Ni nitrilotriacetic acid agarose resin

E. coli BL21 transformed with pTrcHisB-GLD-2 were cultured in LB medium containing 100 μg/ml ampicillin. Cells were cultured at 37°C to 0.8 OD at 600 nm, and protein expression was induced at 25°C by the addition of 1 mM isopropyl-β-d-thiogalactopyranoside for 12 h. Cell pellets from a 2-l culture were washed once with phosphate-buffered saline (PBS) and resuspended in 40 ml of binding buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10 mM β-mercaptoethanol, 10% glycerol, 1% Nonidet P-40). Cells were sonicated on ice and centrifuged at 35,000 × g for 15 min. After centrifugation, the supernatant was bound to Ni-nitrilotriacetic acid agarose resin (Qiagen, Hilden, Germany) pre-equilibrated with the binding buffer. Bound proteins were eluted with the binding buffer containing imidazole (50–500 mM). Fractions containing GLD-2 were then dialyzed against buffer A [50 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol (DTT), 50 mM NaCl, 5 mM MgCl₂, and 10% glycerol], and aliquots were stored at −80°C.

The wt NA and mutant NA (I223R/H275Y) genes derived from A/H1N1 2009 pandemic influenza virus were amplified by PCR. The PCR products were cloned into the pAcGP67A vector (BD Biosciences) and transfected into the Sf9 insect cell line to produce recombinant proteins. The culture medium was collected, clarified by centrifugation at 110 × g, and subjected to further viral propagation. The cultured Sf9 cells were inoculated with recombinant baculovirus at a multiplicity of infection of 10 and incubated at 27 °C for 96 h. The cells were collected after centrifugation, and the cell pellet was resuspended in cell lysis buffer (50 mM Tris-HCl, pH 8.5, 5 mM 2-mercaptoethanol, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride), sonicated, and centrifuged at 16,000 × g for 15 min at 4 °C. After the cell lysate was sonicated to reduce its viscosity, the cell debris was removed by centrifugation for 1 h at 16,000 × g. The soluble protein from the cell supernatant was applied to Ni-nitrilotriacetic acid agarose resin (Qiagen), washed, and eluted with buffer (50 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, pH 8.0). The purified proteins were dialyzed against phosphate-buffered saline (PBS) and further purified by Q-Sepharose anion-exchange chromatography (GE Healthcare).