D in various organisms are 1516647 sucrose, glycerol, D-trehalose, D-mannose or D-sorbitol [27]. For lysozyme, D-mannitol was found to prevent aggregation, sucrose acted against deamidation and lactose reduced oxidation [28]. We have analyzed the compatibility of glycerol, sucrose, Dsorbitol, D-trehalose and D-mannose for our CF system by monitoring fluorescent sGFP expression (Table 3). D-sorbitol, Dtrehalose and D-mannose were dose dependent inhibitors of fluorescent sGFP production starting already at 1 final concentration in the reaction (Fig. 4A). In contrast, sucrose and glycerol are tolerated up to 8 and 4 final concentration, respectively. Both compounds could thus be considered as potential CF additives in the determined tolerated concentration ranges. Amino acids can have a dual role in CF expression systems as they primarily serve as substrater for translation, but also could help to stabilize the expression machinery and/or the synthesized target protein. Proteinogenic amino acids such as L-arginine and L-glutamic acid in addition to some non-proteinogenic amino acids such as trans-OH-L-proline, N-acetyl-L-lysine and Lcarnitine are known as Biotin-NHS site protein stabilizers in vitro [29] and the concentration ranges compatible to the CF system were determined by fluorescent sGFP monitoring (Fig. 4B). Overall, all tested amino acids showed beneficial effects with some 10?0 increased 
sGFP fluorescence. The concentration optima were different and ranging from 50?0 mM for glutamic acid, 20?90 mM for trans-OH-L-proline, 20?0 mM for L-arginine, 30?50 mM for N-acetyl-L-lysine, 30?0 mM for L-carnitine and 50?70 mM for sarcosine. In particular N-acetyl-L-lysine and Lcarnitine rapidly inhibit sGFP expression above their optimal concentrations while the concentration optima of the other amino acids have a more Gaussian appearance. The polyions betaine, choline and ectoine are synthesized by organisms living in extremophile environments for the stabilization of cytoplasmic BTZ-043 proteins. However, even E. coli is able to synthesize 
high amounts of betaine under some conditions [30]. Stabilizing effects have been shown with the inhibition of the in vitro insulin amyloid formation by ectoine or betaine [25]. For betaine and ectoine, a high tolerance of up to approximately 150 mM and 100 mM was determined in the CF system (Fig. 4C). However, neither compound had a positive effect on sGFP fluorescence. In contrast, an approximately 30 increased sGFP fluorescence was measured in presence of 4?4 mM choline. The generalFigure 4. Effect of potential protein stabilizers on fluorescent sGFP expression in the CF batch configuration. The first bar of each set indicates the control without added compound and with sGFP production of approximately 500 mg/ml reaction. Data 15900046 are averages of at least three determinations. A: Polyols; B: Amino acids; C: Polyions. doi:10.1371/journal.pone.0056637.gChemical Chaperones for Improving Protein QualityFigure 5. Effect of potential stabilizers on the quality of CF expressed sGFP and GNA1-sGFP. A: Choline or L-arginine were added at final concentrations of 10 mM each. Controls without any additives were taken as 100 . Soluble protein expression was measured by sGFP fluorescence, total protein production was quantified by 35S-Met incorporation and functional folding of GNA1 was analyzed by enzymatic activity. F, fluorescence; T, total protein production; E, enzymatic activity. B: Correlated screening of PEG 8,000 and choline for fluoresce.D in various organisms are 1516647 sucrose, glycerol, D-trehalose, D-mannose or D-sorbitol [27]. For lysozyme, D-mannitol was found to prevent aggregation, sucrose acted against deamidation and lactose reduced oxidation [28]. We have analyzed the compatibility of glycerol, sucrose, Dsorbitol, D-trehalose and D-mannose for our CF system by monitoring fluorescent sGFP expression (Table 3). D-sorbitol, Dtrehalose and D-mannose were dose dependent inhibitors of fluorescent sGFP production starting already at 1 final concentration in the reaction (Fig. 4A). In contrast, sucrose and glycerol are tolerated up to 8 and 4 final concentration, respectively. Both compounds could thus be considered as potential CF additives in the determined tolerated concentration ranges. Amino acids can have a dual role in CF expression systems as they primarily serve as substrater for translation, but also could help to stabilize the expression machinery and/or the synthesized target protein. Proteinogenic amino acids such as L-arginine and L-glutamic acid in addition to some non-proteinogenic amino acids such as trans-OH-L-proline, N-acetyl-L-lysine and Lcarnitine are known as protein stabilizers in vitro [29] and the concentration ranges compatible to the CF system were determined by fluorescent sGFP monitoring (Fig. 4B). Overall, all tested amino acids showed beneficial effects with some 10?0 increased sGFP fluorescence. The concentration optima were different and ranging from 50?0 mM for glutamic acid, 20?90 mM for trans-OH-L-proline, 20?0 mM for L-arginine, 30?50 mM for N-acetyl-L-lysine, 30?0 mM for L-carnitine and 50?70 mM for sarcosine. In particular N-acetyl-L-lysine and Lcarnitine rapidly inhibit sGFP expression above their optimal concentrations while the concentration optima of the other amino acids have a more Gaussian appearance. The polyions betaine, choline and ectoine are synthesized by organisms living in extremophile environments for the stabilization of cytoplasmic proteins. However, even E. coli is able to synthesize high amounts of betaine under some conditions [30]. Stabilizing effects have been shown with the inhibition of the in vitro insulin amyloid formation by ectoine or betaine [25]. For betaine and ectoine, a high tolerance of up to approximately 150 mM and 100 mM was determined in the CF system (Fig. 4C). However, neither compound had a positive effect on sGFP fluorescence. In contrast, an approximately 30 increased sGFP fluorescence was measured in presence of 4?4 mM choline. The generalFigure 4. Effect of potential protein stabilizers on fluorescent sGFP expression in the CF batch configuration. The first bar of each set indicates the control without added compound and with sGFP production of approximately 500 mg/ml reaction. Data 15900046 are averages of at least three determinations. A: Polyols; B: Amino acids; C: Polyions. doi:10.1371/journal.pone.0056637.gChemical Chaperones for Improving Protein QualityFigure 5. Effect of potential stabilizers on the quality of CF expressed sGFP and GNA1-sGFP. A: Choline or L-arginine were added at final concentrations of 10 mM each. Controls without any additives were taken as 100 . Soluble protein expression was measured by sGFP fluorescence, total protein production was quantified by 35S-Met incorporation and functional folding of GNA1 was analyzed by enzymatic activity. F, fluorescence; T, total protein production; E, enzymatic activity. B: Correlated screening of PEG 8,000 and choline for fluoresce.
