Amicon ultra 30 filters

The expression and purification of proteins from baculovirus system were performed as previously described (38 (link),40 (link)). Briefly, Sf9 cells were infected with the recombinant baculoviruses and harvested at 3 days post-infection. Cell pellets were re-suspended, lysed by sonication and subject to centrifugation for 30 min at 11 000 g to remove debris. To get rid of the possible contaminant co-purified from MBP–VP35 via binding to RNA, the supernatant was treated with RNase A (Omega) at the final concentration of 0.1 μg/μl for 4 h. Then, the protein in the supernatant was purified using amylase affinity chromatography (New England BioLabs, Ipswich, MA, USA) according to the manufacturer's protocol. For His6-fusion protein, the protein in the supernatant was purified by Ni-NTA agarose column (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. All the purified proteins were concentrated using Amicon Ultra-30 filters (Millipore, Schwalbach, Germany). After that, the store buffer was exchanged to 50 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)–KOH (pH8.0). All proteins were quantified by the Bradford method and stored at –80°C in aliquots. Proteins were separated on 10% SDS-PAGE and visualized by Coomassie blue.

The expression and purification of proteins from the Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, CA, USA) were performed as previously described with minor modification (Li et al., 2018 (link); Shu et al., 2019 (link), 2020 (link)). Firstly, Sf9 cells were infected with the recombinant baculoviruses encoding MBP-CVB3 2C or MBP-CVB5 2C. The infected Sf9 cells were harvested three days after infection by centrifugation at 12,000×g for 15 ​min. Then, the cell pellets were re-suspended in PBS (Invitrogen, China), followed by a short sonication at a frequency of 20 ​kHz for cell lysis preparation. The cell lysis was then subject to centrifugation at 12,000×g for 15 ​min to remove debris. Subsequently, protein purification from the supernatant was performed using amylase affinity chromatography (New England BioLabs, Ipswich, MA, USA) as stated by the manufacturer. The purified proteins were then concentrated using Amicon Ultra-30 filters (Millipore, Schwalbach, Germany). Following protein concentration, the store buffer was replaced with 50 ​mmol/L 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)-KOH (pH 8.0). Protein qualifications were determined using the Bradford assay (Thermo scientific, Rockford, lL, USA), and the proteins were stored at −80 ​°C in aliquots.