title: A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants.
abstract: Despite the well-known crucial role of intradomain disulfide bridges for immunoglobulin folding and stability, the single-chain variable fragment of the anti-viral antibody F8 is functionally expressed when targeted to the reducing environment of the plant cytoplasm. We show here that this antibody fragment is also functionally expressed in the cytoplasm of Escherichia coli. A gel shift assay revealed that the single-chain variable fragment (scFv) accumulating in the plant and bacterial cytoplasm bears free sulfhydryl groups. Guanidinium chloride denaturation/renaturation studies indicated that refolding occurs even in a reducing environment, producing a functional molecule with the same spectral properties of the native scFv(F8). Taken together, these results suggest that folding and functionality of this antibody fragment are not prevented in a reducing environment. This antibody fragment could therefore represent a suitable framework for engineering recombinant antibodies to be targeted to the cytoplasm.
authors.affiliation: ENEA, Dipartimento Innovazione, Divisione Biotecnologie e Agricoltura, Rome, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Tavladoraki
authors.forename: A
authors.initials: A
authors.lastname: Girotti
authors.forename: M
authors.initials: M
authors.lastname: Donini
authors.forename: F J
authors.initials: FJ
authors.lastname: Arias
authors.forename: C
authors.initials: C
authors.lastname: Mancini
authors.forename: V
authors.initials: V
authors.lastname: Morea
authors.forename: R
authors.initials: R
authors.lastname: Chiaraluce
authors.forename: V
authors.initials: V
authors.lastname: Consalvi
authors.forename: E
authors.initials: E
authors.lastname: Benvenuto
mesh_terms: D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000276:immunology*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D030821:Plants, Genetically Modified / Q000276:immunology*; D011489:Protein Denaturation; D021382:Protein Sorting Signals / Q000737:chemistry
chemical_list: D004220:Disulfides; D007135:Immunoglobulin Variable Region; D021382:Protein Sorting Signals; D003545:Cysteine