1 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 Na- Phosphate 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 SDS- PAGE 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 variants were pooled and dialyzed against 1X phosphate buffered saline (PBS). Measurement of Fluorescence Whole cell fluorescence assay was performed on cells with a 0.1 OD600, suspended in 1X PBS, by measuring fluorescence intensity by exciting at 485 nm and collecting emission at 515 nm with excitation/emission slits of 5.0 nm using Perkin Elmer/Wallac Victor 2 Multilabel Counter (1420-011). The protein samples were excited at 490 nm and emissions collected at 511 nm with excitation/emission slits of 5.0 nm were recorded on Hitachi FL spectrophotometer (F-4500) equipped with FL solution program for analysis of the spectra. Denaturation and Refolding of GFP Variants Each purified GFP variant (30 mM) was unfolded at 95uC for 5 min in 1X PBS containing 8 M urea and 5 mM DTT. Refolding was carried out by 100-fold dilution of urea denatured samples at room temperature into 1X PBS containing 5 mM DTT. The concentrations of denatured proteins were adjusted to 0.3 mM and recovered fluorescence was measured using Hitachi FL spectrophotometer (490 nm excitation, 511 nm emission, 10 nm excitation/emission slit) for 30 min with an interval of 3 sec. The recovered fluorescence was normalized by dividing final fluorescence after 24 h value. The normalized values were fitted with Sigma Plot (Systat Software Inc., CA) using equations as described by previous report [19]. Renaturation Equilibrium Measurement Each purified GFP variant (100 mM) was denatured at 95uC for 5 min in 9 M urea in TNG buffer (25 mM Tris pH 7.5, 0.2 M NaCl, 5% Glycerol, 1 mM DTT). Equilibrium fluores- cence values were measured by diluting the urea denatured proteins into refolding buffer (TNG) containing 5 mM DTT to various final concentration of urea (1–6 M), and allowing the refolding to proceed up to 24 h at 15uC. Fluorescence was measured by exciting at 485 nm and emission at 515 nm with excitation/emission slits of 5.0 nm and was recorded on Perkin Elmer/Wallac Victor 2 Multilabel Counter (1420-011). C0.5 was calculated by measuring the concentration of urea at which the 50% of initial fluorescence was recovered after 24 h of incubation, and the values were determined by sigmoidal fit using sigma plot (Systat Software Inc., CA) [19]. Protein-protein Conjugation Copper (I)-catalyzed cycloaddition reaction between Hpg incorporated GFPhs-r5M and Aha incorporated GFPhs-r5M was performed in a reaction mixture composed of 0.5 mg/ml of each protein (250 ml), 50 mM Tris.HCl pH 8 (100 ml), 50 mM CuSO4 (50 ml), 50 mM L-ascorbic acid (50 ml), and H2O (300 ml). Control was prepared in similar way without the addition of CuSO4 and L-ascorbic acid. The reaction mixture was shaken for In Vivo N-Terminal Functionalization of Protein 24 h at 4uC, and dialyzed against 1X PBS thrice at 4uC. Finally, the reaction mixtures were analyzed by SDS-PAGE. Results Structural Role of Methionine Residues in GFP The wild-type GFP is a 27 kDa protein with a single chain polypeptide containing 238 amino acids. The protein folds into an 11 stranded b-barrel with a single a-helix running through the barrel to form a chromophore. The chromophore cyclization reaction begins once the GFP achieves its near-native like structure [20]. The fluorescent chromophore is directly correlated with the folding status of the protein, which makes GFP an excellent model protein for protein engineering study [21]. In the present study, GFPmut3.1b with a fast folding property [22] bearing one N- terminal Met and five internal Met (M78, M88, M153, M218, and M233) was used as a native GFP variant, designated as GFPnt. Among the five internal Met residues, three Met residues (M78, M88, and M218) are located in the hydrophobic core of the protein and the remaining Met residues (M153 and M233) are exposed to solvent. The buried internal Met218 plays a major role in folding process by interacting with single Trp57 through sulfur- aromatic interactions, which is essential for the GFP folding process [23]. Engineering of Internal Met-free GFP Sequences To generate the internal Met-free GFP sequence, mutations of internal Met residues were attempted by considering the Met locations in the GFP structure. First, mutation of the surface exposed two Met residues (M153 & M233) in GFP was performed. Since the amino acid residues on the protein surface are relatively insensitive to mutations [24,25], the two Met residues were mutated simply based on previous results instead of rigorous mutation studies. It was reported that the M153T mutation of GFP suppressed the aggregation of GFP [26]. For the M233 residue, our previous study informed that it was tolerable to the mutation of Lys [16]. Thus, the M153T and M233K variant of GFPnt, designated as GFPnt-r2M, was generated and the effect of mutation on the GFP activity and productivity were examined. GFPnt-r2M showed similar whole cell fluorescence (Figure 2) and soluble expression level (Figure S1A) with GFPnt, indicating that the mutations did not affect the GFP folding and activity significantly as expected. As next step, we attempted to replace the remaining three internal Met residues (M78, M88, and M218) of GFPnt-r2M in the core hydrophobic region with other amino acids. Actually it was reported that the three Met residues can be mutated into other residues without affecting significantly the fluorescence of cells expressing the GFP variant (GFPrm_AM), in which M78, M88, and M218 were changed into Leu, Phe, and Ala respectively [12]. Therefore, we introduced the three mutations into GFPnt-r2M, but the fluorescence of cells expressing the GFP variant (M78L, M88F, M153T, M218A, and M233K) was much lower than that of cells expressing GFPnt-r2M. Since different GFP sequences were used as template to generate GFPrm_AM and the Met-free GFP in this study, we decided to change the three Met residues of GFPnt-r2M step by step. The M218 residue that plays an important role in folding [23] was randomized into hydrophobic amino acids (Leu, Ile, Val, Phe, and Ala) using oligonucleotides having degenerate codons. The clones showing fluorescence were selected manually based on their fluorescence, and a GFPnt-r2M variant having the M218A mutation, designated as GFPnt-r3M, showed the highest fluorescent among the mutants obtained. The whole cell fluorescence of the GFPnt-r3M was approximately 5 PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46741