Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate

Research output: Contribution to journalArticle

Abstract

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N,N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production. (C) 2004 Wiley Periodicals, Inc.

Details

Authors
  • Per-Olof Wahlund
  • Per-Erik Gustavsson
  • Vladimir Izumrudov
  • Per-Olof Larsson
  • Igor Galaev
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biochemistry and Molecular Biology
  • Industrial Biotechnology
Original languageEnglish
Pages (from-to)675-684
JournalBiotechnology and Bioengineering
Volume87
Issue number5
Publication statusPublished - 2004
Publication categoryResearch
Peer-reviewedYes