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What methods exist to remove endotoxin contamination of plasmid DNA?
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
(1-3). In some cases endotoxin contamination can be as high as 1,000-1,500EU/µg
of plasmid. 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.
EU or Endotoxin Units is a standard
unit of measurement for endotoxin levels and the value is determined by the limulus
clotting assay (4). |
We recommend the Wizard® PureFection
Plasmid DNA Purification System(*) (Cat.# A2150) to produce
DNA suitable for transfection. The system includes a proprietary Endotoxin Removal Resin,
based on a novel magnetic capture technology, to purify plasmid DNA to low levels of
endotoxin contamination.
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 (5). 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.
See also the Transfection Guide
for
alternative methods to produce transfection-grade DNA.
(Chapter 2, page 11, "Preparation of DNA for Transfection.")
References
- 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.
- 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.
- Wicks, I. et al. (1995) Bacterial lipopolysaccharide copurifies with
plasmid DNA: implications for animal models and human gene therapy. Hum. Gene Ther.
6, 317.
- Iwanaga, S. (1993) The limulus clotting reaction. Curr. Opin. Immunol. 5,
74.
- Schindler, M. and Osborn, M.J. (1979) Interaction of divalent cations and
polymyxin B with lipopolysaccharide. Biochemistry 18, 4425.
Wizard is a trademark of Promega Corporation and is registered with the U.S. Patent
and Trademark Office.
Triton is a registered trademark of Union Carbide Chemicals and Plastics Co, Inc.
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
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