title: Bacterial cytoplasm production of an EGFP-labeled single-chain Fv antibody specific for the HER2 human receptor.
abstract: The human epidermal growth factor receptor 2 (HER2) is the main diagnostic marker of breast and ovary cancers. Here, to obtain a rapid and sensitive immunodiagnostic tool a single-chain antibody (scFv800E6) specific for the HER2 was fused to the N-terminus of the enhanced green fluorescent protein (EGFP) by a flexible linker. The soluble production of the novel scFv800E6-EGFP protein in the cytoplasm of Escherichia coli was investigated at different induction temperatures (25, 30 and 37°C); the intrinsic fluorescent properties and the binding activity to HER2 positive tumour cells of the fusion protein were analysed. Western blotting and fluorescence analysis of SDS-PAGE revealed the presence of two scFv800E6-EGFP forms, with different mobility and optical properties, their ratio depending on the induction temperature. The fluorescent form maintained the optical fluorescence properties of EGFP and exhibited a binding activity to the HER2-expressing cells comparable to that of the non-fused scFv800E6. In addition, to provide an insight into the effect of the induction temperature on the molecular structure, the folding of the fusion protein was assessed at atomic level by performing molecular dynamics simulations of the homology-derived model of scFv800E6-EGFP at 300 K and 310 K. The comparison of the data collected at these two temperatures revealed that the higher temperature affects specific structural elements. To improve the production of the soluble and functional scFv800E6-EGFP protein, "in silico" results could be utilised for ad hoc design of the molecular structure.
authors: Institute of Biology and Agrarian Biotechnology (IBBA), National Research Council, via Bassini 15, 20133 Milan, Italy.
authors: Alessio
authors: A
authors: Lombardi
authors: Giulio
authors: G
authors: Gianese
authors: Caterina
authors: C
authors: Arcangeli
authors: Patrizia
authors: P
authors: Galeffi
authors: Maria
authors: M
authors: Sperandei
mesh_terms: D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D056004:Molecular Dynamics Simulation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry*; D013696:Temperature
chemical_list: D049452:Green Fluorescent Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C485184:enhanced green fluorescent protein; C508053:ERBB2 protein, human