Kta pure 25 m1

FPLC (ÄKTA pure 25 M1, Cytiva) was used to determine the structural integrity and purity of the CPMV, CPMV-H6, and CPMV-G3 samples. The samples were diluted to a final concentration of 0.3 mg mL⁻¹ in 500 μL of 10 mM KP and run through a Superose 6 size exclusion column with dimensions of 10 × 300 mm. The flow rate was set to 0.5 mg mL⁻¹ with an isocratic elution profile for a total elution volume of 50 mL, and absorbance measurements were taken at 260 and 280 nm to measure the nucleic acid and protein concentrations, respectively. The ratio of the absorbance values at 260 and 280 nm at the elution peak was calculated and compared to the UV–vis values to ensure the structural integrity of the VNPs.

To generate variable populations of states A and B, S-2P (100 μL, 0.18 mg/mL) was incubated at either 4 °C or 37 °C for four days. Post-incubation, and prior to SEC-MALS injection, an aliquot from each sample was taken and a one-minute pulse-labeling experiment was performed (see above). The resulting bimodal peptide distributions were used to calculate a fraction state A and B. Samples were filtered (0.22 um um hydrophilic polyvinylidene fluoride Ultrafree-MC GV centrifugal filter) prior to SEC-MALS injection. 90 μL of sample (0.18 mg/mL) was then injected onto a Superose 6 increase 10/300 (GE Healthcare) preequilibrated in filtered (0.1 um polyethersulfone Nalgene Rapid-Flow vacuum filter) PBS flowing at 0.5 ml/min at 4 °C. The ÄKTA Pure 25 M1 (Cytiva) chromatography system was coupled to an 18-angle light scattering Wyatt Dawn detector and Wyatt Optilab refractive index detector (Wyatt Technology). Data analysis was carried out using the program ASTRA 7.1.4.8.