title: Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography.
abstract: In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use.
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Caramelo-Nunes
authors.forename: M F
authors.initials: MF
authors.lastname: Gabriel
authors.forename: P
authors.initials: P
authors.lastname: Almeida
authors.forename: J C
authors.initials: JC
authors.lastname: Marcos
authors.forename: C T
authors.initials: CT
authors.lastname: Tomaz
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004133:Diminazene / Q000031:analogs & derivatives* / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D012965:Sodium Chloride / Q000737:chemistry
chemical_list: D012965:Sodium Chloride; D004247:DNA; C003915:diminazene aceturate; D004133:Diminazene