title: Single-chain Fv fragment antibodies selected from an intrabody library as effective mono- or bivalent reagents for in vitro protein detection.
abstract: In spite of their many potential applications, recombinant antibody molecules selected by phage display are rarely available commercially, one reason being the absence of robust bacterial expression systems that yield sufficient quantities of reagents for routine applications. We previously described the construction and validation of an intrabody library that allows the selection of single-chain Fv (scFv) fragments solubly expressed in the cytoplasm. Here, we show that it is possible to obtain monomeric scFvs binding specifically to human papillomavirus type 16 E6 and cellular gankyrin oncoproteins in quantities higher than 0.5 g/L of shake-flask culture in E. coli cytoplasm after auto-induction. In addition, stable bivalent scFvs of increased avidity were produced by tagging the scFvs with the dimeric glutathione-S-transferase enzyme (GST). These minibody-like molecules were further engineered by fusion with green fluorescent protein (GFPuv), leading to high yield of functional bivalent fluorescent antibody fragments. Our results demonstrate that scFvs selected from an intrabody library can be engineered into cost-effective bivalent reagents suitable for many biomedical and industrial applications.
authors.affiliation: Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, CNRS/UdS, boulevard Sébastien Brant, Illkirch, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Desplancq
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Rinaldi
authors.forename: Audrey
authors.initials: A
authors.lastname: Stoessel
authors.forename: Annie-Paule
authors.initials: AP
authors.lastname: Sibler
authors.forename: Didier
authors.initials: D
authors.lastname: Busso
authors.forename: Mustapha
authors.initials: M
authors.lastname: Oulad-Abdelghani
authors.forename: Marc H
authors.initials: MH
authors.lastname: Van Regenmortel
authors.forename: Etienne
authors.initials: E
authors.lastname: Weiss
mesh_terms: D006367:HeLa Cells; D052162:Human papillomavirus 16 / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D009856:Oncogene Proteins, Viral / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D046988:Proteasome Endopeptidase Complex / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D020836:Protein Structure, Quaternary; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D012097:Repressor Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D057127:Single-Chain Antibodies / Q000032:analysis / Q000737:chemistry / Q000276:immunology*
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; C403591:PSMD10 protein, human; D019151:Peptide Library; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D057127:Single-Chain Antibodies; D046988:Proteasome Endopeptidase Complex