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

Finally we devised a semi-quantitative capping enzyme assay to determine the capping efficiency of C3P3-G1 transcripts. Since capping by C3P3 dramatically increased translation of T7p-derived mRNAs, we reasoned that if mR-NAs remained uncapped by C3P3, then supplementation with auxiliary NP868R enzyme should further increase translation over C3P3 alone. To direct auxiliary NP868R to luciferase reporter mRNAs, we fused NP868R to the N peptide of -phage which binds boxB RNA hairpins at nanomolar affinity (7), and added four BoxBr hairpins to the 3 UTR of the firefly luciferase gene ( Figure 4 ). As expected, N-NP868R plus T7RNAP caused higher luciferase expression than T7RNAP alone, demonstrating the functionality N-NP868R and also the value of capping. N-NP868R also complemented expression by cappingdead mutant (K282N) C3P3-G1. However, addition of N-NP868R with C3P3-G1 marginally increased luciferase expression over C3P3 alone, suggesting that capping was already nearly saturated by C3P3. Similar findings we obtained with transcripts produced by standard nuclear expression system using CMV-driven promoter. These findings support that a majority of transcripts are efficiently capped by C3P3.