Prep scale tff cartridge

Microbial fractions were separated through a combination of serial filtration approaches. Following pre-filtration in situ, water was filtered through a 1-μm Envirochek HV (Pall Corporation, Ann Harbor, MI) sampling capsule to capture eukaryotic-sized particles, followed by filtration through a 0.2-μm 142-mm Supor-200 membrane disc filters (Pall Corporation, Ann Harbor, MI) to capture the bacterial-sized particles. To remove any remaining bacterial cells, the permeate was filtered again using a 0.2-μm Supor Acropak 200 sterile cartridge (Pall Corporation, Ann Harbor, MI) prior to tangential flow filtration (TFF). Viral-sized particles were concentrated to approximately 450 mL as described by Suttle et al. [32 (link)] and Culley et al. [26 (link)], using a regenerated cellulose Prep/Scale TFF cartridge (Millipore Corporation, Billerica, MA) with a 30-kDa molecular-weight cutoff and nominal filter area of 0.23 m².

High Five insect cells were cultured at 27 °C in serum-free Express Five medium (Gibco) supplemented with 0.75% fetal bovine serum (Gibco). Infection of High Five cells with recombinant baculovirus was carried out in 2-liter Delong flasks at a cell density of 1.8–2 × 10⁶ cells/ml, followed by gentle shaking at 27 °C for 70 h. The culture medium (3 liters) was centrifuged (6500 × g, 25 min, 4 °C), and the supernatants were supplemented with 10% glycerol followed by concentration to 300 ml (10-fold) using a Prep/scale-TFF cartridge (Millipore, Burlington, MA). The concentrated supernatant was dialyzed overnight in 50 mm Tris-HCl, pH 8.0, 10 mm imidazole, 10% glycerol, and 300 mm NaCl. The supernatant was then loaded onto a nickel-nitrilotriacetic acid column and washed several times with 50 mm Tris-HCl, pH 8.0, 30 mm imidazole, 300 mm NaCl. The protein was eluted with 3–5 ml of 50 mm Tris-HCl, pH 8.0, 250 mm imidazole, 10% glycerol, 300 mm NaCl. The eluate was immediately supplemented with 1 mm DTT, followed by purification on a Superdex 200 gel filtration column by FPLC. The purified protein in 20 mm Tris-HCl, pH 7.5, 10% glycerol, 150 mm NaCl, 1 mm DTT was kept at −80 °C.