title: Histamine monolith versatility to purify supercoiled plasmid deoxyribonucleic acid from Escherichia coli lysate.
abstract: Preparation of high quantities of supercoiled plasmid DNA of pharmaceutical grade purity is a research area where intensive investigation is being performed. From this standpoint, several downstream methods have been proposed, among them the monolithic chromatographic strategies owing to excellent mass transfer properties of monolithic supports and their high binding capacity for large biomolecules. The present study explores the physicochemical properties of histamine ligand in a supercoiled plasmid DNA purification process from an Escherichia coli clarified lysate, where the emphasis is given to the elution strategy that allows higher selectivity and efficient removal of other impurities besides the open circular isoform. The combination of high NaCl concentration and acidic pH allowed the elimination of 89% of RNA during the preparative loading of the lysate sample. The results of the purification strategy with ascending sodium chloride gradient revealed that 97% of supercoiled plasmid DNA was recovered with a purity degree of 99%. In addition, using a combined purification strategy with ascending sodium chloride (capture step) and then descending ammonium sulfate (polishing step) gradient, it was achieved a lower supercoiled plasmid DNA recovery yield of 79% with a purity degree of 92%, although the dynamic binding capacity under these conditions was higher than in the previous strategy. A significant reduction of host contents, such as proteins, RNA and genomic DNA, was obtained in both purification strategies. Accordingly, histamine is a useful and versatile ligand that allows the desirable supercoiled plasmid purification with high yield and purity level.
authors.affiliation: CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. Electronic address: angela@fcsaude.ubi.pt.
authors.forename: A
authors.initials: A
authors.lastname: Sousa
authors.affiliation: CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: A M
authors.initials: AM
authors.lastname: Almeida
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: U
authors.initials: U
authors.lastname: Černigoj
authors.affiliation: CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: F
authors.initials: F
authors.lastname: Sousa
authors.affiliation: CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: J A
authors.initials: JA
authors.lastname: Queiroz
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D004278:DNA, Superhelical / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry*; D006632:Histamine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D010957:Plasmids / Q000302:isolation & purification*
chemical_list: D004278:DNA, Superhelical; D008024:Ligands; D006632:Histamine; D000645:Ammonium Sulfate
keywords: Histamine chromatography; Monoliths; Selectivity; Supercoiled plasmid DNA purification