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Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

Schwarz H, Schmittner M, Duschl A, Horejs-Hoeck J - PLoS ONE (2014)

Bottom Line: When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher.When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data.We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.

ABSTRACT
Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

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Related in: MedlinePlus

Activation of NF-κB by endotoxin impurities in commercial preparations of a recombinant protein.NF-κB activation in HEK293 cells transfected with an NF-κB luciferase reporter plasmid and plasmids encoding LPS-receptor components (TLR4, CD14, MD-2) is shown. Cells were exposed to recombinant protein 1 from supplier 1 or 2, LPS, or solvent, in the amounts stated. 20 h after induction, luciferase activity was measured. Results show mean and standard deviations of four independent experiments.
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pone-0113840-g002: Activation of NF-κB by endotoxin impurities in commercial preparations of a recombinant protein.NF-κB activation in HEK293 cells transfected with an NF-κB luciferase reporter plasmid and plasmids encoding LPS-receptor components (TLR4, CD14, MD-2) is shown. Cells were exposed to recombinant protein 1 from supplier 1 or 2, LPS, or solvent, in the amounts stated. 20 h after induction, luciferase activity was measured. Results show mean and standard deviations of four independent experiments.

Mentions: To assesses whether these small amounts of LPS are capable of activating NF-κB-signalling, we generated a highly LPS-responsive cell system modified from Peters and colleagues [11] by co-transfecting HEK293 cells with expression plasmids encoding the LPS receptor subunits TLR4, CD14 and MD-2 along with an NF-κB luciferase reporter plasmid. These cells were exposed to different concentrations of recombinant protein 1 from suppliers 1 and 2, as well as to different amounts of LPS. As shown in Figure 2, the recombinant protein from supplier 1 induced an increase in NF-κB activity, whereas the protein from supplier 2 did not activate NF-κB (even at 400 ng/ml, twice as high as the maximum protein concentration tested from supplier 1). Interestingly, the protein from supplier 1 induced NF-κB activation in the same range as 0.02 ng/ml LPS. Of note, this LPS concentration is approximately equivalent to the amount of contamination in 100 ng of recombinant protein, as measured in the LAL test above. This experiment clearly shows that even the small amounts of endotoxin contamination found in commercially available recombinant proteins are sufficient to activate NF-κB in a highly sensitive cell system.


Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

Schwarz H, Schmittner M, Duschl A, Horejs-Hoeck J - PLoS ONE (2014)

Activation of NF-κB by endotoxin impurities in commercial preparations of a recombinant protein.NF-κB activation in HEK293 cells transfected with an NF-κB luciferase reporter plasmid and plasmids encoding LPS-receptor components (TLR4, CD14, MD-2) is shown. Cells were exposed to recombinant protein 1 from supplier 1 or 2, LPS, or solvent, in the amounts stated. 20 h after induction, luciferase activity was measured. Results show mean and standard deviations of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257590&req=5

pone-0113840-g002: Activation of NF-κB by endotoxin impurities in commercial preparations of a recombinant protein.NF-κB activation in HEK293 cells transfected with an NF-κB luciferase reporter plasmid and plasmids encoding LPS-receptor components (TLR4, CD14, MD-2) is shown. Cells were exposed to recombinant protein 1 from supplier 1 or 2, LPS, or solvent, in the amounts stated. 20 h after induction, luciferase activity was measured. Results show mean and standard deviations of four independent experiments.
Mentions: To assesses whether these small amounts of LPS are capable of activating NF-κB-signalling, we generated a highly LPS-responsive cell system modified from Peters and colleagues [11] by co-transfecting HEK293 cells with expression plasmids encoding the LPS receptor subunits TLR4, CD14 and MD-2 along with an NF-κB luciferase reporter plasmid. These cells were exposed to different concentrations of recombinant protein 1 from suppliers 1 and 2, as well as to different amounts of LPS. As shown in Figure 2, the recombinant protein from supplier 1 induced an increase in NF-κB activity, whereas the protein from supplier 2 did not activate NF-κB (even at 400 ng/ml, twice as high as the maximum protein concentration tested from supplier 1). Interestingly, the protein from supplier 1 induced NF-κB activation in the same range as 0.02 ng/ml LPS. Of note, this LPS concentration is approximately equivalent to the amount of contamination in 100 ng of recombinant protein, as measured in the LAL test above. This experiment clearly shows that even the small amounts of endotoxin contamination found in commercially available recombinant proteins are sufficient to activate NF-κB in a highly sensitive cell system.

Bottom Line: When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher.When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data.We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.

ABSTRACT
Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

Show MeSH
Related in: MedlinePlus