Menu ccc

Products Description

ＣlearColi特色:

• 專為需要 endotoxin-free 的需要而設計的勝任細胞。

• 不在需要昂貴的 endotoxin-free kits,精減實驗步驟。

Lucigen 獨家販售的 ClearColi® cells 直接對 E. coli 表面的 lipopolysaccharide (LPS) 的 7 個基因進行 突變,將其修飾為 Lipid IVA 且減少 lipid A 的 Acyl Chains,使其無法活化 hTLR4/MD-2 pathway 進而 抑制 NF-kB 路徑及促發炎反應的 cytokines 產生。在移除了複雜的 LPS 的 ClearColi® 中, 純化蛋白質毋需再使用昂貴的 Endotoxin-free kits,即可得到幾乎無內毒性的 proteins。因此特別適合應用於人體 免疫和毒性測試、臨床醫用蛋白等的研發及製造 。
 

Figure 1. The LPS of a normal E. coli cell compared to the genetically modified Lipid IVA from ClearColi cells.  In ClearColi, the oligosaccharide chain has been deleted, and two of the six acyl chains have been removed to disable the endotoxin signal.

Protein Expression with ClearColi

Figure 2. Comparison of protein expression in ClearColi BL21(DE3) and Lucigen's E. Cloni® EXPRESS BL21(DE3) competent cells.Cells containing a T7 expression plasmid harboring 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 analyzed by SDS-PAGE on a 4% - 20% polyacrylamide gradient gel.

Endotoxicity Assay Results In most cases, a simple nickel column purification is sufficient for endotoxic shock response negative recombinant proteins suitable or downstream eukaryotic cellular response  assays.  Researchers can now assay target protein effects without concern that immune response triggers are a result of LPS endotoxin contamination.  To demonstrate, ApoA1 protein expressed from ClearColi BL21(DE3) competent cells was Ni-column purified and tested for endotoxicity using both the HEK-Blue cell line from Invivogen (see Fig 3)  and the LAL assay (see Fig. 4).

Figure 3. Comparison of endotoxic response from protein derived from ClearColi BL21(DE3) and traditional BL21(DE3) competent cells

Figure 4. 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.