title: Formation of disulfide bridges by a single-chain Fv antibody in the reducing ectopic environment of the plant cytosol.
abstract: Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event and is mostly linked to inactivation of protein activity. In this report the in vivo redox state of a single-chain Fv (scFv) antibody fragment in the plant cytosol was investigated. The scFv antibody fragment consists of the variable light and heavy chain domains from a mouse IgG antibody, which are connected by a flexible linker peptide. In each domain one disulfide bridge is present. The functionality of antibodies, which are normally secreted via the oxidizing environment of the endoplasmic reticulum, depends on the formation of intramolecular disulfide bridges. We demonstrate that a scFv can form intramolecular disulfide bridges and is functionally expressed in the cytosol of stably transformed plants. In addition, the formation of intermolecular disulfide bridges through a cysteine present in the linker peptide was observed. In contrast, transient expression in tobacco protoplasts resulted in a cytosolic scFv lacking disulfide bridges, which had a substantially reduced affinity for the antigen. This indicates that functionality rather than stability is determined by the presence of disulfide bridges in the in planta-expressed scFv antibody. The controversial observation of disulfide bond formation in the cytosol is discussed.
authors.affiliation: Department of Plant Sciences, Wageningen University, P. O. Box 8123, 6700 ES Wageningen, The Netherlands. sander.schouten@fyto.dpw.wau.nl
authors.forename: Alexander
authors.initials: A
authors.lastname: Schouten
authors.forename: Jan
authors.initials: J
authors.lastname: Roosien
authors.forename: Jaap
authors.initials: J
authors.lastname: Bakker
authors.forename: Arjen
authors.initials: A
authors.lastname: Schots
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010944:Plants / Q000276:immunology; D017434:Protein Structure, Tertiary; D014026:Nicotiana / Q000378:metabolism; D014162:Transfection
chemical_list: D004220:Disulfides; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D003545:Cysteine; D010100:Oxygen