Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography.
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 micrograms/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.
Prazeres
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DM
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Lisbon, Portugal.
Schluep
T
T
Cooney
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C
D000838:Anions; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004278:DNA, Superhelical / Q000302:isolation & purification*; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial
D000838:Anions; D004278:DNA, Superhelical; D015247:Deoxyribonuclease HindIII; C061568:endodeoxyribonuclease SspI; D015252:Deoxyribonucleases, Type II Site-Specific