Recombinant H6 Hemagglutinin Protein Production

The genes encoding the ectodomains of A/Taiwan/2/2013(H6N1) (TW H6) and A/chicken/Guangdong/S1311/2010(H6N6) (GD H6) HA proteins were assembled [57 (link)] from oligos ordered from Integrated DNA Technologies. The H6 HA genes were inserted into pFastBac1 vector that was modified to contain a C-terminal T4 fibritin (foldon) and His₆-tag [58 (link),59 (link)]. The recombinant baculoviruses were made according to the manufacturer’s instruction (Invitrogen). High Five cells were infected at a multiplicity of infection of ~1.0 for 40 hours and the culture was harvested. The supernatant was dialyzed against 20 mM Tris, pH 7.5, 50 mM NaCl followed by incubating with Ni-NTA resin (Thermo Scientific). The HA-bound resin was washed with wash buffer (20 mM Tris, pH 7.5, 50 mM NaCl and 15 mM imidazole) and digested by trypsin (at a weight ratio of about 1:1000) at room temperature overnight to remove the C-terminal foldon and His₆-tag, which also cleaved HA into HA₁ and HA₂. The cleaved HA was further purified by Mono Q 4.6/100 PE and Superdex 200 10/300 GL (GE Healthcare).