Affinity purification of plasmid DNA directly from crude bacterial cell lysates
Darby, Richard A.J., Forde, Gareth M., Slater, Nigel K.H., & Hine, Anna V. (2007) Affinity purification of plasmid DNA directly from crude bacterial cell lysates. Biotechnology and Bioengineering, 98(5), pp. 1103-1108.
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.
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|Item Type:||Journal Article|
|Keywords:||Affinity chromatography, Affinity ligands, LacO-LacI binding, Plasmid DNA, Protein-DNA interaction, Ligands, Optimization, Proteins, Purification, RNA, DNA, bacterium lysate, genomic DNA, ribonuclease A, article, bacterial cell, cell lysate, controlled study, DNA purification, immobilized metal affinity chromatography, methodology, nonhuman, room temperature, Aspartic Acid, Bacteria, Bacterial Proteins, Chromatography, Affinity, DNA, Superhelical, Escherichia coli, Freeze Drying, Green Fluorescent Proteins, Histidine, Isopropyl Thiogalactoside, Plasmids, Recombinant Fusion Proteins, Repressor Proteins, Reproducibility of Results, Ribonuclease, Pancreatic, Sepharose, Bacteria (microorganisms)|
|Divisions:||Current > Schools > School of Chemistry, Physics & Mechanical Engineering
Current > QUT Faculties and Divisions > Science & Engineering Faculty
|Deposited On:||05 Feb 2015 22:54|
|Last Modified:||11 Feb 2015 05:18|
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