Ferritin

Analytical size-exclusion chromatography (SEC) of the full-length and trypsin-processed Vpb4Da2 was performed on an AKTA pure™ using a Superdex-200 10/300 GL increase (Cytiva) equilibrated at 4°C with 25 mM Na carbonate pH 9.5 and 100 mM NaCl. Protein samples were injected at the flow rate of 0.5 mL/min. Superdex-200 10/300 was calibrated using Ferritin (440, 000 Da), Aldolase (158, 000 Da), Conalbumin (75,000 Da), Ovalbumin (44, 000 Da), and Blue dextran 2000, which were all from Cytiva. Kav = (Ve-Vo)/Vc-Vo), with Ve = elution volume, Vo = void volume (8.07 mL), and Vc = Column volume (24 mL) were used. Calibration curve equation, Y = -03896x + 1.2061 (R² = 0.9225) was used to calculate the molecular weight for full length and trypsin processed Vpb4Da2.
Thermal stability of the purified wild-type Vpb4Da2 and variants in protein storage buffer was performed in triplicate reactions using 25 μL of 0.5 mg/mL protein and 5 μL of 10x SYPRO™ Orange (Thermo Scientific). The CFX96™ real-time PCR detection system (Bio-Rad) was used to follow a thermal denaturation gradient of 20°C to 95°C with an increment of 0.5°C per 5 Sec. Fluorescence data were fit using the CFX manager 2.1 (Bio-Rad).

EcOpgD (0.5 mg/mL) and EcOpgG (0.5 mg/mL) were loaded onto a Superdex^TM 200GL column (24 ml; Cytiva) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, and then the target enzymes were eluted with the same buffer at the flow rate of 0.3 mL/min. Ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa) (Cytiva) were used as molecular weight markers. Blue dextran 2000 (2000 kDa) was used to determine the void volume of the column. The molecular weights of EcOpgD and EcOpgG were calculated using Eq. 1, $K_{\mathrm{av}}=\left(V_{\mathrm{e}}-V_{\mathrm{o}}\right)/\left(V_{\mathrm{t}}-V_{\mathrm{o}}\right)$ where K_av is the gel-phase distribution coefficient; V_e is the volume required to elute each protein; V_o is the volume required to elute blue dextran 2000; and V_t is the bed volume of the column.

The biophysical characterization of SARS-CoV-2 HexaPro S protein was conducted using SDS-PAGE analysis (4–12% NuPage™ SDS gel, ThermoFisher, Victoria, Australia) and size-exclusion chromatography (SEC) on a Superose 6 Increase 10/300 GL column (Cytiva, Marlborough, MA, USA) with Gel Filtration Calibration Kits containing a mixture of proteins (thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, carbonic anhydrase, Cytiva, Marlborough, MA, USA) as standards. The accuracy of the antigenic structure of HexaPro S protein was probed by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of S-specific monoclonal antibodies (1047, 2M-10B11, CR3022, S309, hACE2, 2-17, 1-22, mAb 2.8, and mAb 18C2) [47 (link),48 (link),49 (link),50 (link),51 (link),52 (link)].