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

The full-length ORF from the NP868R capping enzyme was optimized for codon usage in Sf9 cells (Spodoptera frugiperda 9). The resulting sequence was synthesized into the F8 donor plasmid (GenScript, Piscataway, NJ), then transferred into the Bacmid shuttle DNA through sitespecific recombination using the Escherichia coli DH10Bac strain (Invitrogen). The recombinant Bacmid DNA was transfected into Sf9 cells and incubated in serum-free culture medium Sf-900 II SFM (Life Technologies) for 5 days at 27 • C before harvest of the budded virus particles released into the medium. The recombinant baculovirus was further amplified by propagation for three days post-infection in Sf9 cells. Cells were then lysed by sonication in presence of protease inhibitor. The HISx10-EK-NP868R recombinant protein was purified from cell extracts on nickelnitrilotriacetic acid columns using a standard imidazole stepwise elution protocol. The samples were analyzed by SDS-PAGE and western-blot using an anti-His antibody, then stored at -20 • C in 50% glycerol buffer. The yields of the baculovirus/Sf9 expression assay were of 4 mg/l of cell culture with a 72% purity rate, as estimated by Coomassie gel staining.