Expression and Purification of VP40 Mutants

VP40 mutations were generated by Gene Universal (Newark, DE) and confirmed by DNA sequencing. VP40 was expressed in Rosetta^TM BL21DE3 cells and purified as previously described (Johnson et al., 2021 (link); Narkhede et al., 2023 (link)) using affinity and size exclusion chromatography. Briefly, cell pellets were lysed for 30 min on ice in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) containing 20 μg/mL lysozyme, 1 μg/mL ribonuclease A, 30 μM PMSF, and 1x HALT protease inhibitor cocktail. The lysate was then subjected to sonication (10 sec on, 59 sec off for 10 times on ice). The lysate was cleared by centrifugation at 50,000 x g for 30 min. The cleared lysate was filtered and incubated with Ni‐NTA beads for 20 min at 4°C in an orbital shaker. The beads were then washed with wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 30 mM imidazole, pH 8.0) and VP40 protein was eluted with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0). Size exclusion chromatography (HiLoad 16/600 Superdex 200 pg column) was performed on the eluted protein fractions by running in storage buffer (10 mM HEPES, pH 8.0 containing 150 mM NaCl)) using an AKTA PURE Protein Purification System (Cytiva, Marlborough, MA) to separate and detect VP40 dimer and octamer fractions for WT VP40 and respective mutations.