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.

Highlights

  • HNTX-IV is a 35 amino-acid neuronal toxin with an inhibitory cystine knot (ICK) motif isolated from the venom of the Chinese bird spider Selenocosmia hainana [1]

  • HNTX-IV was traditionally obtained through natural sources [2] or solid-phase peptide synthesis [3]

  • The cDNA of HNTX-IV [12] and glutathione S-transferase (GST)-tag were kept in our laboratory

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Summary

Introduction

HNTX-IV is a 35 amino-acid neuronal toxin with an inhibitory cystine knot (ICK) motif isolated from the venom of the Chinese bird spider Selenocosmia hainana [1]. The toxin can inhibit TTX-S voltage-gated sodium channels with an IC50 value of 34.0 nM in adult rat dorsal root ganglion (DRG) neurons. Because of its compact ICK motif and robust activity, HNTXIV can act as a potential scaffold for drug design. HNTX-IV was traditionally obtained through natural sources [2] or solid-phase peptide synthesis [3]. Its low abundance in venom produces a low yield, and the correct folding of disulfide-rich peptides by chemical synthesis is always time-consuming and difficult

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