Recombinant Expression and In Vitro Characterisation of Active Huwentoxin-IV

Abstract

Huwentoxin-IV (HwTx-IV) is a 35-residue neurotoxin peptide with potential application as a novel analgesic. It is a member of the inhibitory cystine knot (ICK) peptide family, characterised by a compact globular structure maintained by three intramolecular disulfide bonds. Here we describe a novel strategy for producing non-tagged, fully folded ICK-toxin in a bacterial system. HwTx-IV was expressed as a cleavable fusion to small ubiquitin-related modifier (SUMO) in the cytoplasm of the SHuffle T7 Express lysY Escherichia coli strain, which allows cytosolic disulfide bond formation. Purification by IMAC with selective elution of monomeric SUMO fusion followed by proteolytic cleavage and polishing chromatographic steps yielded pure homogeneous toxin. Recombinant HwTx-IV is produced with a C-terminal acid, whereas the native peptide is C-terminally amidated. HwTx-IV(acid) inhibited Nav1.7 in a dose dependent manner (IC50 = 463-727 nM). In comparison to HwTx-IV(amide) (IC50 = 11 ± 3 nM), the carboxylate was ~50 fold less potent on Nav1.7, which highlights the impact of the C-terminus. As the amide bond of an additional amino acid may mimic the carboxamide, we expressed the glycine-extended analogue HwTx-IVG36(acid) in the SUMO/SHuffle system. The peptide was approximately three fold more potent on Nav1.7 in comparison to HwTx-IV(acid) (IC50 = 190 nM). In conclusion, we have established a novel system for expression and purification of fully folded and active HwTx-IV(acid) in bacteria, which could be applicable to other structurally complex and cysteine rich peptides. Furthermore, we discovered that glycine extension of HwTx-IV(acid) restores some of the potency of the native carboxamide. This finding may also apply to other C-terminally amidated peptides produced recombinantly.

Modulatory features of the novel spider toxin μ‐TRTX‐Df1a isolated from the venom of the spider Davus fasciatus

“…C‐terminal amidation has been reported to significantly increase the potency of other NaSpTx peptides. The amidated form of huwentoxin‐IV, a toxin isolated from the spider Haplopelma schmidti , is 42‐fold more potent against Na V 1.7 (Sermadiras et al, ). The C‐terminally amidated form of ProTx‐III, a toxin that we recently isolated from the spider Thrixopelma pruriens , is 4.6‐fold more potent against Na V 1.7 and 8.9‐ and 3.5‐fold more potent against Na V 1.1 and Na V 1.3 respectively (Cardoso et al, ).…”

Expression in Escherichia coli of fusion protein comprising α‐conotoxin TxIB and preservation of selectivity to nicotinic acetylcholine receptors in the purified product

“…Intracellular environment of E. coli can help to keep the structure of the inclusion body stable, which also would be helpful for the purification of recombinant protein. 8 Biological toxicity on insects [33] Conkunitzin-S1 Voltage-gated potassium channels (VGPCs) [34] GeXIVAWT VGSCs [40] cytoplasm. There are following advantages at the methodology in this work.…”

Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus

“…Knottin peptides can be challenging to produce in a recombinant manner owing to the large number of disulphide bonds (Sermadiras et al, 2013;Zhang et al, 2015). Some L. intermedia knottin peptides have 10 cysteine residues that are predicted to form up to five disulphide bonds, for which there are 945 theoretically possible disulphide isomers.…”