Endotoxins, also called lipopolysaccharides (LPS), constitute the cell membrane elements of gram-negative bacteria, such as E. coli. The outer layer's lipid segment of the bacterial outer membrane is entirely made up of endotoxin molecules (see figure Schematic diagram of the E. coli envelope).
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A single E. coli cell contains around 2 million LPS (lipopolysaccharide) molecules, each composed of a hydrophobic lipid A region, complex sugar residues, and negatively charged phosphate groups. This unique structure provides endotoxins with hydrophobic, hydrophilic, and charged domains, influencing their interactions with other molecules.
During bacterial growth, endotoxins are shed in small amounts, but when cells lyse such as during plasmid preparation large quantities are released from the outer membrane into the lysate. These molecules are invisible on agarose gels, undetectable by optical density, and can negatively affect reproducibility in molecular biology experiments.
Why Endotoxins Matter in Transfection
Endotoxins are a major uncontrolled variable in transfection experiments. They can:
- Reduce transfection efficiency in endotoxin-sensitive cell lines.
- Compete with plasmid DNA for free transfection reagents, lowering DNA uptake.
- Impact reproducibility, making results harder to interpret and compare.
Measuring Endotoxins
Historically, endotoxins were measured via a clotting reaction in amoebocytes of the horseshoe crab (Limulus polyphemus). Today, highly sensitive photometric LAL (Limulus Amebocyte Lysate) assays are used, often with colorimetric substrates. Endotoxin levels are expressed in endotoxin units (EU), where typically 1 ng LPS = 1–10 EU.
Endotoxin Contamination in Plasmid DNA
The structural properties of endotoxins allow them to copurify with plasmid DNA:
- On size-exclusion resins, micellar endotoxins behave like large DNA molecules.
- In anion-exchange chromatography, their negative charges interact with the resin.
This means contamination levels depend on the purification method:
| Plasmid purification method | Endotoxin (EU/µg DNA) | Relative transfection efficiency |
|---|---|---|
| Endotoxin-free purification | <0.1 | 154% |
| Low-endotoxin plasmid prep | <1.0 | 100% |
| Standard plasmid prep | ~9.3 | 100% |
| Silica slurry method | ~1230 | 24% |
How to Remove Endotoxins from Plasmid DNA
Effective endotoxin removal strategies include:
- Using specialized purification protocols with endotoxin removal buffers that block LPS binding.
- Employing endotoxin-free plasticware and reagents to prevent recontamination.
- Treating glassware at 180°C overnight, as standard autoclaving does not eliminate endotoxins.
Endotoxin-free plasmid DNA is essential for sensitive applications such as transfection, gene therapy research, and in vivo studies.