Figure 3. CrPV IGR IRES-mediated translation in coupled eukaryotic cell-free expression systems.

A) Time course analysis of cell-free synthesized LUC within 4 h of incubation using optimized reaction conditions. B) Impact of the optimization process on CrPV IGR IRES-mediated protein synthesis. Reaction temperature, incubation time, template concentration and ion concentrations (Mg(OAc)₂ and KOAc) were adapted to CrPV IGR IRES-directed translation. C) Comparison of the IRES-independent translation at standard conditions and CrPV IGR IRES-dependent translation at optimized conditions in coupled eukaryotic cell-free systems. Translations were carried out in the presence of 0.33 mM m⁷GpppG cap analogue. D) CrPV IGR IRES-mediated translation in a wheat germ CECF system within 24 h of incubation at 24°C. 100 mM KOAc was added to each dialysis reaction. Relative light units were measured using a LUC reporter assay and the corresponding yields of active LUC in µg/mL were calculated based on a calibration curve. Yields of active LUC were determined from three independent experiments and the corresponding standard deviations were calculated.