HIGH-LEVEL EXPRESSION IN INSECT CELLS AND PURIFICATION OF SECRETED MONOMERIC SINGLE-CHAIN FV ANTIBODIES

We have constructed a recombinant baculovirus encoding an anti-(phenyl -oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the bac ulovirus GP67 secretion signal sequence. 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one an d cultured in a bioreactor for 72 h. The dialyzed supernatant was subj ected to cation exchange chromatography at pH 6.0 followed by size exc lusion 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 sc Fv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yi elded a half maximum inhibitory concentration of 3.4 x 10(-7) M which marches the earlier measured K-D 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. (1 992) 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 express ion.