S were pooled and dialyzed against 1X phosphate buffered saline (PBS).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 GFPThe 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 117793 excellent model protein for protein engineering study [21]. In the present study, GFPmut3.1b with a fast folding property [22] bearing one Nterminal 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 sulfuraromatic interactions, which is essential for the GFP folding process [23].Measurement of FluorescenceWhole 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 VariantsEach purified GFP variant (30 15755315 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 AKT inhibitor 2 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].Engineering of Internal Met-free GFP SequencesTo 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 fl.S were pooled and dialyzed against 1X phosphate buffered saline (PBS).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 GFPThe 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 Nterminal 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 sulfuraromatic interactions, which is essential for the GFP folding process [23].Measurement of FluorescenceWhole 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 VariantsEach purified GFP variant (30 15755315 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].Engineering of Internal Met-free GFP SequencesTo 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 fl.
