In the present study, we used Escherichia coli to produce recombinant Hainantoxin-III (rHNTX-III), a 33-amino acid peptic toxin from the tarantula spider Haplopelma hainanum. The toxin has three pairs of disulfide bonds. A pET-HS-HNTX-III vector was constructed and transformed into the E. coli strain SHuffleTM. rHNTX-III was expressed using auto-induction medium. After using a Ni-NTA column, the expressed fusion protein was digested using SUMO protease (ULP1) to remove the HIS-SUMO tag, and then RP-HPLC and ultrafiltration were used for further purification. Then the rHNTX-III was identified by MALDI-TOF/TOF mass spectrometry. The purified rHNTX-III was further analyzed using a whole-cell patch-clamp assay. It was shown that the rHNTX-III was able to block currents generated by human Nav1.7 (hNav1.7) at an IC50 of 225 nM and also have high selectivity for different voltage-gated sodium channels. Therefore, it has very similar activity to the natural one.
Keywords: Activity; IC50; expression; hNav1.7; purification; rHNTX-III.