title: An efficient strategy for heterologous expression and purification of active peptide hainantoxin-IV.
abstract: Hainantoxin-IV (HNTX-IV) from the venom of the spider Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S) sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK) motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF) cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST) tag and a small ubiquitin-related modifier (SUMO) tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA) extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV) is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) analysis. The recombinant toxin has similar activity (IC50 value of 120 nM) on the tetrodotoxin-sensitive (TTX-S) sodium channels in adult rat dorsal root ganglion (DRG) neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.
authors: Key Laboratory of Protein Chemistry and Developmental Biology of the Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors: Hui
authors: H
authors: Zhang
authors: Key Laboratory of Protein Chemistry and Developmental Biology of the Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors: Peng-Fei
authors: PF
authors: Huang
authors: Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.
authors: Er
authors: E
authors: Meng
authors: Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.
authors: Wen-Ying
authors: WY
authors: Li
authors: Key Laboratory of Protein Chemistry and Developmental Biology of the Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors: Lu
authors: L
authors: Zhou
authors: Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.
authors: Ling-Yun
authors: LY
authors: Zhu
authors: Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.
authors: Lei
authors: L
authors: Wu
authors: Key Laboratory of Protein Chemistry and Developmental Biology of the Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors: Meng-Jie
authors: MJ
authors: Li
authors: Key Laboratory of Protein Chemistry and Developmental Biology of the Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors: Song-Ping
authors: SP
authors: Liang
authors: Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.
authors: Dong-Yi
authors: DY
authors: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005727:Ganglia, Spinal / Q000187:drug effects / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013111:Spider Venoms / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D013112:Spiders / Q000737:chemistry / Q000235:genetics*
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D013111:Spider Venoms; C474037:hainantoxin-IV, Seleconosmia hainana
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