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What methods exist to remove endotoxin contamination of plasmid DNA?

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Endotoxin (lipopolysaccharide or LPS) is a cell wall component of all Gram negative bacteria such as E. coli. Endotoxin is a potent stimulator of the mammalian immune system in vivo, and it may decrease tissue culture cell viability and inhibit transfection efficiency in vitro. Significant amounts of endotoxin are released from E. coli during plasmid purification. Due to the large size of the micelles formed by endotoxin and the negative charges associated with it, endotoxin can carry over with plasmid DNA isolated by CsCl banding, anion-exchange and silica-gel purification methods(1) (2) (3) . In some cases endotoxin contamination can be as high as 1,000-1,500EU/µg of plasmid. (EU or Endotoxin Units is a standard unit of measurement for endotoxin levels and the value is determined by the limulus clotting assay(4) .) For maximum consistency and when transfecting a broad variety of cell lines, it is best to use plasmid DNA containing as little endotoxin as possible.

In the PureYield™ Plasmid Systems, there is an Endotoxin Removal Wash solution that reduces the amount of endotoxin, proteins and other contaminants eluted with the plasmid DNA. For many common cell lines like 293 and HeLa, the amount of endotoxin present for routine transfections has a minimal effect on the efficiency of transfection(5) .

For supposedly purified plasmid DNA, methods have been described to remove endotoxin from these samples. The simplest method is to perform a salt/alcohol precipitation of DNA from solution. Precipitation removes salt, guanidine and endotoxin from DNA in solution. Although not a quantitative removal, this method may be sufficient to improve transfection, especially for cells not extremely sensitive to endotoxin.

More stringent endotoxin removal from purified DNA uses either extraction or chromatography. A simple and effective extraction uses Triton® X-114 detergent(1) . Triton® X-114 can be used to remove endotoxin from solutions containing protein as well as DNA(2) (3) . At temperatures below 20°C, Triton® X-114 detergent will dissolve in aqueous solutions. At temperatures above 20°C, the detergent separates into two phases where lipophilic endotoxin partitions to the organic phase. Repeated extractions followed by isopropanol precipitation of the collected aqueous phase results in a reduction of endotoxin levels to <0.1% for the collected DNA (typically <0.2EU/µg of plasmid).

An alternative method involves chromatography against polymyxin B sulfate (PMB), a cyclic fungal peptide that binds to LPS with high affinity(6) . Resin chromatography can be used to remove endotoxin from solutions containing DNA or proteins(1) (2) . Column chromatography is commonly accomplished using a peristaltic pump to add DNA in TE (pH 7.4) or 100mM NaCl to packed PMB-agarose. The DNA/endotoxin-containing solution is recirculated through the equilibrated column for 12–24 hours. The eluate is collected and DNA is precipitated with salt and alcohol. This method removes similar amounts of endotoxin as extraction with Triton® X-114 but is more time consuming.

Also see the Transfection Chapter of the online Protocols and Applications Guide for more information about optimizing transfection efficiencies.

References

  1. Aida, Y. and Pabst, M.J. (1990) Removal of endotoxin from protein solutions by phase separation using Triton X-114. J. Immunol. Meth. 132, 191–5.
  2. Cotton, M. et al. (1994) Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus. Gene Ther. 1, 239–46.
  3. Wicks, I. et al. (1995) Bacterial lipopolysaccharide copurifies with plasmid DNA: Implications for animal models and human gene therapy. Hum. Gene Ther. 6, 317–23.
  4. Iwanaga, S. (1993) The limulus clotting reaction. Curr. Opin. Immunol. 5, 74–82.
  5. Butahs, K.A. et al. (2000) Reexamination of the effect of endotoxin on cell proliferation and transfection efficiency. BioTechniques 29, 610–9.
  6. Schindler, M. and Osborn, M.J. (1979) Interaction of divalent cations and polymyxin B with lipopolysaccharide. Biochemistry 18, 4425–30.

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