Functional expression of Bacillus thuringiensis Cry proteins on the surface of Bacillus subtilis spores

The Cry1Fa insecticidal protein from Bacillus thuringiensis (Bt) was expressed on the surface of Bacillus subtilis (Bs) spores to create transgenic Bs spores referred to as Spore-Cry1Fa. Cry1Fa, along with its leader sequence, was connected to the carboxyl end of a Bs spore outercoat protein, CotC, through a flexible linker. The Arg-27 residue of the Cry1Fa protein was mutated to Leu to prevent detachment from the spores due to protease digestion. The expression of the Cry protein on the Bs spores was confirmed by fluorescence microscopy using an anti-Cry1Fa antibody. The Cry protein, tightly anchored to the spore surface, appeared to be functional in terms of receptor binding. Spore-Cry1Fa bound to Sf9 cells expressing Spodoptera frugiperda (Sf) ABCC2 transporter and killed the cells within 60 min. Additionally, nano-lipid particles of SfABCC2 were produced using styrene-maleic acid (SMA) to demonstrate the binding to Spore-Cry1Fa.