Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c+ Dendritic Cells

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.

Hepatitis B Virus Surface Antigen Activates Myeloid Dendritic Cells via a Soluble CD14-Dependent Mechanism

“…ϩ DC (12). Unexpectedly, patient-derived HBsAg used at 1 g/ml contained low endotoxin impurities (Endolisa result, 1.4 EU/ml), yet at 100 ng/ml, which was used in most experiments, pHBsAg endotoxin levels were Ͻ0.1 EU/ml, which is below the reported detection limit for BDCA1…”

“…Since many commercially obtained recombinant proteins contain endotoxin impurities (12), we carefully selected preparations with the highest purity. Furthermore, all proteins were tested at used concentrations for endotoxin impurities by Endolisa (Hyglos GmbH, Bernried, Germany), a specific and sensitive method to detect endotoxins (12), according to the manufacturer's instructions. Recombinant HBsAg and beta-NGF had endotoxin levels of Ͻ0.1 endotoxin units (EU)/ml, which is below the detection limit for BDCA1…”

“…ϩ DC (12). Heat treatment of 3ϫ concentrated preparations of pHBsAg and lipopolysaccharide (LPS; Ultrapure, from Salmonella enterica serovar Minnesota mutant R595; Invivogen) was performed by boiling for 30 min at 100°C in a heat block (13).…”

Prerequisites for Functional Interleukin 31 Signaling and Its Feedback Regulation by Suppressor of Cytokine Signaling 3 (SOCS3)

“…The day after the transfection, cells were induced with IL-31 (30 ng/ml if not stated otherwise). Luciferase activity was assessed after 1 day of stimulation as described previously (18). As an internal control for transfection efficiency, in some experiments a Renilla-luciferase expression plasmid was cotransfected, and cell lysates were analyzed using the dual-luciferase assay reporter system (Promega, Mannheim, Germany).…”

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen

“…Furthermore, the purification procedure resulted in low levels of contamination with endotoxin compatible with human use (55,56). The average recovery yield of the protein (approximately 8 mg per liter of induced culture) was 2-to 14-fold higher than those previously reported (51,52).…”