title: Construction of Escherichia coli Mutant with Decreased Endotoxic Activity by Modifying Lipid A Structure.
abstract: Escherichia coli BL21 (DE3) and its derivatives are widely used for the production of recombinant proteins, but these purified proteins are always contaminated with lipopolysaccharide (LPS). LPS is recognized by the toll-like receptor 4 and myeloid differentiation factor 2 complex of mammalian immune cells and leads to release of pro-inflammatory cytokines. It is a vital step to remove LPS from the proteins before use for therapeutic purpose. In this study, we constructed BL21 (DE3) ∆msbB28 ∆pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity. The plasmids harboring pagL and/or lpxE were then introduced into this mutant to further modify the LPS. The new strain (S004) carrying plasmid pQK004 (pagL and lpxE) produced mono-phosphoryated tetra-acylated lipid A, which induces markedly less production of tumor necrosis factor-α in the RAW264.7 and IL-12 in the THP1, but still retains ability to produce recombinant proteins. This study provides a strategy to decrease endotoxic activity of recombinant proteins purified from E. coli BL21 backgrounds and a feasible approach to modify lipid A structure for alternative purposes such as mono-phosphoryl lipid A (MPL) as vaccine adjuvants.
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China. p19890528@126.com.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China. yanyanli@jiangnan.edu.cn.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China. xxinzhao@163.com.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China. yx1207@foxmail.com.
authors.forename: Xue
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Bioengineering, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China. qing.liu.2@sicau.edu.cn.
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China. qingke.kong@sicau.edu.cn.
authors.forename: Qingke
authors.initials: Q
authors.lastname: Kong
mesh_terms: D000818:Animals; D002460:Cell Line; D004731:Endotoxins / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005240:Feasibility Studies; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D008050:Lipid A / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000633:toxicity; D051379:Mice; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000633:toxicity; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D004731:Endotoxins; D018836:Inflammation Mediators; D008050:Lipid A; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12
keywords: Escherichia coli BL21 (DE3); lipid A; lpxE; pagL; protein expression