Nuclease-Free Scalable Purification of Physalis Mottle Virus-Like Particles Expressed in Escherichia coli.

Abstract

Plant virus-like particles (VLPs) are promising nanotechnology platforms, but their translation requires purification strategies suitable for large-scale production. Here, we developed a nuclease-free downstream process for Physalis mottle virus (PhMV) VLPs expressed in Escherichia coli, combining ammonium sulfate precipitation, low-pH extraction, ultrafiltration/diafiltration (UF/DF), and flow-through anion-exchange (AEX) chromatography. Ammonium sulfate precipitation separated the VLPs from bulk host nucleic acids, whereas extraction at low pH removed most host cell proteins (HCPs) while retaining the VLPs in solution. Subsequent UF/DF was used to concentrate VLPs and simultaneously achieve buffer exchange for downstream AEX chromatography, which removed remaining HCPs while the PhMV VLPs were collected in the flow-through fraction. This combined workflow produced PhMV VLPs with > 95% purity and a recovery of ~68%. Compared with conventional workflows based on ultracentrifugation and size-exclusion chromatography, the new process reduced the purification time from ~3 days to 1 day and hands-on processing time from ~18 h to 8 h, also increasing the average yield from ~80 to ~130 mg PhMV VLPs per liter of culture. The resulting particles were intact and monodisperse, with a lower content of encapsulated host nucleic acid than VLPs purified by ultracentrifugation. The VLPs also had increased chemical addressability of lysine and cysteine residues. These results establish a scale-up compatible, nuclease-free purification strategy for PhMV VLPs expressed in bacteria and provide a framework for the downstream processing of plant VLP platforms produced in microbial systems.