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

Since C3P3-G1 was thus far optimized for expression of luciferase, it was critical to determine whether C3P3-G1 promotes efficient expression, processing, and localization of various proteins. All experiments were performed using DNA templates, which were optimized in both HEK-293 and CHO-K1 cells with similar results (data not shown). As judged by immunofluorescence microcopy, C3P3-G1 drives expression and localization of fluorescent proteins tagged for transport in the expected nuclear, mitochondrial, and endosomal compartments (Figure 7) . The localization of these fluorescent proteins to the endosomal and mitochondrial compartments was confirmed by the coexpression of EGFP fused to the human LDLR and rat OCT sorting signals, respectively (Supplementary Figure S13) . Noticeably, the morphological patterns of these fluorescent proteins appear similar when expressed using a standard nuclear expression system, therefore suggesting that the C3P3-G1 does not impair protein sorting (Figure 7) . To further confirm that the proteins produced by the C3P3-G1 system were properly processed, we assayed in culture medium the functional activity of the released hEPO, a N-and Oglycosylated secreted protein (60). Its activity was similar when produced by C3P3-G1 versus standard nuclear expression systems, showing normal protein folding and disulfide bond formation (Figure 8 ). Taken together, the findings confirm that the expression of C3P3 transcripts generates normal proteins.