High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies

Autor: Otto Zingel, Bénédicte Vonach, Laurent Aoustin, Harry Towbin, Marcello Marangi, Titus Kretzschmar, Martin Geiser
Rok vydání: 1996
Předmět:
Zdroj: Journal of Immunological Methods. 195:93-101
ISSN: 0022-1759
Popis: 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 × 10 −4 M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 × 10 −7 M which matches the earlier measured K D for the anti-phOx-scFv (3.2–5.3 × 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.
Databáze: OpenAIRE
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