Purity of Recombinant Proteins: Research Effects of Endotoxins
October 21, 2020
The purity of your recombinant proteins is critical to the success of your research. A number of contaminants can affect both in vitro and in vivo systems. Some of the results are predictable, but others are not. All can affect your experimental data.
This article will focus on one of the most common contaminants: endotoxins or lipopolysaccharides. List Labs provides certificates of analysis showing the very low levels of endotoxins in our products.
What Are Endotoxins?
Lipopolysaccharides, also known as endotoxins, are a class of complex hydrophobic molecules found in the cell membranes of Gram-negative bacteria such as Escherichia coli. They are released in large quantities following cell death and during cell division, so they are a common component of recombinant protein production.
The general structure of an endotoxin is one or more Lipid A molecules bonded to one end of a short polysaccharide oligomer. The oligomer has polysaccharide side chains that carry O-antigen. Endotoxins are generally not inactivated by heat and must be removed during purification.
Endotoxins have a variety of deleterious effects on mammalian systems. These can vary widely even in similar systems. In fact, at least one case is known in which two insensitive T-cell lines were cloned from an endotoxin-sensitive parent line. One factor seems to be the presence of the CD14 receptor protein on the surface of the affected cells. Higher expression of CD14 seems to correlate with greater endotoxin sensitivity. In especially sensitive systems, even picomolar concentrations of endotoxin can lead to anomalous experimental results.
Effects in vitro
Documented effects in vitro include:
- Distortion of cell membranes by interactions with Lipid A, up to and including cell death.
- Lesser membrane distortions still affect gene expression and protein production, with specifics varying by different types of cells.
- Stimulation of leukocyte cultures to produce tissue factors.
- Inducing production of IL-6 in equine macrophages.
- Activating primary human immune cells, especially CD1c+ dendritic cells, to produce inflammatory cytokines. Sensitive cells can be activated by endotoxin concentrations as low as 0.02 ng/ml.
- Enhancing production of prostaglandins, acid phosphatase, fibrinolytic inhibitor, collagenase, nerve growth factor, and adhesion molecule 1, depending on the cell type.
- Inhibiting angiotensin-converting enzyme activity, synthesis of proteoglycan, and synthesis of alpha2 macroglobulin.
Effects in vivo
In live animals, endotoxins produce an inflammatory response in almost all tissues that are exposed to them. The pyrogenic nature of endotoxins produces effects ranging from fever to fatal septic shock.
All of the above effects, both in vivo and in vitro, may be synergistic with other contaminants or (in live animals) endogenous products. This, combined with widely varying cell sensitivity, make the experimental effects of endotoxin contamination difficult to predict.
Purification to Remove Endotoxins
The standard method of purifying recombinant proteins and removing endotoxins is affinity chromatography, using affinity tags on the target proteins, eluting the bound target, and then cleaving the tags in post-processing. Affinity chromatography is the method of choice at List Labs and gives our recombinant products exceptional purity.
Endotoxin contamination is a potentially serious problem in recombinant proteins, with highly variable and difficult to predict experimental effects. Even low levels of contamination may produce anomalous effects, which may vary across different cell lines and test subjects. Only reliable purification can prevent contamination. List Labs provides certified products with known purity and very low levels of endotoxin.
Sigma Aldrich, “Cell Culture FAQs: Bacterial Endotoxin Contamination;”
- Dawson, “The Significance of Endotoxin to Cell Culture and Biotechnology;” LAL Update, March 1998, Vol 16, No. 1, p. 1-4; Associates of Cape Cod Incorporated; https://www.acciusa.com/pdfs/newsletter/updt0398.pdf
- Schwarz, M. Schmittner, A. Duschl, and J. Horejs-Hoeck, “Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c+ Dendritic Cells;” PLoS One. 2014; 9(12): e113840; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257590/