Table 2. Reaction conditions were adapted to CrPV IGR IRES-mediated translation in eukaryotic cell-free systems.
Coupled cell-free systems | Sf21 cell extract | CHO cell extract | K562 cell extract |
Expression vector | 60 nM/60 nM | 60 nM/20 nM | 60 nM/20 nM |
KOAc | 75 mM/120 mM | 75 mM/150 mM | 75 mM/120 mM |
Mg(OAc)2 | 2.9 mM/3.4 mM | 2.9 mM/3.9 mM | 2.9 mM/3.4 mM |
Incubation temperature | 27°C/27°C | 30°C/33°C | 30°C/33°C |
Reaction parameters used for cap-dependent translation in coupled reactions (left) were optimized for CrPV IGR IRES-mediated translation (right). Optimization was performed by cell-free expression of active LUC using the pIX3.0-CrPV IGR IRES (GCT) vector as a template. Reactions were incubated for 3 h in a thermomixer.