A bacterial single-chain Fv antibody fragment that inhibits binding of its parental anti- E-Selectin monoclonal antibody to activated human endothelial cells

• Published in: Journal of biotechnology Vol. 72; no. 1; pp. 1 - 12
• Main Authors: Casalvilla, Racmar, Dueñas, Marta, Ayala, Marta, Cruz, Silian, Cruz, Liset, Buurman, Wim A., Gavilondo, Jorge V.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 11.06.1999; Amsterdam Elsevier; New York, NY
• Subjects: Affinity chromatography; Amino Acid Sequence; Animals; Antibodies, Monoclonal - immunology; Antibody fragments; Bacteria; Base Sequence; Binding Sites, Antibody; Bioassay; Biological and medical sciences; Biotechnology; Cells, Cultured; Cloning, Molecular; DNA, Recombinant; E-Selectin; E-Selectin - immunology; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - immunology; Enzyme-Linked Immunosorbent Assay; Enzymes; Escherichia coli - genetics; Fractionation; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Humans; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Industrial applications and implications. Economical aspects; Mice; Molecular Sequence Data; Monoclonal antibodies; Polymerase Chain Reaction; Production of active biomolecules; Recombinant Proteins - genetics; Recombinant Proteins - immunology; ScFv; Solubility; Transmission electron microscopy; Tumor Necrosis Factor-alpha - pharmacology; Antibody fragments; E-Selectin; ScFv; E Selectin; Escherichia coli; Tumor progression; Bacteria; Fv-Fragment; Recombinant protein; Gene expression; Carcinogenesis; Biological activity; Enterobacteriaceae; Single chain antibody
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/S0168-1656(99)00005-X

Using the polymerase chain reaction, we cloned, modified, and linked antibody variable (V) region coding genes from a mouse hybridoma, and produced a bacterial single-chain Fv (scFv) antibody fragment specific for E-Selectin. A vector of pBR322 origin, bearing the tryptophan promoter and the ompA bacterial signal peptide, was used to direct scFv expression to periplasm. The vector included a six-histidine coding sequence 5′ to the scFv for the purification of the expressed protein using immobilized metal affinity chromatography (IMAC). We found that the V H-Linker-V L 32–33 kDa scFv remained insoluble after cellular fractionation, and transmission electron microscopy showed the new protein to be present in the periplasm as inclusion bodies. The scFv was solubilized using urea, purified using IMAC, and renatured to its active form. In a competitive enzyme-linked immunosorbent assay with activated human vein endothelial cells in the solid phase, the scFv competed for binding with the original monoclonal antibody.