title: High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies.
abstract: We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.
authors.affiliation: Ciba-Geigy Ltd., Basei, Switzerland.
authors.forename: T
authors.initials: T
authors.lastname: Kretzschmar
authors.forename: L
authors.initials: L
authors.lastname: Aoustin
authors.forename: O
authors.initials: O
authors.lastname: Zingel
authors.forename: M
authors.initials: M
authors.lastname: Marangi
authors.forename: B
authors.initials: B
authors.lastname: Vonach
authors.forename: H
authors.initials: H
authors.lastname: Towbin
authors.forename: M
authors.initials: M
authors.lastname: Geiser
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D016367:Baculoviridae / Q000276:immunology*; D002460:Cell Line; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007313:Insecta; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv