An Engineered Bivalent Single-Chain Antibody Fragment That Increases Antigen Binding Activity

• Published in: Journal of biochemistry (Tokyo) Vol. 121; no. 5; pp. 831 - 834
• Main Authors: Luo, D., Geng, M., Noujaim, A.A., Madiyalakan, R.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.05.1997
• Subjects: Amino Acid Sequence; Animals; Antibodies, Bispecific - physiology; antibody engineering; Base Sequence; bivalent scFv; Cysteine - metabolism; Dimerization; Epitopes - physiology; Humans; Immunoglobulin Fragments - biosynthesis; Immunoglobulin Fragments - immunology; Molecular Sequence Data; Pichia pastoris; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - immunology; single-chain Fv
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a021661

Bivalent single chain Fv (scFv) was constructed by fusing a polypeptide extension containing one or two cysteines to the COOH-terminus of an scFv antibody fragment. The scFv protein was expressed and secreted in a recombinant Pichia pastoris system as a dimer with a C-terminal disulfide bridge, as determined by Western blot analysis under non-reducing conditions. We found that the scFv construct with one cysteine in the C-extension (scFv-1Cys) exhibited a much higher dimer/monomer ratio than the two cysteine counterpart (scFv-2Cys). Binding activity measurements performed by means of a competitive radioimmunoassay showed that scFv-1Cys exhibited specific antigen binding activity, which was almost the same as that of the parental MAb, and approximately four- and fortyfold higher than those of the control scFv monomer and scFv-2Cys.