Vivaspin 20 column

Thrombin cleavage was performed to remove the tag from purified RBD protein. Ten microlitres of thrombin (1U/μL) were added to the 500 μL sample of purified RBD protein and incubated at 22°C for 20 hours. To remove the cleaved 3 kDa tag from the final product, the reaction was passed through a Strep-Tactin resin column (5 mL bed volume) as described above. Flowthrough fractions corresponding to the peak were collected and concentrated to 500 μL using a Vivaspin 20 column (GE Healthcare, Mississauga, Canada). Protein concentration was quantified using the Bio-Rad Protein Assay and used BSA for standard curve preparation. Protein purity was analyzed using SDS-PAGE and protein was visualized using Coomassie blue staining solution.

Fractions corresponding to the protein peak from the HisPur Ni-NTA resin were pooled and protein was further purified using a Strep-Tactin resin column (5 mL bed volume; Cytiva, Marlborough, US) using the AKTA Pure system. The Strep-Tactin column was pre-equilibrated with five bed volumes of binding buffer (PBS, pH 7.4) prior to sample loading, and the column was then washed with 10 bed volumes of binding buffer (PBS, pH 7.4). Protein was eluted using six bed volumes of StrepTrap elution buffer (PBS, 5 mM d-Desthiobiotin, pH 7.4) at a flow rate of 2 mL/minute. Samples were collected as 0.5 mL fractions. In initial experiments, aliquots of each fraction were immunoblotted (probed with anti-His antibody) and protein was also visualized via SDS-PAGE gels stained with Coomassie blue staining solution. We reproducibly detected RBD within fractions 22 to 33, which corresponded to the visible peak eluting from the column. Peak samples were collected, pooled, and concentrated to 500 μL using a Vivaspin 20 column (GE Healthcare; Mississauga, Canada). Plant and mammalian RBD were run on an SDS-PAGE gel and Coomassie-stained for analysis. Both proteins were also immunoblotted with anti-S1 (1: 2000) antibody (Cat: PA5-81795, Invitrogen) and a goat anti-rabbit secondary antibody (1: 10 000).

For CD analyses, Vivaspin 20 columns (10 kDa molecular weight cutoff, GE Healthcare) were used to exchange purified McpB LBD into CD buffer (20 mM sodium phosphate, 100 mM NaCl, pH 7.0). CD experiments were performed on a Jasco J-820 CD spectrometer (Tokyo, Japan) equipped with a 1-mm path length cuvette using 20 μM McpB LBD in the absence and presence of 100 μM boric acid. CD spectra (190–260 nm) were recorded at 25 °C. For thermal denaturation experiments, CD at 222 nm was monitored from 20 to 70 °C.