Figure 1. IRES-mediated translation in eukaryotic cell-free systems.

LUC encoding expression constructs harboring different IRESs in the EasyXpress pIX3.0 vector backbone were investigated. Cell-free protein synthesis was performed in linked and coupled transcription-translation systems using (A) Sf21, (B) CHO and (C) K562 cell extracts. Vectors containing a specific IRES are indicated by the name of the IRES insert. In the case of the CrPV IGR IRES, the influence of an AUG-to-GCU mutation of the initiation codon was investigated. Reactions were performed at standard conditions in the absence of a cap analogue. 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.