title: Characterization of three different single chain antibodies recognizing non-reducing terminal mannose residues expressed in Escherichia coli by an inducible T7 expression system.
abstract: We previously isolated phage antibodies from a phage library displaying human single chain antibodies (scFvs) by screening with a mannotriose (Man3)-bearing lipid. Of four independent scFv genes originally characterized, 5A3 gene products were purified as fusion proteins such as a scFv-human IgG1 Fc form, but stable clones secreting 1A4 and 1G4 scFv-Fc proteins had never been established. Thus, bacterial expression systems were used to purify 1A4 and 1G4 scFv gene products as soluble forms. Purification of 1A4 and 1G4 scFv proteins from inclusion bodies was also carried out together with purification of 5A3 scFv protein in order to compare their Man3-binding abilities. The present studies demonstrated that 1A4 and 1G4 scFv proteins have a higher affinity for Man3 than 5A3 scFv protein, which may determine whether scFv-Fc proteins expressed in mammalian cells are retained in the ER or secreted. Furthermore, the inhibitory effects of anti-Man3 1G4 scFv and anti-Tn antigen scFv proteins on MCF-7 cell growth were evaluated. Despite the fact that no obvious difference was detected in cell growth, microscopic observations revealed inhibition of foci formation in cells grown in the presence of the anti-carbohydrate scFv proteins. This finding provides a basis for the development of cancer therapeutics.
authors.affiliation: Department of Applied Biochemistry, Tokai University School of Engineering, Kanagawa 259-1292, Japan.
authors.forename: Ayano
authors.initials: A
authors.lastname: Matsumoto-Takasaki
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Yuasa
authors.forename: Daiichi
authors.initials: D
authors.lastname: Katagiri
authors.forename: Tsubasa
authors.initials: T
authors.lastname: Koyama
authors.forename: Keiko
authors.initials: K
authors.lastname: Sakai
authors.forename: Normaiza
authors.initials: N
authors.lastname: Zamri
authors.forename: Sheryl
authors.initials: S
authors.lastname: Phung
authors.forename: Shiuan
authors.initials: S
authors.lastname: Chen
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakada
authors.forename: Munehiro
authors.initials: M
authors.lastname: Nakata
authors.forename: Yoko
authors.initials: Y
authors.lastname: Fujita-Yamaguchi
mesh_terms: D000970:Antineoplastic Agents / Q000032:analysis / Q000494:pharmacology; D017123:Bacteriophage T7 / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008358:Mannose / Q000737:chemistry / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000032:analysis / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D057127:Single-Chain Antibodies; D008358:Mannose