title: Dynamic binding capacity and specificity of 3,8-diamino-6-phenylphenanthridine-Sepharose support for purification of supercoiled plasmid deoxyribonucleic acid.
abstract: Affinity chromatography represents a sole technique in purification of different biomolecules. The specific recognition between affinity ligands and target biomolecules has a major role in the specificity of the process. Therefore, choosing the right ligand is a crucial step for the development of a successful purification system. This work describes the application of the DNA intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as a chromatographic affinity ligand for the specific separation and purification of supercoiled plasmid DNA (pDNA). The support was prepared by coupling DAPP onto an epoxy-activated Sepharose matrix, using mild conditions and resulting in a ligand density of 0.15mmolDAPP/g derivatized Sepharose. The characterization of DAPP-Sepharose support in terms of dynamic binding capacity was achieved by studying the effect of plasmid DNA concentration and flow rate on pDNA adsorption. The maximum capacity value of 336.75μgpDNA/mL gel was obtained at 1mL/min with a pDNA concentration of 150μg/mL. Moreover, the values did not vary significantly with the variation of flow rate. In addition, the DAPP-Sepharose showed a high affinity towards pDNA as quantified by the respective dissociation constant (Kd=2.29±0.195×10(-7)M). The support was also tested for the purification of two plasmid molecules with different sizes (pVAX1-LacZ and pCAMBIA-1303, with 6.05kbp and 12.361kbp, respectively) from clarified Escherichia coli lysate solutions. Total retention of all lysate components was achieved without any added salt to the eluent buffer. The selective elution of impurities and supercoiled pDNA was accomplished simply by the addition of small amounts of salt to the buffer solution. The yield for pCAMBIA-1303 was 65% and for pVAX1-LacZ was 94%, with all host impurity levels in accordance with the requirements established by the regulatory agencies.
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. catcnunes@gmail.com
authors.forename: C
authors.initials: C
authors.lastname: Caramelo-Nunes
authors.forename: D
authors.initials: D
authors.lastname: Bicho
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*; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004996:Ethidium / Q000031:analogs & derivatives; D010617:Phenanthridines / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D004278:DNA, Superhelical; D010617:Phenanthridines; C029154:3,8-diamino-6-phenyl-phenanthridine; D012685:Sepharose; D004996:Ethidium
keywords: Affinity chromatography; DAPP-Sepharose; Dissociation constant; Dynamic binding capacity; Supercoiled pDNA purification