Fully active recombinant Bont/E purified from E. coli in high yield

Both rHis6-BoNT/E and rBoNT/E-His6 were produced from a codon optimized gene within Escherichia coli. Similar to native BoNT/E isolated from Clostridium botulinum, the recombinant proteins were isolated as single-chain molecules. The His6 affinity tag facilitated a two-step IMAC-IEX purification. While reasonable yields were obtained, single-chain rHis6-BoNT/E was produced less efficiently than was rBoNT/E-His6. Treatment with recombinant trypsin efficiently converted both purified rBoNT/E constructs to the activated dichain forms, with concomitant removal of the His6 tag, and the nicking profile for each rBoNT/E molecule matched that of native BoNT/E. Following nicking with trypsin, it was observed that the resultant rHis6-BoNT/E dichain displayed reduced in vivo potency (mouse potency assay) compared to activated native BoNT/E dichain. In contrast, activated rBoNT/E-His6 displayed a specific potency equivalent to that of trypsin-nicked native BoNT/E in the mouse potency assay. These results demonstrate that rBoNT/E, containing a C-terminal His6 affinity tag, can be expressed and purified in high yield, with the activated molecule displaying equivalent in vivo potency to activated native BoNT/E.