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.

Fig 1. Construction of pET-GS-HNTX-IV expression vector.

A) Schematic diagram of RF-cloning strategy. F1: forward primer. R1: reverse primer. The portion complementary with the target gene is marked gray, and the portion complementary to the recipient vector is marked black. B) Map of pET-GS-HNTX-IV expression vector.

Fig 2. Expression of GST-SUMO tagged HNTX-IV in BL21(DE3).

A) Solubility analysis at 37°C and 16°C. M: protein molecular weight marker; S: soluble fraction. P: precipitates. The target protein is denoted by an arrow. B) The expression level under different IPTG conditions.

Fig 3. Purification of rHNTX-IV.

A) Purification by glutathione column. B) Affinity chromatography independent purification by TCA, acetonitrile and acetone. T: the total cell extract digested by SUMO protease; P: the precipitated fraction; S: the soluble fraction.

Fig 4. Affinity chromatography independent purification of rHNTX-IV by TCA.

A) M: Protein molecular weight marker; 1–10: fractions extracted by TCA from 1% to 10%; P: precipitates. B) Relative purity (compared with lane 10) and relative yield (compared with lane 1) of rHNTX-IV under different TCA concentrations. The value was calculated by grayscale by Quantity One software. The data points (means ± S.E.) come from three independent experiments. C) Purification by RP-HPLC.

Fig 5. Identification of rHNTX-IV.

A) Comparison of rHNTX-IV and native HNTX-IV on RP-HPLC. B) MALDI-TOF analysis of rHNTX-IV.

Fig 6. Functional characterization of the venom on voltage-gated sodium channels.

A) Typical current traces from TTX-S Na⁺ channel before and after application of 100 nM and 10 μM rHNTX- IV. B) The I-V curve of TTX-S I _Na with 100 nM and 10 μM rHNTX- IV. C) Typical current traces from TTX-R Na⁺ channel before and after application of 10 μM rHNTX- IV. D) The I-V curve of TTX-R I _Na with 100 nM rHNTX- IV. The data points (means ± S.E.) come from at least five cells.

Fig 7. The kinetics curves of rHNTX-IV.

A) The dose-effects curve of rHNTX-IV. The data points (means ± S.E.) are fit to the Hill equation. B) The steady-state inactivation and activation curves for TTX-S I _Na with 100 nM rHNTX-IV. The data points (means ± S.E.) come from at least five cells and are fit to the Boltzmann equation.

Fig 8. Functional characterization of the venom on voltage-gated calcium channels.

A) Typical current traces from an L-type Ca²⁺ channel before and after application of 10 μM native HNTX-IV; B. Typical current traces from a T-type Ca²⁺ channel before and after application of 10 μM native HNTX- IV. C) Typical current traces from an L-type Ca²⁺ channel before and after application of 10 μM rHNTX- IV. D) Typical current traces from a T-type Ca²⁺ channel before and after application of 10 μM rHNTX-IV.