Affinity purification of plasmid DNA directly from crude bacterial cell lysates

Richard A.J. Darby, Gareth M. Forde, Nigel K. H. Slater, Anna V. Hine*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required. © 2007 Wiley Periodicals, Inc.

Original languageEnglish
Pages (from-to)1103-1108
Number of pages6
JournalBiotechnology and Bioengineering
Volume98
Issue number5
DOIs
Publication statusPublished - Dec 2007

Keywords

  • affinity chromatography
  • affinity ligands
  • lacO-LacI binding
  • plasmid DNA
  • protein-DNA interaction

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