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

Having found that NP868R can boost protein expression when coupled to T7RNAP, we asked if NP868R could function with other bacteriophage RNAPs. We selected 54 complete bacteriophage genome sequences, which were analyzed for candidate phage-promoter sequences using the PHIRE software (55) . Twenty-nine RNAPs with wellcharacterized promoters were then subjected to BLAST+ analysis (56) , and clustered into 12 subgroups having <80% amino-acid identity (Supplementary Figure S9) . One member of each subgroup was randomly selected and fused to the carboxyl-end of NP868R via (G 4 S) 4 linker (Supplementary Table S5 ). For each RNAP, the putative phage promoter from the major capsid protein was inserted upstream of the luciferase gene and co-transfected with the corresponding C3P3-G1 enzyme plasmids (Supplementary Table S5 ). While highest luciferase expression was achieved with SP6RNAP moiety (Figure 3D ), additional phage RNAPs having >80% amino-acid identity with SP6RNAP were examined in a second optimization step. The two closely related K1ERNAP and K1.5RNAP, which share 99% aminoacid identity together and 85% with SP6RNAP (57), were identified and fused in-frame with NP868R with best results given by K1ERNAP ( Figure 3D ). Overall these studies show that coupling of NP868R to prokaryotic RNAPs generates a novel cytoplasmic expression system. The tethered NP868R enzyme increases the expression of uncapped reporter mRNA produced by the T7RNAP or by the capping-dead mutant (K282N) C3P3-G1 system. In contrast, the expression of capped reporter mRNA produced by the C3P3-G1 system remains virtually unchanged, which suggests that most of C3P3-G1 transcripts are efficiently capped.