibility of bio-conjugation using the N-terminal specific functionalized GFPs produced in vivo. The purified GFPhsr5M-Hpg and 478-01-3 GFPhs-r5M-Aha containing an alkyne and azideIn Vivo N-Terminal Functionalization of ProteinFigure 4. Relative specific fluorescene of GFPnt, GFPhs-r5M, GFPhs-r5M-Hpg and GFPhs-r5M-Aha of purified proteins. Relative specific activity (in arbitrary units) is the fluorescence of purified protein compared with the fluorescence of purified GFPhs-r5M-Hpg. doi:10.1371/journal.pone.0046741.ggroups, respectively, on their N-termini were incubated in the presence of CuSO4 and L-ascorbic acid to carry out the cycloaddition (Figure 7A). The bio-conjugation was analyzed by SDS-PAGE. Formation of the GFPhs-r5M 15900046 dimer, 55 kDa in size, was observed with a yield 
of approximately 50 (Figure 7B), whereas the control reaction performed without CuSO4 and L-ascorbic acid did not produce such dimer band.This suggests that the protein-protein conjugation between the N-terminal specific functionalized proteins was achieved in a site-specific manner. In the protein-protein conjugation reaction, we could observe some protein aggregation and this hampered further characterization of the conjugated protein. This problem should be solved to use the protein-protein conjugation method more efficiently.Figure 5. Refolding kinetics of the variants GFPnt, GFPhs-r5M, GFPhs-r5M-Hpg and GFPhs-r5M-Aha. Refolding kinetics was measured aft.Obustness were higher than GFPnt. This suggests that the functionalized unnatural proteins produced were active and stable enough for further study such as bio-conjugation through the introduced functional groups. The results also support the possibility that the introduced mutations for GFP folding enhancement provided the GFP sequence with sufficient folding robustness to withstand the cumulative effects of internal Met-free mutations.Protein-protein Conjugation Using the Functionalized ProteinsFigure 3. Effect of Met analogue incorporation on the productivity of active protein. (A) Protein expression profile of GFPhs-r5M incorporated with Met analogue in E. coli M15A. GFPhs-r5M was expressed without Met (GFPhs-r5M-Neg) or with 
Met (GFPhs-r5MPos) or with Met analogues such as Hpg (GFPhs-r5M-Hpg) and Aha (GFPhs-r5M-Aha) in minimal media containing 19 amino acids. The expected size is indicated by an arrow (S, soluble fraction; I, insoluble fraction; M, molecular weight marker). (B) Whole cell fluorescence of GFPhs-r5M expressed in minimal medium without Met (GFPhs-r5MNeg) or with Met (GFPhs-r5M-Pos) or with Met analogues such as Hpg (GFPhs-r5M-Hpg) and Aha (GFPhs-r5M-Aha). The relative fluorescence (in arbitrary units) is the fluorescence of whole cells compared with the fluorescence of cells expressing GFPhs-r5M-Pos. doi:10.1371/journal.pone.0046741.gAzides and alkynes are highly energetic functional groups with a particularly narrow distribution of reactivity. In addition, the copper (I) catalyzed cycloaddition reaction of azide and alkyne yields 1,4-disubstituted 1,2,3-triazole linked conjugates under very mild conditions such as room temperature and in an aqueous buffer. Moreover, the reaction is highly regiospecific, chemoselective and tolerant to a wide range of functional groups [1,30,31]. These outstanding features of click chemistry have been extended to various bio-conjugation applications. Protein-protein bio-conjugation reaction based on the click chemistry described above was performed to evaluate the possibility of bio-conjugation using the N-terminal specific functionalized GFPs produced in vivo. The purified GFPhsr5M-Hpg and GFPhs-r5M-Aha containing an alkyne and azideIn Vivo N-Terminal Functionalization of ProteinFigure 4. Relative specific fluorescene of GFPnt, GFPhs-r5M, GFPhs-r5M-Hpg and GFPhs-r5M-Aha of purified proteins. Relative specific activity (in arbitrary units) is the fluorescence of purified protein compared with the fluorescence of purified GFPhs-r5M-Hpg. doi:10.1371/journal.pone.0046741.ggroups, respectively, on their N-termini were incubated in the presence of CuSO4 and L-ascorbic acid to carry out the cycloaddition (Figure 7A). The bio-conjugation was analyzed by SDS-PAGE. Formation of the GFPhs-r5M 15900046 dimer, 55 kDa in size, was observed with a yield of approximately 50 (Figure 7B), whereas the control reaction performed without CuSO4 and L-ascorbic acid did not produce such dimer band.This suggests that the protein-protein conjugation between the N-terminal specific functionalized proteins was achieved in a site-specific manner. In the protein-protein conjugation reaction, we could observe some protein aggregation and this hampered further characterization of the conjugated protein. This problem should be solved to use the protein-protein conjugation method more efficiently.Figure 5. Refolding kinetics of the variants GFPnt, GFPhs-r5M, GFPhs-r5M-Hpg and GFPhs-r5M-Aha. Refolding kinetics was measured aft.