Expression and characterization of scFv-6009FV in Pichia pastoris with improved ability to neutralize the neurotoxin...

Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % β-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC₅₀) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.