title: Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography.
abstract: The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification.
authors.affiliation: CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal.
authors.forename: Catherine
authors.initials: C
authors.lastname: Nunes
authors.forename: Angela
authors.initials: A
authors.lastname: Sousa
authors.forename: José C
authors.initials: JC
authors.lastname: Nunes
authors.forename: António M
authors.initials: AM
authors.lastname: Morão
authors.forename: Fani
authors.initials: F
authors.lastname: Sousa
authors.forename: João A
authors.initials: JA
authors.lastname: Queiroz
mesh_terms: D000327:Adsorption; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry; D008567:Membranes, Artificial; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial; D004278:DNA, Superhelical; D008567:Membranes, Artificial
keywords: Microfiltration; Monolithic chromatography; Plasmid DNA; Purification