C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya

In Nature, eukaryotic RNAPs are commonly coupled with their corresponding capping enzymes, which is likely required for selective capping of transcripts. In contrast, the absence of coupling between the T7RNAP and capping enzyme of the hybrid vaccinia virus-T7RNAP expression system possibly accounts for the poor capping rate of T7transcripts (30) . Hence, we chose to increase the proximity of T7RNAP and vaccinia virus capping enzyme by directly coupling these enzymes together. This approach therefore differs from the strategy used by others (37) (38) (39) , in which nuclear-directed-T7RNAP was fused with the carboxyl terminal domain from POLR2A (40) , which interacts with the host-cell nuclear capping enzyme (41) . Although this fusion was sought to bring these enzymes together, it generates neither capped nor spliced transcripts but instead promotes formation of DNA:RNA hybrids (37, 38) . T7RNAP and vaccinia virus capping enzyme were coupled using EE 1234 L and RR 1234 L leucine-zippers. These amphipathic ␣-helices form an antiparallel heterodimer with binding affinity of ∼10 −15 M (42). RR 1234 L was fused inframe to the amino-terminal ends of either D1R or D12L vaccinia virus capping enzyme subunits, while EE 1234 L was fused in-frame to the amino-terminal end of T7RNAP. Two heterotrimeric enzymes were therefore generated by co-transfection: EE 1234 L-T7RNAP that binds to RR 1234 L-D1R and D12L duplex, as well as EE 1234 L-T7RNAP that binds to RR 1234 L-D12L and D1R duplex. The constructs were transfected in HEK-293 cells and assayed for luciferase expression. In comparison to uncoupled enzymes, leucinezippers on D1R or D12L increased luciferase expression by 7.6-and 5.1-fold respectively ( Figure 2) .