Recombinant Protein Purification and Analysis

Transduced 293-F cells (at least 1 week post-transduction) were seeded at 5 × 10⁵ cells/ml in a 1-l vented shaker flask (Nalgene) in 100 ml of 293 Expression media (Gibco). Twenty-five milliliters of fresh media was added 2–3 days later, when cells reached densities of 2–3 × 10⁶ cells/ml. The media was harvested after 5 days of total incubation after measuring final cell concentration and viability. Culture supernatants were harvested by low-speed centrifugation to remove cells and filtered through a 0.22-micron Centricon ultrafilter (Millipore). NaCl was added to a final concentration of 250 mM and the supernatants were concentrated to final volumes of ~5 ml using a Vivacell-100 centrifugal concentrator (Sartorius Stedim). Recombinant proteins were separated from media by size-exclusion chromatography (SEC) on a Superdex 75 column (GE Healthsciences). Proteins were transferred to PVDF-FL (Millipore) membranes for western blot analysis with mouse anti-STREP primary (IBA) and an anti-mouse-Alexa 680 (Molecular Probes) secondary antibody, with results visualized using Li-COR fluorescent detection system (Odyssey). Endotoxin levels were measured by the Pyrogene endotoxin detection system (Lonza) following the manufacturer's protocols.