Artefactual band patterns by SDS-PAGE of the Vip3Af protein in the presence of proteases mask the extremely high stability of this protein.

Vip3 proteins are secretable proteins from Bacillus thuringiensis with important characteristics for the microbiological control of agricultural pests. The exact details of their mode of action are yet to be disclosed and the crystallographic structure is still unknown. Vip3 proteins are expressed as protoxins that have to be activated by the insect gut proteases. A previous study on the peptidase processing of Vip3Aa revealed that the protoxin produced artefactual band patterns by SDS-PAGE due to the differential stability of this protein and the peptidases to SDS and heating (Bel et al., 2017 Toxins 9:131). To determine whether this phenomenon also applies to other Vip3A proteins, here we chose a different Vip3A protein (Vip3Af) and subjected it to commercial trypsin and midgut juice from a target insect species (Spodoptera frugiperda). The misleading degradation patterns were also observed with Vip3Af, both with trypsin and midgut juice. However, gel filtration chromatography showed that, under native conditions, Vip3Af is found as a tetramer and that peptidases only act upon primary cleavage sites. The proteolytic cleavage renders two fragments of approximately 20 kDa and 65 kDa which remain together in the tretameric structure and that are no further processed even at high peptidase concentrations.