title: Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli.
abstract: Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.
authors.affiliation: Institut für Industrielle Genetik, Universität Stuttgart, 70569 Stuttgart, Germany.
authors.forename: J F
authors.initials: JF
authors.lastname: Rippmann
authors.forename: M
authors.initials: M
authors.lastname: Klein
authors.forename: C
authors.initials: C
authors.lastname: Hoischen
authors.forename: B
authors.initials: B
authors.lastname: Brocks
authors.forename: W J
authors.initials: WJ
authors.lastname: Rettig
authors.forename: J
authors.initials: J
authors.lastname: Gumpert
authors.forename: K
authors.initials: K
authors.lastname: Pfizenmaier
authors.forename: R
authors.initials: R
authors.lastname: Mattes
authors.forename: D
authors.initials: D
authors.lastname: Moosmayer
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex; D015703:Antigens, CD / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011513:Proteus mirabilis / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D018124:Receptors, Tumor Necrosis Factor / Q000276:immunology; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
chemical_list: D000936:Antigen-Antibody Complex; D015703:Antigens, CD; D007135:Immunoglobulin Variable Region; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins