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Here, we developed a nuclease\u2010free downstream process for Physalis mottle virus (PhMV) VLPs expressed in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    , combining ammonium sulfate precipitation, low\u2010pH extraction, ultrafiltration\/diafiltration (UF\/DF), and flow\u2010through anion\u2010exchange (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\u2010through fraction. This combined workflow produced PhMV VLPs with &gt;\u200995% purity and a recovery of ~68%. Compared with conventional workflows based on ultracentrifugation and size\u2010exclusion chromatography, the new process reduced the purification time from ~3 days to 1 day and hands\u2010on processing time from ~18\u2009h to 8\u2009h, also increasing the average yield from ~80 to ~130\u2009mg 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\u00a0chemical addressability of lysine and cysteine residues. These results establish a scale\u2010up compatible, nuclease\u2010free purification strategy for PhMV VLPs expressed in bacteria and provide a framework for the downstream processing of plant VLP platforms produced in microbial systems.\n                  <\/jats:p>","DOI":"10.1002\/bit.70412","type":"journal-article","created":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T10:47:39Z","timestamp":1792666059000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Nuclease\u2010Free Scalable Purification of Physalis Mottle Virus\u2010Like Particles Expressed in\n                    <i>Escherichia coli<\/i>"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4592-703X","authenticated-orcid":false,"given":"Sean K.","family":"Hsu","sequence":"first","affiliation":[{"name":"Aiiso Yufeng Li Family Department of Chemical and Nano Engineering University of California San Diego California La Jolla USA"},{"name":"Center for Nano\u2010Immuno Engineering University of California San Diego California La Jolla USA"},{"name":"Shu and K.C. Chien and Peter Farrell Collaboratory University of California San Diego California La Jolla USA"},{"name":"School of Biological Sciences University of California San Diego California La Jolla USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4068-0843","authenticated-orcid":false,"given":"Patrick","family":"Opdensteinen","sequence":"additional","affiliation":[{"name":"Aiiso Yufeng Li Family Department of Chemical and Nano Engineering University of California San Diego California La Jolla USA"},{"name":"Center for Nano\u2010Immuno Engineering University of California San Diego California La Jolla USA"},{"name":"Shu and K.C. Chien and Peter Farrell Collaboratory University of California San Diego California La Jolla USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-2285-472X","authenticated-orcid":false,"given":"Tariq","family":"Hussain","sequence":"additional","affiliation":[{"name":"Aiiso Yufeng Li Family Department of Chemical and Nano Engineering University of California San Diego California La Jolla USA"},{"name":"Center for Nano\u2010Immuno Engineering University of California San Diego California La Jolla USA"},{"name":"Shu and K.C. Chien and Peter Farrell Collaboratory University of California San Diego California La Jolla USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0130-0481","authenticated-orcid":false,"given":"Nicole F.","family":"Steinmetz","sequence":"additional","affiliation":[{"name":"Aiiso Yufeng Li Family Department of Chemical and Nano Engineering University of California San Diego California La Jolla USA"},{"name":"Center for Nano\u2010Immuno Engineering University of California San Diego California La Jolla USA"},{"name":"Shu and K.C. 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