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

Polysome fractionation was performed as described elsewhere with minor modifications (21) . A single 75 cm 2 tissue culture flask of HEK-293 transfected cells was used for each sucrose gradient. The culture medium was removed 24 h after transfection and replaced with fresh medium. After overnight incubation, the medium was changed again and 2 h later, cycloheximide at 100 g/ml was added for 10 min. Cells were washed with PBS, collected by trypsinization, and pelleted. The dry cell pellet was resuspended in 500 l of lysis buffer (50 mM Tris-HCl at pH 7.4, 300 mM KCl, 10 mM Mg-Acetate, 1 mM DTT, 0.05% Nonidet P40) containing 200 units/ml of SUPERaseIn RNAse inhibitor (Invitrogen) and 100 g/ml of cycloheximide, and lyzed by incubation on ice for 10 min with occasional shaking. Nuclei and cell debris were removed by centrifugation at 1000 × g for 10 min and 400 l of supernatant was layered directly onto a 12 ml 15-50% (w/v) sucrose gradient in 50 mM Tris-acetate (pH 7.5), 50 mM NH 4 Cl, 12 mM MgCl 2 and 1 mM DTT. The gradient was centrifuged at 39 000 rpm in a SW41 Beckman rotor for 2.75 h at 4 • C. After centrifugation, optical density (O.D.) at 254 nm was monitored by pumping the gradient through a Retriever 500 (Teledyne Isco) fraction collector.