ClearColi® BL21(DE3) Electrocompetent Cells/ClearColi® BL21(DE3) 电感受态细胞
货号：60810-1,60810-2
规格：12 rxns (DUOs),24 rxns (DUOs)
价格：4618,8406
产品类型：感受态细胞
品牌：Lucigen
| ClearColi® BL21(DE3) cells are the first commercially available competent cells with a modified LPS
(Lipid IVA) that does not trigger the endotoxic response in human cells. ClearColi cells lack
outer membrane agonists for hTLR4/MD-2 activation; therefore, activation of hTLR4/MD-2 signaling by
ClearColi® is several orders of magnitude lower as compared with E. coli wild-type cells. Heterologous
proteins prepared from ClearColi® are virtually free of endotoxic activity. After minimal purification from
ClearColi cells, proteins or plasmids (which may contain Lipid IVA) can be used in most applications
without eliciting an endotoxic response in human cells. In ClearColi cells, two of the secondary acyl chains of the normally hexa-acylated LPS have been
deleted, eliminating a key determinant of endotoxicity in eukaryotic cells. The six acyl chains of the LPS
are the trigger which is recognized 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.
The deletion of the two secondary acyl chains results in lipid IVA, which does not induce formation of the
activated heterotetrameric TLR4/MD-2 complex and thus does not trigger the endotoxic response. In addition, the oligosaccharide chain is deleted, making it easier to remove the resulting lipid IVA from any
downstream product.ClearColi®BL21（DE3）细胞是第一种商业化的感受态细胞，具有修饰的LPS（脂质IVA），不会触发人细胞的内毒素反应。 ClearColi细胞缺乏用于hTLR4 / MD-2激活的外膜激动剂； 因此，与大肠杆菌野生型细胞相比，ClearColi®对hTLR4 / MD-2信号的激活作用要低几个数量级。 由ClearColi®制备的异源蛋白几乎没有内毒素活性。从ClearColi细胞中最少次纯化，蛋白质或质粒（可能包含脂质IVA）可用于大多数应用，而不会引起人细胞内毒素反应。在ClearColi细胞中，通常被六酰基化的LPS的两条次级酰基链已被删除，从而消除了真核细胞内毒性的关键决定因素。 LPS的六个酰基链是触发因子，与髓样分化因子2（MD-2）结合时，被Toll样受体4（TLR4）识别，从而引起NF-κB的活化和促炎细胞因子的产生。 两个二级酰基链的缺失导致脂质IVA，它不会诱导活化的异四聚体TLR4 / MD-2复合物的形成，因此不会触发内毒素反应。 另外，寡糖链被删除，使得更容易从任何下游产物中除去所得的脂质IVA。 |
| 优点： |
| ▪ Genetically modified LPS does not trigger endotoxic response in human cells；▪ Ideal for mammalian cell immunogenicity testing, toxicity assays, and therapeutic protein drug discovery；▪ Protein expression similar to BL21(DE3) cells without requiring downstream endotoxin removal. - 转基因的LPS不会在人体细胞中触发内毒素反应- 哺乳动物细胞免疫原性测试，毒性测定和治疗性蛋白药物发现的理想选择- 蛋白质表达类似于BL21（DE3）细胞，无需下游内毒素清除 |
| 数据： |
| Figure 1. The LPS of a normalE. colicell compared to the genetically modified Lipid IVAfrom 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.图 1.正常大肠杆菌细胞的LPS与ClearColi细胞的基因修饰的脂质IVA相比。 在ClearColi中，寡糖链已被删除，六个酰基链中的两个已被删除以禁用内毒素信号。Figure. 2. Comparison of relative NF-κB induction in HEK-Blue Cells using purified LPS from a K-12E. colistrain or from pure, synthetically manufactured Lipid IVA. 图 2.使用来自K-12大肠杆菌菌株或纯合成生产的脂质IVA的纯化LPS比较HEK-Blue细胞中相对NF-κB诱导。 Figure 3. 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 OD600of 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.图 3.在ClearColi BL21（DE3）和Lucigen's E.Cloni®EXPRESS BL21（DE3）感受态细胞中蛋白质表达的比较。含有T7表达质粒的细胞在LB Miller培养基中生长，该质粒带有编码人载脂蛋白A1（ApoA1）的基因。 在37°C下。 当培养物的OD600达到0.6至0.8时，通过加入0.4mM IPTG诱导表达，并继续孵育3小时。 通过在Laemmli缓冲液中加热裂解等效数量的未诱导（-）和诱导（+）细胞，并在4％-20％聚丙烯酰胺梯度凝胶上通过SDS-PAGE分析样品。 Figure 4. Comparison of fluorescent protein expression in ClearColi BL21(DE3) andE. cloniEXPRESS BL21(DE3) cells.A pET expression vector harboring a gene encoding a yellow fluorescent protein (LucY) under the control of a T7 promoter was transformed into both ClearColi BL21(DE3) and E. cloni EXPRESS BL21(DE3). Colonies were inoculated into LB-Miller medium and grown at 37°C for induction. Samples of induced and uninduced cells containing equivalent numbers of cells were centrifuged, and cell pellets were photographed under long-wavelength UV illumination.图 4.在ClearColi BL21（DE3）和克隆的大肠杆菌EXPRESS BL21（DE3）细胞中荧光蛋白表达的比较。将含有编码黄色荧光蛋白（LucY）的基因的pET表达载体在T7启动子的控制下转化为 ClearColi BL21（DE3）和大肠杆菌克隆EXPRESS BL21（DE3）。 将菌落接种到LB-Miller培养基中，并在37℃下生长用于诱导。 离心包含相等数量细胞的诱导和未诱导细胞样品，并在长波紫外线照射下拍摄细胞沉淀。 Figure 5. Comparison of endotoxic response from protein derived from ClearColi BL21(DE3) and traditional BL21(DE3) competent cells.图 5.来自ClearColi BL21（DE3）和传统BL21（DE3）感受态细胞的蛋白质的内毒素反应比较。 Figure 6. 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.图 6.用LAL测定法测定的内毒素单位的比较，该测定使用在ClearColi BL21（DE3）（红色柱）和大肠杆菌克隆BL21（DE3）（灰色柱）感受态细胞中表达的镍柱纯化的重组ApoA1和HSP蛋白。 从ClearColi表达的蛋白质无需去除内毒素即可证明EU / mg显着降低。 Figure 7. Comparison of growth rates for ClearColi BL21(DE3) vs.E. cloniEXPRESS BL21(DE3) cells.Cells were inoculated to an initial OD600of ~0.003 in 200 ml of LB Miller medium and grown at 37° C with shaking at 210 rpm. The OD600of the cultures was recorded hourly.图 7.ClearColi BL21（DE3）与克隆大肠杆菌EXPRESS BL21（DE3）细胞的生长速率比较。 将细胞接种到200 ml LB Miller培养基中的初始OD600约为0.003，并在210 rpm摇动下于37°C生长。 每小时记录培养物的OD600。 |
| 相关产品： |
| ▪CJ236 Electrocompetent Cells, 12 rxns (DUOs) (#60701)▪E. cloni® 5-alpha Chemically Competent Cells, 12 rxns (DUOs) (#60602)▪MC1061 F- Electrocompetent Cells, 12 rxns (DUOs) (#60514) |
技术参数 产品优点 - Genetically modified LPS does not trigger endotoxic response in human cells- Ideal for mammalian cell immunogenicity testing, toxicity assays, and therapeutic protein drug discovery- Useful for membrane and lipid binding protein production- Protein expression similar to BL21(DE3) cells without requiring downstream endotoxin removal- Reduce false positives in cytokine assays, improve confidence in your results- 转基因的LPS不会在人体细胞中触发内毒素反应- 哺乳动物细胞免疫原性测试，毒性测定和治疗性蛋白药物发现的理想选择- 用于膜和脂质结合蛋白的生产- 蛋白质表达类似于BL21（DE3）细胞，无需下游内毒素清除- 减少细胞因子测定中的假阳性，提高结果可靠性
产品应用 - Eliminate endotoxin at the source- 从源头消除内毒素
组成成分 Each ClearColi® BL21(DE3) Electocompetent Cell Kit contains: ClearColi BL21(DE3) Electocompetent Cells in DUO packaging (2 transformations per tube), Expression Recovery Medium (lactose minus), pUC19 Positive Control Plasmid, and complete protocols.
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