Direct Transfection without Endotoxin Cleanup
With the original source of endotoxin eliminated, it is now possible to transfect plasmid DNA prepared with standard Qiagen Maxiprep kits directly into human or other mammalian cell lines without concern for cell viability, altered cellular responses or poor protein expression.
To illustrate this, a plasmid (pME-HA) containing a fluorescent protein was transformed into both ClearColi K-12 and E. coli DH10B. The plasmid was then isolated from ClearColi using the standard Qiagen Plasmid Maxi Kit method, or from DH10B using Qiagen’s EndoFree Plasmid Maxi Kit. The resulting plasmids were transfected into HEK293T cells and evaluated for fluorescent protein expression (Figure 2). Using a standard (nonendo-free) plasmid prep method in combination with ClearColi, no difference in cell viability or protein expression levels are seen in comparison to endo-free preps from DH10B cells.
These results are especially significant in light of recent efforts to scale up mammalian protein expression via transient transfection. The large-scale transfection of mammalian cells allows moderate (milligram to gram) amounts of recombinant proteins to be obtained for fundamental or clinical research. The method is based on a DNA delivery step performed at high cell density by direct addition of DNA and polyethylenimine to the culture.
One of the major bottlenecks in this procedure is the production of large amounts of endotoxin-free DNA. Isolation of plasmid DNA from ClearColi can alleviate this limitation, making it more economical and producing higher yields. It should be noted that while lipid IVA is known not to induce an endotoxic response in human LPS-responsive cells, species-specific differences in the structures of TLR4 and MD-2 may allow varying degrees of sensitivity to lipid IVA in other mammalian cells.
Rapid protein expression experiments are critical to speeding the overall process of drug discovery, as is the need to produce endotoxin-free plasmids and proteins. The ability to achieve this without the need for endotoxin-removal procedures will enable researchers to reduce false positives in toxicity assays while increasing confidence in their results. Simultaneously, plasmid purification costs can be reduced by 90% or more by eliminating the need for endo-free maxiprep kits.
In addition to the plasmid production strain described here, a ClearColi BL21(DE3) strain is also available for E. coli-based protein expression, which is ideal for producing low endotoxin proteins for downstream toxicity testing. Both of these strains allow scientists to remove time-consuming and expensive cleanup steps that may affect yield, solubility, and functionality of the end product.