title: Functional expression of a single-chain antibody specific for the HER2 human oncogene in a bacterial reducing environment.
abstract: Recombinant antibody fragments represent useful tools for cancer diagnosis and therapy. Aberrant expression of the HER2 receptor is implicated in metastatic breast and ovary cancers, two malignancies with a high prevalence in young women. In this study, we focussed on a single-chain fragment of variable antibody regions specific for HER2 (scFv800E6) that can be expressed in a functional form in the cytoplasm of Escherichia coli. ScFv800E6 was extracted from bacterial cultures following induction at different temperatures and purified. The yield of both soluble and insoluble forms was measured. We found that scFv800E6 was functional when expressed in the soluble fraction in the bacteria cytosol. In addition, scFv800E6 extracted from inclusion bodies was easily refolded and largely recovered its functionality. Thus, scFv800E6 is intrinsically capable of efficient and functional folding in a reducing environment and represents one of the few described antibody fragments with a framework well adapted for cytoplasmic expression.
authors.affiliation: ENEA, BIOTEC-GEN Unit, CR Casaccia, Via Anguillarese 301, 00060 Rome, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Lombardi
authors.forename: Maria
authors.initials: M
authors.lastname: Sperandei
authors.forename: Cristina
authors.initials: C
authors.lastname: Cantale
authors.forename: Patrizio
authors.initials: P
authors.lastname: Giacomini
authors.forename: Patrizia
authors.initials: P
authors.lastname: Galeffi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D010084:Oxidation-Reduction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins