title: Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli.
abstract: Production of intracellular antibodies in Escherichia coli has been thought unlikely owing to an inability to form stable disulfide bonds in the cytoplasm, a necessary step in the folding of most immunoglobulin (Ig) domains. This work investigates whether E. coli strains carrying mutations in the major intracellular disulfide bond-reduction systems (i.e. the thioredoxin and the glutathione/glutaredoxin pathways) allow the oxidation and folding of single chain variable fragment (scFv) antibodies in the cytoplasm. The effect of the co-expression of disulfide bond chaperones in these cells was also examined. An scFv that recognizes the alternative sigma factor sigma(54) was used as a model to investigate disulfide bond formation and the folding of Ig domains in E. coli. The results demonstrate that functional intrabodies, with oxidized disulfide bonds in their Ig domains, are produced efficiently in E. coli cells carrying mutations in the glutathione oxidoreductase (gor) and the thioredoxin reductase (trxB) genes and co-expressing a signal-sequence-less derivative of the disulfide-bond isomerase DsbC ((Delta)ssDsbC). We obtained evidence indicating that (Delta)ssDsbC acts as a chaperone promoting the correct folding and oxidation of scFvs.
authors.affiliation: Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain.
authors.forename: Paola
authors.initials: P
authors.lastname: Jurado
authors.forename: Daniel
authors.initials: D
authors.lastname: Ritz
authors.forename: Jon
authors.initials: J
authors.lastname: Beckwith
authors.forename: Víctor
authors.initials: V
authors.lastname: de Lorenzo
authors.forename: Luis A
authors.initials: LA
authors.lastname: Fernández
mesh_terms: D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018832:Molecular Chaperones; D011994:Recombinant Proteins