LPS-Free BL21(DE3) BioVector NTCC质粒载体菌种细胞基因保藏中心

LPS-Free BL21(DE3) BioVector NTCC质粒载体菌种细胞基因保藏中心 LPS-Free BL21(DE3) cells are the first commercially available competent cells with a modified LPS that does not trigger the endotoxic response in human cells. LPS-Free cells lack outer membrane agonists for hTLR4/MD-2 activation; therefore, activation of hTLR4/MD-2 signaling by LPS-Free is several orders of magnitude lower as compared with E. coli wild-type cells. Heterologous proteins prepared from LPS-Free are virtually free of endotoxic activity. After minimal purification from LPS-Free cells, proteins or plasmids (which may contain Lipid IVA) can be used in most applications without eliciting an endotoxic response in human cells In LPS-Free 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. LPS-Free competent cells have a genetically modified lipopolysaccharide (LPS) that disables the trigger for endotoxic response in mammalian cells. This was accomplished by incorporating seven genetic deletions that modify LPS to Lipid IVA, while one additional compensating mutation (msbA148) enables the cells to maintain viability in the presence of the LPS precursor lipid IVA. LPS-Free BL21(DE3) cells are BL21(DE3) derived cells with several key mutations which result in significantly reduced endotoxicity. The specific set of mutations made to LPS-Free cells is as follows: msbA148 gutQ kdsD lpxL lpxM pagP lpxP eptA Growth/Colony Characteristics of LPS-Free BL21(DE3) Cells: LPS-Free BL21(DE3) cells grow at approximately 50% of the rate of normal BL21(DE3) cells. Users should expect to see very small colonies for the first 24 hours after plating transformants. BioVector recommends incubating plates for 32-40 hours before picking colonies. for future experiments. Longer growth times are necessary to reach desired cell densities prior to inducing protein expression. In addition, users may observe some variation in colony size when plating LPS-Free cells. A small portion (<2%) of colonies may be larger than the general population. These larger colonies have similar protein expression levels and endotoxin levels as the average size colonies. LPS-Free cells have a specially engineered membrane composition and require a modified medium formulation compared to most E. coli strains. Consider the following: IMPORTANT Information About Medium Formulation • LPS-Free cells are osmosensitive and require 1% NaCl in their growth medium. We strongly recommend you use high salt LB-Miller Medium to achieve optimal growth. LB-Miller differs from LB-Lennox. See p.11 for the LB-Miller recipe. • We do not recommend growing LPS-Free cells in LB-Lennox or Super broth medium. These media have resulted in slow and suboptimal cell growth. • Do not include Mg2+ and Ca2+ in your medium. They have been shown to inhibit growth of LPS-Free cells. • LPS-Free cells tend to aggregate when grown in liquid culture. We suggest that you vortex the cell solution before measuring OD600. Genotype Information LPS-Free BL21(DE3) F– ompT hsdSB (rB- mB-) gal dcm lon λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) msbA148 gutQ kdsD lpxL lpxM pagP lpxP eptA Eliminate endotoxin at the source • 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 BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心www.biovector.net [Supplier来源] http://www.biovector.net