Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography

• Published in: Journal of Chromatography A 1998-05, Vol.806 (1), p.31-45
• Main Authors: Prazeres, Duarte Miguel F, Schluep, Thomas, Cooney, Charles
• Format: Article
• Language: English
• Subjects: Anions; Chromatography, High Pressure Liquid - methods; Chromatography, Ion Exchange - methods; Deoxyribonuclease HindIII - metabolism; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA, Superhelical - isolation & purification; Electrophoresis, Agar Gel; Escherichia coli - genetics; Gene therapy; Plasmid DNA; Plasmids - genetics; Transformation, Bacterial
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(97)01254-5

Large scale manufacturing of gene vectors such as plasmid DNA is an important issue in gene therapy. Anion-exchange chromatography is fundamental in the downstream processing of plasmids both as a process and analytical technique. This work reports the use of Q-Sepharose columns (1, 10 and 40 ml) for the preparative purification of plasmid pUC18. NaCl gradient elution enabled the isolation of supercoiled plasmid from low- M r RNA, cDNA and plasmid variants. A compact covalently closed, supercoiled form of denatured plasmid carrying large stretches of single-stranded DNA was identified as one of the major contaminants. Anion-exchange HPLC on a Poros QE 20 column was used to quantify plasmid yield. Supercoiled plasmid was recovered in a single fraction with a 62±8% yield. Loadings higher than 40 μg/ml gel could be used but at the expense of a loss of resolution between open circular and supercoiled forms. Plasmid quality was evaluated by gel electrophoresis, restriction analysis, transformation experiments and protein assays.