Plant‐based production can result in covalent cross‐linking of proteins

To investigate potential differences between plant and insect cell‐derived procapsids, genes encoding the identical amino acid sequence of the α‐peptide were expressed in N. benthamiana leaves and insect cells using plasmids pEAQ‐HT‐NωV and pFastBac‐NωV, respectively (Agrawal and Johnson, 1995; Berardi et al., 2020; Castells‐Graells, 2019); in both cases, VLPs were extracted in the procapsid form as previously described. Since identical conditions were used to extract and purify the procapsids and the denaturing conditions used for the SDS‐PAGE analysis were the same in each case, the formation of the oligomers must be a specific consequence of using plants for expression. Western blot analysis (Figure 1b) showed that incubation of insect cell‐produced procapsids in plant extracts results in the appearance of dimers and additional high molecular bands (lanes 2 to 4), not seen when the procapsids were incubated in buffer alone (lane 1).