Recombinant RSV F Protein Production

Prefusogenic F (BV1184), postfusion F (BV2129), prefusion F DS (BV2267), Cav1 (BV2279), and DS-Cav1 (BV2280) were produced by infecting Sf9 cells (MOI = 0.5 pfu/cell) with recombinant BV for 67 h at 27 °C. Cells were harvested by centrifugation and detergent-lysed with 25 mM Tris-HCL (pH 8.0), 50 mM NaCl, 0.5% (v/v) Tergitol™ NP-9, and 2.0 µg mL⁻¹ leupeptin (Sigma-Aldrich, St. Louis, MO, USA), then RSV F proteins were extracted from cell membranes. RSV F proteins were purified by a combination of anion exchange, lentil lectin affinity, and cation ion exchange chromatography [30 (link)]. For soluble postfusion F (BV2128), Sf9 cells were infected with recombinant BV for 67 h and supernatants were clarified by centrifugation. RSV F (BV2128) was purified with an immobilized metal affinity column (IMAC) and ion exchange chromatography [31 (link)]. Purified RSV F proteins (0.33-1.5 mg mL⁻¹) were formulated in 25 mM phosphate buffer (pH 6.8), 150 mM NaCl, 0.032% (v/v) polysorbate-80 (PS80), and 1% (w/v) histidine.