Anti-digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

• Published in: FEBS letters Vol. 423; no. 2; pp. 159 - 166
• Main Authors: Lemeulle, Corinne, Chardès, Thierry, Montavon, Céline, Chaabihi, Hassan, Mani, Jean-Claude, Pugnière, Martine, Cerutti, Martine, Devauchelle, Gérard, Pau, Bernard, Biard-Piechaczyk, Martine
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 20-02-1998; Wiley
• Subjects: Affinity; Amino Acid Sequence; Animals; Antibody Specificity; Baculoviridae - genetics; Baculovirus; Base Sequence; BCA, bicinchoninic acid; Cellular Biology; Cloning, Molecular - methods; CNBr, cyanogen bromide; Digoxin; Digoxin - immunology; EDTA, ethylenediaminetetraacetic acid; Escherichia coli; Escherichia coli - genetics; Gene Expression; Genetic Vectors - genetics; IMAC, ion metal chelate affinity chromatography; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - immunology; Insecta - cytology; IPTG, isopropyl-thiogalactoside; Life Sciences; Molecular Sequence Data; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; Protein expression; Sequence Alignment; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Single-chain variable fragment; SN, supernatant; Specificity; SPR analysis; Digoxin; EDTA, ethylenediaminetetraacetic acid; Escherichia coli; BCA, bicinchoninic acid; Baculovirus; IMAC, ion metal chelate affinity chromatography; CNBr, cyanogen bromide; Specificity; SPR analysis; PBS, phosphate-buffered saline; IPTG, isopropyl-thiogalactoside; Single-chain variable fragment; Affinity; Protein expression; PCR, polymerase chain reaction; SN, supernatant
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)00029-5

A gene encoding a single-chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did
Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 F
ab, whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half-lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.