Researchers uncover new potential for botulinum neurotoxin E in therapeutic and cosmetic applications NatureComms
. All mutations were generated by two-step PCR and verified by DNA sequencing. BoNT/E and mutants carrying C-terminal His6-tag were purified on Co-Talon matrix and eluted with 50 mM Tris–HCl , 150 mM NaCl, and 250 mM imidazole. For proteolytic activation and removal of an affinity tag, BoNT/E was incubated for 16 h at room temperature with 0.01 U bovine thrombin per µg of BoNT. Subsequent gel filtration was performed in phosphate-buffered saline .
For the HE–G6 complex, the best crystals were obtained in a reservoir containing 0.1 M HEPES, pH 7.0, 0.2 M NaCl, and 18% PEG 3350, and the crystals were cryoprotected in the mother liquor supplemented with 20% ethylene glycol. The best crystals for the HE–SV2Ac–G6 complex were obtained in a reservoir containing 0.1 M HEPES, pH 7.5, 0.2 M potassium sulfate, 20% PEG 3350, and 5% PEG 400. Streak seeding was necessary to obtain single crystals.
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