Expression of recombinant proteins in a lipid A mutant of Escherichia coli BL21 with a strongly reduced capacity to induce dendritic cell activation and maturation
Expression of recombinant proteins in a lipid A mutant of Escherichia coli BL21 with a strongly reduced capacity to induce dendritic cell activation and maturation
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DOI：
10.1016/s0022-1759(02)00506-9
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发表时间：
2003-01-15
影响因子：
2.2
通讯作者：
Gauchat, JF
中科院分区：
文献类型：
--
作者：
Cognet, I;de Coignac, AB;Gauchat, JF
大肠杆菌（E. coli）和沙门氏菌lpxM基因的突变已被证明能产生正常生长的菌株，并能产生一种内毒性大大降低的非肉豆荚酰基脂多糖（nmLPS）。通过同源重组，我们灭活了广泛用于重组蛋白生产的菌株BL21 （DE3）的lpxM基因。这导致衍生物不受其支持重组蛋白生产能力的影响。通过核因子κ B （nf - κ B）的核易位、tnf - α和IL-8的产生或CD86的表达来评估，该新菌株表达非豆芽糖酰基化LPS，诱导单核细胞衍生的树突状细胞（DC）的激活和成熟明显减少。细胞外LPS的主要信号转导受体Toll样受体（TLR） 4与可溶性辅助蛋白MD-2的激活也显著降低。改良的BL21菌株代表了lpxM失活的新应用，用于在树突状细胞或其他LPS敏感细胞/受体复合物上检测蛋白的表达。这可能有助于鉴定激活先天免疫反应的新蛋白，并降低治疗性重组蛋白中低水平内毒素污染的风险。(C) 2002 Elsevier Science B.V.版权所有
Mutations in the Escherichia coli (E. coli) and Salmonella lpxM gene have been shown to result in strains which grow normally and which produce a non-myristoylated lipopolysaccharide (nmLPS) with strongly reduced endotoxicity. Using homologous recombination, we inactivated the lpxM gene in BL21 (DE3), a strain widely used for the production of recombinant proteins. This led to a derivative unaffected in its capacity to support the production of recombinant proteins. This new strain expresses non-myristoylated LPS that induces markedly less activation and maturation of monocyte-derived dendritic cells (DC), as assessed by nuclear translocation of nuclear factor kappa B (NF-kappaB), production of TNF-alpha and IL-8 or expression of CD86. Activation of the main signal transducing receptor for extracellular LPS, Toll like receptor (TLR) 4 in conjunction with the soluble accessory protein MD-2 was also markedly decreased.The modified BL21 strain represents a new application of lpxM inactivation for the expression of proteins to be tested on dendritic cells or other LPS sensitive cells/receptor complexes. It is likely to be useful for the identification of new proteins activating the innate immune response and to reducing the risk linked with low level of endotoxin contamination in therapeutic recombinant proteins. (C) 2002 Elsevier Science B.V. All rights reserved.