title: 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.
abstract: 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.
authors.affiliation: Centre d'Immunologie Pierre-Fabre, 5 avenue Napoléon III, Saint-Julien en, Genevois, 74164, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Cognet
authors.forename: Amélie Benoit
authors.initials: AB
authors.lastname: de Coignac
authors.forename: Giovanni
authors.initials: G
authors.lastname: Magistrelli
authors.forename: Pascale
authors.initials: P
authors.lastname: Jeannin
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Aubry
authors.forename: Karine
authors.initials: K
authors.lastname: Maisnier-Patin
authors.forename: Gersende
authors.initials: G
authors.lastname: Caron
authors.forename: Sylvie
authors.initials: S
authors.lastname: Chevalier
authors.forename: Frédéric
authors.initials: F
authors.lastname: Humbert
authors.forename: Thien
authors.initials: T
authors.lastname: Nguyen
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.forename: Dominique
authors.initials: D
authors.lastname: Velin
authors.forename: Yves
authors.initials: Y
authors.lastname: Delneste
authors.forename: Martine
authors.initials: M
authors.lastname: Malissard
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Gauchat
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D051940:B7-2 Antigen; D001483:Base Sequence; D002454:Cell Differentiation; D004269:DNA, Bacterial / Q000235:genetics; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D005798:Genes, Bacterial; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000096:biosynthesis; D008050:Lipid A / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D015703:Antigens, CD; D000954:Antigens, Surface; D051940:B7-2 Antigen; C497964:CD86 protein, human; D004269:DNA, Bacterial; D029721:Drosophila Proteins; D016209:Interleukin-8; C495377:LY96 protein, human; D008050:Lipid A; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha