Superdex 200 sec column

ITC studies were performed using a MicroCal PEAQ-ITC instrument (Malvern, UK). Before performing any experiment, a system suitability test was performed as per the manufacturer’s recommendation (Figures S7 and S8 and Table S3). The samples were buffer-exchanged into PBS, pH 7.4, using a Cytiva Superdex-200 SEC column and degassed by incubating in a vacuum for 20 min. The concentration of the analytes was optimized to reach saturation at least four injections before titration ended. A stirring speed of 750 rpm, temperature 25 ℃, reference power 10 μcal/s, high feedback, and an initial delay of 60 s was used for all experiments. The first priming injection was 0.4 μL, and the rest were 2 μL with 150 s intervals between injections. All calorimetric measurements were performed in triplicate. The data were analyzed using MicroCal PEAQ-ITC analysis software (Version 1.1.0.1262).

To compare the thermal stability of the HBc and NoC constructs, 100µl lysate supernatant samples were incubated for 1 hour at the indicated temperatures. Afterwards, samples were centrifuged at 15,000xg for 10min, the supernatant was carefully collected, and the pellet was resuspended in PBS as to reach the original final volume. Gel protein analysis was performed as previously described. To assess chemical stability, pure HBc and NoC VLPs were precipitated by adding a saturated ammonium sulphate (AMS) to reach 40% v/v. Afterwards, the mixture was centrifuged at 4°C and 15,000xg for 30 min to drive VLP precipitation and the pellet was re-suspended in the indicated urea concentrations in 50mM phosphate buffer (pH 8.5) + 1mM DTT. After overnight incubation at 37°C, samples were run in a Superdex 200 SEC column (Cytiva). For the NoC construct, fractions containing the disassembled monomer fraction after SEC were collected and combined. For re-assembly, 200µl of the disassembled NoC monomer were dialyzed overnight against 50mM Tris (pH 7) + 800mM (NaCl). The resulting sample was then concentrated by AMS precipitation before SEC separation in PBS buffer (pH 7.4) to assess VLP re-assembly.