Ip. Wicks et al., BACTERIAL LIPOPOLYSACCHARIDE COPURIFIES WITH PLASMID DNA - IMPLICATIONS FOR ANIMAL-MODELS AND HUMAN GENE-THERAPY, Human gene therapy, 6(3), 1995, pp. 317-323
During the course of gene therapy experiments in rodents, using intram
uscular injections of plasmid DNA derived from Escherichia coil, we no
ted dose-related toxicity. This observation prompted a search for poss
ible contaminants of DNA samples. We used the highly specific and sens
itive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioa
ctivity in DNA samples, and found plasmid DNA derived from standard E.
coil bacterial strains, using traditional DNA isolation protocols, to
be heavily contaminated with endotoxin, or lipopolysaccharide (LPA).
Standard DNA isolation procedures result in the copurification of up t
o 500 mu g/ml of LPS. LPS is a potent inducer of cytokines and other i
nflammatory mediators, and may complicate the use of naked DNA in gene
therapy. The copurification of endotoxin with plasmid DNA also has im
portant implications for in vitro transfection studies and microinject
ion of DNA into embryos. A simple and efficient protocol to reduce LPS
contamination of plasmid DNA was developed. The conversion of intact
bacteria to spheroplasts prior to the isolation of plasmid DNA, incuba
tion with lysozyme, treatment with the detergent n-octyl-beta-D-thiogl
ucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the
isolation of plasmid DNA containing less than 50 ng/ml LPS. This repr
esents a 10,000-fold reduction in LPS contamination, compared to conve
ntional methods of plasmid DNA purification, avoids potentially toxic
reagents such as ethidium bromide, and produces a higher yield of plas
mid DNA.