Surface display of GFP using CotX as a molecular vector on Bacillus subtilis spores

Abstract:

Spore coat proteins, such as CotB, CotC, CotG et al, are able to efficiently display exogenous protein on spore surface for preparing oral vaccines or enzymes. CotX is another structural protein of spore coats of Bacillus subtilis. To investigate whether CotX could carry target protein onto the spore surface, we constructed a recombinant integrative plasmid, designated as pJS749, which carries a recombinant cotX-gfp gene under the control of the cotX promoter. We transformed pJS749 into Bacillus subtilis 168(trp-), an α-amylase inactivated mutant DRJS749 was selected and confirmed to be a double crossover integrant, where cotX-gfp fragment was integrated into the chromosome. After induction of spore formation, significant green fluorescence was observed on spore surface of strain DRJS749 under fluorescent microcopy. This suggests that CotX is associated with the outer part of the coat. CotX can therefore be used as a molecular vehicle for spore surface display of exogenous proteins.