Table 1.
Construction of DNA templates.
Table 2.
CF reaction protocol for compound screening.
Figure 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.
Figure 2.
Correlated concentration screens with Mg2+ ions.
Expression efficiency was determined by sGFP fluorescence. A: NTP mix/Mg2+; B: PEP/Mg2+.
Figure 3.
Effect of PEG and alcohols on fluorescent sGFP expression in the CF batch configuration.
The first bar of each set indicates the control without added compound. Data are averages of at least three determinations. A: Screening of PEGs of different molecular weight. The sGFP protein control was 600–750 µg/ml. B: Effect of alcohols. The sGFP protein control was approximately 500 µg/ml.
Figure 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 µg/ml reaction. Data are averages of at least three determinations. A: Polyols; B: Amino acids; C: Polyions.
Table 3.
Compatibility of protein stabilizing compounds to the CF system.
Figure 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 fluorescent expression of GNA1-sGFP. Controls without any additives were taken as 100%. Black, 160–180%; Dots, 120–160%; Lines, 80–120%; Gray, 0–80%.
Figure 6.
Effect of protein stabilizers on the soluble expression of CurA halogenase.
The CurA halogenase domain was expressed in the batch configuration with different additives. Protein production was quantified by immunoblotting. The results were normalized with the control as 100% corresponding to a protein concentration of 80 ng/µl. A: Immunoblot with anti-penta-His antibody. M, marker proteins in kDa; P, positive control for quantification (Positope™, invitrogene). B: Quantification of band intensity. 1, control; 2, 6% D-trehalose; 3, 10 mM L-arginine; 4, 10 mM choline.