title: Negative pseudo-affinity chromatography for plasmid DNA purification using berenil as ligand.
abstract: The present study, reports the utilization of berenil as ligand in a negative pseudo-affinity chromatographic step to purify the plasmid pVAX1-LacZ from Escherichia coli clarified lysates. The chromatographic support was prepared by coupling berenil to epoxy-activated Sepharose and was qualitatively and quantitatively characterized using scanning electron microscopy, Fourier transformed infrared spectroscopy and elemental analysis. The clarified lysate was loaded onto the berenil-Sepharose support with 0.55M of ammonium sulphate in the eluent, achieving the immediate elution of plasmid DNA. The impurities tightly bound to the support, were eluted after decreasing the salt concentration to 0M. The overall process enabled the recovery of 87% of loaded plasmid DNA with a HPLC purity of ≫99% and according to FDA specifications. This method represents an alternative approach to the previous utilization of the same chromatographic pseudo-affinity support in a positive mode. It uses lower amounts of salt and one-step chromatographic procedure, resulting in smaller operating time and costs and representing an alternate procedure for plasmid DNA purification.
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Caramelo-Nunes
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal.
authors.forename: M F
authors.initials: MF
authors.lastname: Gabriel
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal.
authors.forename: P
authors.initials: P
authors.lastname: Almeida
authors.affiliation: Center of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: J C
authors.initials: JC
authors.lastname: Marcos
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal. Electronic address: ctomaz@ubi.pt.
authors.forename: C T
authors.initials: CT
authors.lastname: Tomaz
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004270:DNA, Circular / Q000737:chemistry / Q000302:isolation & purification*; D004133:Diminazene / Q000031:analogs & derivatives* / Q000737:chemistry; D010316:Particle Size; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose / Q000737:chemistry*
chemical_list: D004270:DNA, Circular; D012685:Sepharose; C003915:diminazene aceturate; D004133:Diminazene
keywords: Berenil-Sepharose; Negative pseudo-affinity chromatography; Plasmid purification; Small DNA ligands