by ESI-MS to identify the incorporation of Hpg or Aha. The Hpg or Aha incorporated proteins showed an exact mass shift of 222 and 25 Da corresponding to one Met residue substitution of the respective unnatural amino acids (Figure S3). The ESI-MS data in the Figure S3 also showed an incorporation efficiency of .90%. These results clearly show that active GFP with N-terminal specific functional groups with high yield could be produced using the engineered GFPhs-r5M and Met residue substitution method. Figure 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-r5M- Pos) 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-r5M- Neg) 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.g003 In Vivo N-Terminal Functionalization of Protein Characterization of the Functionalized GFP Variants The specific fluorescence, refolding rate and folding robustness of the N-terminal functionalized GFPhs-r5M with Hpg or Aha (designated as GFPhs-r5M-Hpg and GFPhs-r5M-Aha respective- ly) were compared with those of GFPhs-r5M to examine the biophysical effects of N-terminal functionalization on the protein. The biophysical properties of GFPnt were also examined and compared as a control. As shown in Figure 4, GFPhs-r5M, GFPhs-r5M-Hpg and GFPhs-r5M-Aha exhibited similar specific fluorescence activities, which suggest that the addition of alkyne or azide on the N- terminus of the protein did not affect the protein activity negatively. On the other hand, the specific fluorescent activities of GFPhs-r5M and its derivatives were approximately 1.5–2 fold higher than that of GFPnt. This indicates that the mutations introduced into GFPnt-r5M for folding enhancement had influ- ence on the spectral properties of protein in addition to the folding efficiency. This also suggests that the higher whole cell fluores- cence of GFPhs-r5M than that of GFPnt in Figure 2 was caused by an enhancement of the specific fluorescent activity as well as by an increase in the soluble expression level. Figure 5 shows the refolding kinetics of the GFPnt, GFPhs- r5M, and GFPhs-r5M with Hpg or Aha. Both GFPhs-r5M-Hpg and GFPhs-r5M-Aha showed similar folding rates in both the fast and slow phases compared to GFPhs-r5M, which were 4–5 fold higher folding rate compared to GFPnt. These results are correlated with the soluble expressions level of GFPnt and GFPhs-r5M (Figure S1). The study on folding robustness was carried out by estimating the refolding tolerance of the four GFP variants to protein denaturant. The fractions of recovered fluorescence under different urea concentrations were determined after 24 hours and their C0.5 were estimated from the refolding equilibrium plot (Figure 6). The estimated C0.5 values of the GFP variants suggest that the incorporation of the unnatural amino acids has little effect on the folding robustness. Overall, the GFPhs-r5M and its variants with N-terminal specific functional groups showed comparable biophysical prop- erties, and their specific activity, refolding rate and folding robustness 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 Proteins Azides 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, chemoselec- tive 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 GFPhs- r5M-Hpg and GFPhs-r5M-Aha containing an alkyne and azide PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e46741