Tion volume of 25 ml at a temperature of 30uC and with Fruquintinib web gentle shaking. The basic reaction mixture (RM) contained 2.5 mM ATP, 1.7 mM each of GTP, UTP and CTP, 34 mg/ml folinic acid, 170 mg/ml E. coli tRNA mixture (Roche, Penzberg, Germany), 4?5 ng/ml of plasmid template DNA, 10 mg/ml T7 RNA polymerase, 2 mM each of the 20 proteinogenic amino acids, 0.53 mM NAD+, 0.26 mM CoA, 280 mM K+-glutamate, 10 mM NH4+-glutamate, 10 mM Mg2+glutamate, 1.5 mM spermidine, 1.5 mM putrescine, 4 mM Na+oxalate, 1 mM DTT and 0.24 (v/v) of S30 extract in analytical scale reactions or 31 (v/v) in preparative scale reactions (Table 2) [5]. If Mg2+ ions were not analyzed as screening reagent, the final Mg2+ concentration of the reaction was adjusted to 26 mM with Mg2+-glutamate. The 10-fold premix prepared for screening reactions contained 15 mM putrescine, 15 mM spermidine, 2.5 M K+-glutamate, 100 26001275 mM NH4+-glutamate, 100 mM Mg2+glutamate, 40 mM Na+-oxalate, 330 mM Na+-pyruvate, 340 mg/ ml folinic acid, 10 mM DTT, 5.3 mM NAD+(Table 2). The premix could be stored at 220uC and refrozen multiple times without detectable loss of efficiency.Compound ScreeningBatch reactions were pipetted with a Tecan Freedom EVO 200 device equipped with an eight channel liquid handling arm (461,000 ml and 4650 ml syringes) and two transport 
arms (Tecan, Mannedorf/Zurich, Switzerland). The pipetting range ??was in between 300 nl and 800 ml. Stock solutions of chemicals (Sigma-Aldrich, Steinheim, Germany) were prepared in either H2O or 500 mM HEPES-KOH buffer, pH 8.2, and kept on cooling carriers at 4uC upon pipetting. All additives were adjusted prior addition to pH 8.2 by titration with either 500 mM HEPESKOH, pH 8.2, or with 100 mM L-glutamic acid. Linear concentration screening of selected single compounds as well as correlated concentration screening of two compounds was programmed by the custom designed EYES software based on theFigure 1. Linear concentration screens of basic CF batch reaction compounds. Expression efficiency was determined by sGFP fluorescence. A: Basic compounds S30 extract, DTT, NH4+, Mg2+; B: Plasmid templates. doi:10.1371/journal.pone.0056637.gFigure 2. Correlated concentration screens with Mg2+ ions. Expression efficiency was determined by sGFP fluorescence. A: NTP mix/Mg2+; B: PEP/Mg2+. doi:10.1371/journal.pone.0056637.gChemical Chaperones for Improving Protein Qualitywell (Circle): 262; Incubation time: 20 s; Settle time: 20 s. Protein concentration was calculated from the measured sGFP fluorescence according to a calibration curve with purified sGFP. Potential effects of the analyzed chemicals on sGFP were determined by fluorescence measurements after incubating aliquots of 300 mg/ml purified sGFP with corresponding chemicals at 30uC for 4 hrs. Alternatively, immunoblotting using anti-His antibodies or proteins labeled with 35S-methionine were used for quantification. 35 S-methionine mixed with non-labeled amino acids in a ratio of 1:40,000 were added into the reaction. After expression, samples were transferred into reaction tubes, centrifuged at 22,0006g for 10 min and the supernatant was precipitated with 10 SC66 site trichloric acid. After washing, the pellet and the precipitated supernatant were measured for radioactivity. Control experiments without any DNA template were used as background value for the radioassay.Activity Assay of GNA1-sGFPThe 50 ml reactions were transferred into D-tubes (Novagen, Darmstadt, Germany), diluted with 50 ml buffer (50 mM Tri.Tion volume of 25 ml at a temperature of 30uC and with gentle shaking. The basic reaction mixture (RM) contained 2.5 mM ATP, 1.7 mM each of GTP, UTP and CTP, 34 mg/ml folinic acid, 170 mg/ml E. coli tRNA mixture (Roche, Penzberg, Germany), 4?5 ng/ml of plasmid template DNA, 10 mg/ml T7 RNA polymerase, 2 mM each of the 20 proteinogenic amino acids, 0.53 mM NAD+, 0.26 mM CoA, 280 mM K+-glutamate, 10 mM NH4+-glutamate, 10 mM Mg2+glutamate, 1.5 mM spermidine, 1.5 mM putrescine, 4 mM Na+oxalate, 1 mM DTT and 0.24 (v/v) of S30 extract in analytical scale reactions or 31 (v/v) in preparative scale reactions (Table 2) [5]. If Mg2+ ions were not analyzed as screening reagent, the final Mg2+ concentration of the reaction was adjusted to 26 mM with Mg2+-glutamate. The 10-fold premix prepared for screening reactions contained 15 mM putrescine, 15 mM spermidine, 2.5 M K+-glutamate, 100 26001275 mM NH4+-glutamate, 100 mM Mg2+glutamate, 40 mM Na+-oxalate, 330 mM Na+-pyruvate, 340 mg/ ml folinic acid, 10 mM DTT, 5.3 mM NAD+(Table 2). The premix could be stored at 220uC and refrozen multiple times without detectable loss of efficiency.Compound ScreeningBatch reactions were pipetted with a Tecan Freedom EVO 200 device equipped with an eight channel liquid handling arm (461,000 ml and 4650 ml syringes) and two transport arms (Tecan, Mannedorf/Zurich, Switzerland). The pipetting range ??was in between 300 nl and 800 ml. Stock solutions of chemicals (Sigma-Aldrich, Steinheim, Germany) were prepared in either H2O or 500 mM HEPES-KOH buffer, pH 8.2, and kept on cooling carriers at 4uC upon pipetting. All additives were adjusted prior addition to pH 8.2 by titration with either 500 mM HEPESKOH, pH 8.2, or with 100 mM L-glutamic acid. Linear concentration screening of selected single compounds as well as correlated concentration screening of two compounds was programmed by the custom designed EYES software based on theFigure 1. Linear concentration screens of basic CF batch reaction compounds. Expression efficiency was determined by sGFP fluorescence. A: Basic compounds S30 extract, DTT, NH4+, Mg2+; B: Plasmid templates. doi:10.1371/journal.pone.0056637.gFigure 2. Correlated concentration screens with Mg2+ ions. Expression efficiency was determined by sGFP fluorescence. A: NTP mix/Mg2+; B: PEP/Mg2+. doi:10.1371/journal.pone.0056637.gChemical Chaperones for Improving Protein Qualitywell (Circle): 262; Incubation time: 20 s; Settle time: 20 s. Protein concentration was calculated from the measured sGFP fluorescence according to a calibration curve with purified sGFP. Potential effects of the analyzed chemicals on sGFP were determined by fluorescence measurements after incubating aliquots of 300 mg/ml purified sGFP with corresponding chemicals at 30uC for 4 hrs. Alternatively, immunoblotting using anti-His antibodies or proteins labeled with 35S-methionine were used for quantification. 
35 S-methionine mixed with non-labeled amino acids in a ratio of 1:40,000 were added into the reaction. After expression, samples were transferred into reaction tubes, centrifuged at 22,0006g for 10 min and the supernatant was precipitated with 10 trichloric acid. After washing, the pellet and the precipitated supernatant were measured for radioactivity. Control experiments without any DNA template were used as background value for the radioassay.Activity Assay of GNA1-sGFPThe 50 ml reactions were transferred into D-tubes (Novagen, Darmstadt, Germany), diluted with 50 ml buffer (50 mM Tri.
