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Cytotechnology

, Volume 63, Issue 1, pp 7–12 | Cite as

Separation of supercoiled from open circular forms of plasmid DNA, and biological activity detection

  • Huangjin Li
  • Huaben Bo
  • Jinquan Wang
  • Hongwei Shao
  • Shulin HuangEmail author
Method in Cell Science

Abstract

To establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccination, a single anion-exchange chromatography (AEC) step was employed to purify supercoiled plasmid DNA (sc pDNA) from other isoforms and Escherichia coli impurities present in a clarified lysate. Two different size and conformation plasmids were used as model targets, and showed similar elution behavior in this chromatographic operation, in which sc pDNA was effectively separated from open circle plasmid DNA (oc pDNA) in a salt gradient. The process delivered high-purity pDNA of homogeneity of 95 ± 1.1% and almost undetectable levels of endotoxins, genomic DNA, RNA and protein, at a yield of 65 ± 8%. Furthermore, the transfection efficiency (29 ± 0.4%) was significantly higher than that (20 ± 0.1%) of a pDNA control. The present study confirms the possibility of using a single AEC step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.

Keywords

Anion-exchange chromatography Supercoiled plasmid DNA Open circular plasmid DNA Gene therapy 

Notes

Acknowledgments

Supported by National Natural Science Foundation (30572124), Guangdong Natural Science Foundation (5002855), Guangdong Science and Technology Plan Project (2004B31201001) and Guangdong Pharmaceutical University doctor science starting foundation

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Huangjin Li
    • 1
  • Huaben Bo
    • 1
  • Jinquan Wang
    • 1
  • Hongwei Shao
    • 1
  • Shulin Huang
    • 1
    Email author
  1. 1.School of Life Science and Bio-PharmaceuticalGuangdong Pharmaceutical UniversityGuangzhouChina

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