title: Heterologous expression and purification of neurotoxic Hainantoxin-III in E. coli.
abstract: 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 IC50of 225 nM and also have high selectivity for different voltage-gated sodium channels. Therefore, it has very similar activity to the natural one.
authors.affiliation: a Research Center of Biological Information , College of Science, National University of Defense Technology , Changsha , China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wu
authors.affiliation: b Key Laboratory of Protein Chemistry and Developmental Biology of the Ministry of Education , College of Life Sciences, Hunan Normal University , Changsha , China.
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.affiliation: c Beijing Institute of Pharmaceutical Chemistry , Beijing , China.
authors.forename: Hui
authors.initials: H
authors.lastname: Jiang
authors.affiliation: a Research Center of Biological Information , College of Science, National University of Defense Technology , Changsha , China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wu
authors.affiliation: a Research Center of Biological Information , College of Science, National University of Defense Technology , Changsha , China.
authors.forename: Ling-Yun
authors.initials: LY
authors.lastname: Zhu
authors.affiliation: a Research Center of Biological Information , College of Science, National University of Defense Technology , Changsha , China.
authors.forename: Er
authors.initials: E
authors.lastname: Meng
authors.affiliation: a Research Center of Biological Information , College of Science, National University of Defense Technology , Changsha , China.
authors.forename: Dong-Yi
authors.initials: DY
authors.lastname: Zhang
mesh_terms: D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D018408:Patch-Clamp Techniques; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013111:Spider Venoms / Q000235:genetics* / Q000302:isolation & purification; D014462:Ultrafiltration
chemical_list: D013111:Spider Venoms; C488683:hainantoxin III, Seleconosmia hainana
keywords: Activity; IC50; expression; hNav1.7; purification; rHNTX-III