4.3. Baculovirus Expression System and Protein Purification

Recombinant baculovirus were developed for the expression and purification of the extracellular domains of ISAV HE and F proteins, using the baculovirus/Sf21 cells expression system, as previously described by Ojeda et al. [35]. For recombinant protein production, a suspension culture of 300 mL of Sf21 cells at 6 × 10⁶/mL was infected using a multiplicity of infection (MOI) of 10. After 120 h culture at 23 °C, Sf21 cells were pelleted at 500× g, washed twice with Grace’s Insect Medium, and then suspended in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, and 1% Triton X-100). After a 2 h incubation on ice, samples were sonicated using 20% of amplitude in five 30 s repetitions, with 30 s of cooling between each repetition. Samples were then centrifuged at 12,500× g for 30 min to remove cell debris. The supernatant, containing the recombinant proteins, was filtered (0.45 μm) using a low-protein-binding filter (Millipore, Burlington, USA). Recombinant hemagglutinin esterase and fusion proteins (rHE and rF) were purified using the anti-FLAG M2 Magnetic Beads System (Sigma-Aldrich, St. Louis, Missouri, USA) and the Magnetight Separator Stand (Novagen, Darmstadt, Germany) according to the manufacturer’s instructions. Protein concentration for each sample was measured using the BCA Assay Kit and BSA standard (Pierce, Waltham, MA, USA). Approximately 20 µg of each protein was obtained from the infected Sf21 cells.