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Eliminate LPS At Source - ClearColi®

Eliminate LPS At Source - ClearColi®

Cambridge Bioscience offers the first commercially available competent cells with a genetically modified lipopolysaccharide (LPS) that does not trigger an endotoxic response in mammalian cells. Instead of removing LPS contamination from protein or plasmid DNA preparations after production, the ClearColi® cells effectively eliminate LPS at the source, producing functionally clean recombinant proteins and plasmids.

Benefits of Using the ClearColi® BL21(DE3) Cells
• No endotoxic response triggered in mammalian cells
• Functionally clean recombinant proteins & plasmids produced
• Downstream endotoxin removal not required in most cases
• Minimal purification needed to remove lipid IVA
• False positives reduced in cytokine assays
• Protein purification yields maintained - similar to standard strains


ClearColi® BL21 (DE3) Genotype
Modifications to the genotype of the ClearColi® cells consist of seven separate gene deletions ensuring there is no chance of genetic reversion back to wild type and production of normal LPS. These mutations result in the deletion of the oligosaccharide chain from the LPS, making it easier to remove the resulting lipid IVA from the downstream product. The six acyl chains of the LPS are the trigger which is recognised by the Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD-2), causing activation of NF-ƙB and production of proinflammatory cytokines. In ClearColi®, two of the six acyl chains are deleted and the lipid IVA, which contains only four acyl chains, is not recognised by TLR4 and thus does not trigger the endotoxic response. Genotype: F– ompT hsdSB (rB- mB-) gal dcm lon λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) msbA148 ΔgutQ ΔkdsD ΔlpxL ΔlpxM ΔpagP ΔlpxP ΔeptA

Comparison of protein expression in ClearColi BL21(DE3) and Lucigen's E. Cloni® EXPRESS BL21(DE3) competent cells

Comparison of protein expression in ClearColi BL21(DE3) and Lucigen's E. Cloni® EXPRESS BL21(DE3) competent cells

Cells containing a T7 expression plasmid harbouring a gene encoding the human apolipoprotein A1 (ApoA1) were grown in LB Miller medium at 37°C. When cultures reached OD600 of 0.6 to 0.8, expression was induced by the addition of 0.4 mM IPTG and incubation was continued for 3 hours. Equivalent numbers of uninduced (-) and induced (+) cells were lysed by heating in Laemmli buffer and samples were analysed by SDS-PAGE on a 4% - 20% polyacrylamide gradient gel.

Comparison of  endotoxin units as measured by the LAL assay using nickel-column purified recombinant ApoA1 and HSP protein expressed in ClearColi BL21(DE3) (red bars) and E. cloni EXPRESS BL21(DE3) (grey bars) competent cells

Comparison of  endotoxin units as measured by the LAL assay using nickel-column purified recombinant ApoA1 and HSP protein expressed in ClearColi BL21(DE3) (red bars) and E. cloni EXPRESS BL21(DE3) (grey bars) competent cells

Protein expressed from ClearColi demonstrates significant reduction in EU/mg without endotoxin removal steps.

Most Common Methods For Endotoxin Removal & Their Associated Shortcomings

Method Disadvantages
Ultrafiltration • Only useful for small proteins
• Endotoxin monomers may permeate the membrane due to endotoxin/protein interaction
• Inefficient for proteins which can be damaged by physical forces
Activated carbon • Adsorbing activity for both endotoxin and protein
Surfactants • Expensive
• May affect bioactivity of protein
• Difficult to completely remove
• Removal may lead to product loss
Anion-exchange chromatography • High adsorbtion of both endotoxin and acidic protein
• No selectivity to adsorb endotoxin
Histamine- and histidine-immobilised sepharose • Removing capacity dependent on the ionic strength
• Biological activity of histamine
Polymyxin B-immobilised Sepharose • Protein losses due to the ionic interaction between polymyxin B and protein
• Polymixin B is physiologically active

Applications
• Human cell immunogenicity and toxicity assays
• Therapeutic protein production
• Membrane and lipid-binding protein production

Material Available For Download
ClearColi® BL21(DE3) User Manual

ClearColi® Licensing Restrictions
Please note certain licensing restrictions apply to the ClearColi® products. To find out more, please Click Here. If you would like to discuss the licensing restrictions further, please contact us by Clicking Here

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