Protein purification.

A shaking suspension culture of T. ni High Five cells was set up at a density of 0.5 × 10⁶ cells ml⁻¹ in a total volume of 500 ml. Cells were infected with the virus expressing the His-tagged protein at an MOI of 5 and incubated at 28°C. At the required time point, cells were harvested by centrifugation at 10,000 × g for 15 min. The supernatant was removed, and cells were washed with 50 ml of ice-cold PBS. Cells were lysed with the CytoBuster protein extraction reagent (Novagen) and spun at 14,000 × g for 30 min to remove all insoluble material. After centrifugation, the supernatant was filtered through a 0.45-μm membrane to prevent clogging of purification resin in subsequent steps. His-tagged protein purification was carried out by using the His-Bind purification kit (Novagen) according to the manufacturer's instructions. In brief, iminodiacetic acid (IDA) agarose resin was used in a spin column to purify His-tagged proteins. IDA agarose resin was activated with charge buffer (50 mM NiSO₄) and equilibrated with binding buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole [pH 7.9]). Prepared soluble lysates were passed through the spin column. The resin was treated with binding buffer and then wash buffer (0.5 M NaCl, 60 mM imidazole, 20 mM Tris-HCl [pH 7.9]) to remove any nonspecific binding of proteins with the resin. Elution was performed with buffer containing 400 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl at pH 7.9. The purified protein was assessed for purity by Coomassie staining.