In Vivo N-Terminal Functionalization of Protein Figure 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.g004 groups, 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 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 after denaturation in 8M urea followed by renaturation by dilution. Inlet table shows the refolding rates of GFPnt and GFPhs-r5M variants for fast and slow phase of refolding progress. Normalized fluorescence in arbitrary units was plotted against time. doi:10.1371/journal.pone.0046741.g005 PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46741