Cosmogel his accept resin

The expression and purification of B. subtilis GGT have been described previously (Wada et al., 2010 ▶ ). Briefly, E. coli C41(DE3) strain transformed with the plasmid pCold I-His₆-ggt was grown at 310 K in 3.6 l liquid Terrific broth containing ampicillin (50 µg ml⁻¹) to an optical density of 0.6 at 600 nm. At this stage, expression of the N-­terminal His₆-tagged GGT was induced by decreasing the temperature from 310 to 288 K, followed by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After induction, the transformant was cultured at 288 K for 38 h, the cells were collected by centrifugation (2560g) and disrupted. The soluble fraction was subjected to COSMOGEL His-Accept resin (Nacalai Tesque) and the N-­terminal His-tagged GGT was eluted according to the manufacturer’s protocol. Fractions containing the GGT were collected and concentrated. The His₆-GGT was further purified by gel filtration using a HiPrep 16/60 Sephacryl S-200 HR column (GE Healthcare) to homogeneity as checked by SDS gels stained with Coomassie Blue.

For the coexpression of VP6 and VP3, three sets of recombinant baculovirus combinations were used. To copurify VP6 with VP3, nickel affinity chromatography was used as described previously, but with some modifications (22 (link), 55 (link)). Briefly, Sf9 cells were infected with Ac/BTV10/His-VP6 at an MOI of 2.5 together with either Ac/BTV1/HA-VP3 or Ac/BTV1/VP3 at an MOI of 5.0. In parallel, Sf9 cells were infected with Ac/BTV1/His-VP3 (MOI, 5.0) together with Ac/BTV10/VP6 (MOI, 2.5). At 2 days postinfection, the cells were harvested and lysed with VP3 lysis buffer containing 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1.0% (wt/vol) Triton X-100, and protease inhibitor cocktail (Nacalai Tesque). Nonnuclear RNA and DNA were removed by treatment with 10 μg/ml of RNase A (Nippon Gene) and 0.3 U/ml of DNase (Nippon Gene) at 37°C for 15 min. After removal of the nuclei and cell debris, the cell lysate was mixed with Cosmogel His-Accept resin (Nacalai Tesque) for 1 h at 4°C. After the affinity gel was washed with 50 mM sodium phosphate buffer (pH 8.0) containing 10% glycerol, 200 mM NaCl, and 20 mM imidazole, His-tagged proteins and interacted proteins were eluted with 50 mM sodium phosphate buffer (pH 8.0) containing 10% glycerol, 200 mM NaCl, and 250 mM imidazole. Fractions of 1.0 ml each were collected and analyzed by SDS-PAGE.