Isolation and Characterization of Phage-Displayed Single Chain Antibodies Recognizing Nonreducing Terminal Mannose Residues. 2. Expression, Purification, and Characterization of Recombinant Single Chain Antibodies

• Published in: Biochemistry (Easton) 2007-01, Vol.46 (1), p.263-270
• Main Authors: Zhang, Wei, Matsumoto-Takasaki, Ayano, Kusada, Yu, Sakaue, Hiroyuki, Sakai, Keiko, Nakata, Munehiro, Fujita-Yamaguchi, Yoko
• Format: Article
• Language: English
• Subjects: Antibody Specificity; Cells, Cultured; Dimerization; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Immunoglobulin Variable Region - isolation & purification; Mannose - immunology; Molecular Weight; Peptide Library; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; Surface Plasmon Resonance
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0618767

Since phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties, we employed phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as carbohydrate antigens. An accompanying paper in this issue describes how phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues were isolated and characterized. In this study, four independent scFv genes, isolated by a mannotriose (Man3)-bearing lipid as an antigen as previously described, were used to construct expression vectors to produce soluble scFv proteins in quantity. Both bacterial and mammalian expression systems were used to produce glutathione S-transferase-scFv fusion proteins and scFv-human IgG1 Fc conjugates, respectively. The expressed scFv fusion proteins were purified to apparent homogeneity with yields of approximately 1 and 48 mg, from 1 L of bacterial culture and myeloma cell media, respectively. Surface plasmon resonance and ELISA analyses confirmed that purified scFv proteins showed Man3 specificity. The humanized antibody in scFv-Fc form, derived from clone 5A3, was a disulfide-liked dimer with a molecular mass of 108 kDa. According to a bivalent model, the kinetics parameters of its binding to Man3 were determined to be k a = 4.03 × 104 M-1 s-1, k d = 5.77 × 10-4 s-1, K A = 6.98 × 107 M-1, and K D = 1.43 × 10-8 M. This study thus established the foundation for isolation of carbohydrate-specific scFv genes and eventual production of humanized scFv-Fc type antibodies.