Removal Of Bacterial Endotoxins

Schematic diagram of the endotoxin molecule

Reliable endotoxin removal 

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Endotoxins, also known as lipopolysaccharides or LPS, are cell membrane components of Gram-negative bacteria (e.g., E. coli). The lipid portion of the outer layer of the outer membrane is completely composed of endotoxin molecules (see figure Schematic diagram of the envelope of E. coli). A single E. coli cell contains about 2 million LPS molecules, each consisting of a hydrophobic lipid A moiety, a complex array of sugar residues and negatively charged phosphate groups (see figure Schematic diagram of the endotoxin molecule). Therefore, each endotoxin molecule possesses hydrophobic, hydrophilic, and charged regions giving it unique features with respect to possible interactions with other molecules. Bacteria shed small amounts of endotoxins into their surroundings while they are actively growing and large amounts when they die. During lysis of bacterial cells for plasmid preparations, endotoxin molecules are released from the outer membrane into the lysate.

For further information on endotoxins, view the sections below.
Endotoxin contamination of different plasmid preparation methods 
Measuring endotoxins 
Influence of endotoxins on biological applications 
Removal of endotoxins
Endotoxin-free plasticware and glassware
Endotoxin contamination of different plasmid preparation methods 
The chemical structure and properties of endotoxin molecules and their tendency to form micellar structures lead to copurification of endotoxins with plasmid DNA. For example, in CsCl ultracentrifugation, the CsCl-banded DNA is easily contaminated with endotoxin molecules, which have a similar density in CsCl to plasmid–ethidium bromide complexes.

On size exclusion resins, the large size of the micellar form of endotoxin causes the molecule to behave like a large DNA molecule; and in anion-exchange chromatography, the negative charges present on the endotoxin molecule can interact with anion exchange resins, thus leading to copurification of endotoxins with the plasmid DNA. However, the level of endotoxin contamination found in plasmid DNA is dependent on the purification method used . QIAGEN Plasmid Kits and 2x CsCl gradient centrifugation both yield very pure DNA with relatively low levels of endotoxoin. Silica slurry-purified DNA contains significantly higher endotoxin contamination. DNA purified with EndoFree Plasmid Kits contains only negligible amounts of endotoxin (<0.1 EU/µg plasmid DNA) (see table Endotoxin contamination and transfection efficiency using various plasmid preparation methods).

Endotoxin contamination and transfection efficiency using various plasmid preparation methods* 
Plasmid preparation method
 Endotoxin (EU/µg DNA) Endotoxin (EU/µg DNA)
EndoFree Plasmid Kits 0.1 154%
QIAGEN Plasmid Plus Kits <1.0 100%
QIAGEN Plasmid Kits 9.3 100%
2x CsCl 2.6 99%
Silica slurry 1230 24%
* Host strain: DH5α; plasmid: pRSVcat.
1 ng LPS = 1.8 EU.
The transfection efficiency obtained using plasmid prepared with QIAGEN Plasmid Kits was set to 100%. The transfection efficiencies for all other preparation methods were calculated relative to the QIAGEN Plasmid Kit.

 
Measuring endotoxins
Historically, endotoxins were measured in a clotting reaction between the endotoxin and a clottable protein in the amoebocytes of Limulus polyphemus, the horseshoe crab. Today, much more sensitive photometric tests (e.g., Kinetic-QCL Test from BioWhittaker, Inc.) are used, which are based on a Limulus amoebocyte lysate (LAL) and a synthetic color-producing substrate. LPS contamination is usually expressed in endotoxin units (EU). Typically, 1 ng LPS corresponds to 1–10 EU. 

Influence of endotoxins on biological applications
Endotoxins strongly influence transfection of DNA into primary cells and sensitive cultured cells, and increased endotoxin levels lead to sharply reduced transfection efficiencies. Furthermore, it is extremely important to use endotoxin-free plasmid DNA for gene therapy applications, since endotoxins cause fever, endotoxic shock syndrome, and activation of the complement cascade in animals and humans. Endotoxins also interfere with in vitro transfection into immune cells such as macrophages and B cells by causing nonspecific activation of immune responses. These responses include the induced synthesis of immune mediators such as IL-1 and prostaglandin. It is important to make sure that plasticware, media, sera, and plasmid DNA are free of LPS contamination to avoid misinterpretation of experimental results. 

Removal of endotoxins
The patented EndoFree Plasmid procedure integrates endotoxin removal into the standard QIAGEN Plasmid purification procedure. The neutralized bacterial lysate is filtered through a QIAfilter Cartridge and incubated on ice with a specific endotoxin removal buffer (Buffer ER). The endotoxin removal buffer prevents LPS molecules from binding to the resin in the QIAGEN-tips allowing purification of DNA containing less than 0.1 endotoxin units per µg plasmid DNA. 

Endotoxin-free plasticware and glassware
To avoid recontamination of plasmid DNA after initial endotoxin removal, we recommend using only new plasticware that is certified to be pyrogen- or endotoxin-free. Endotoxin-free or pyrogen-free plasticware can be obtained from many different suppliers. Endotoxins adhere strongly to glassware and are difficult to remove completely during washing. Standard laboratory autoclaving procedures have little or no effect on endotoxin levels. Moreover, if the autoclave has previously been used for bacteria, the glassware will become extensively contaminated with endotoxin molecules. Heating glassware at 180°C overnight is recommended to destroy any attached endotoxin molecules. For further reading on endotoxin removal, please refer to the appropriate literature. It is also important not to recontaminate the purified endotoxin-free DNA by using reagents that are not endotoxin-free. All buffers supplied with the EndoFree Plasmid Kits are tested and certified to be endotoxin-free, as are the water for preparation of 70% ethanol and the TE buffer for resuspension.