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

Nine candidate single-unit capping enzymes were selected for analysis (Supplementary Table S4 , Supplementary Figure S2 ), which were inferred by bioinformatic annotation to comprise the three enzymatic domains required for mRNA cap synthesis ( Figure 3B ): a 5'-triphosphatase (RTPase) that removes the ␥ -phosphate residue of 5'triphosphate mRNA ends, a guanylyltransferase (GTase) that transfers GMP from GTP to diphosphate 5 -terminus, and a N7-guanine methyltransferase (N7-MTase) that adds a methyl residue onto nitrogen-7 of guanine to produce m7 GpppN caps (43) . For five of these capping enzymes, at least one of these three enzymatic activities were also previously confirmed biochemically (Bluetongue virus VP4 (44) (45) (46) , Mimivirus R382 (47), ASFV NP868R (48), ORF3 from the yeast cytoplasmic episome pGKL2 (49, 50) , Bamboo Mosaic virus ORF1 (51, 52) ). The remaining four capping enzymes were selected based on having >20% amino-acid identity with NP868R and all three enzymatic capping domains inferred by electronic annotation. The activity of each single-subunit C3P3-G1 enzyme was tested following co-transfection with pT710-Luciferase reporter plasmid (25) . High expression was obtained with NP868R-(G 4 S) 4 -T7RNAP and to a lesser extent R382-(G 4 S) 4 -T7RNAP, relative to T7RNAP alone ( Figure 3C ). Coupling of NP868R with T7RNAP by leucine-zippers also increased luciferase expression in comparison to uncoupled enzymes (Supplementary Figure S3) . Similar results were obtained when the T7RNAP was substituted by T3 or SP6RNAP (Supplementary Figure S4) . Both NP868R-(G 4 S) 4 -T7RNAP and R382-(G 4 S) 4 -T7RNAP were also active, albeit at reduced efficiency, when the 10 promoter was substituted by the low-activity 2.5 promoter that initiates transcription with an adenosine (53) (Supplementary Figure S5) . Together, these results show that ASFV NP868R can significantly enhance protein expression when coupled to various phage RNAPs.