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Recombinant Production, SpeciesSpecific Activity at the TRPA1 Channel, and Significance of the N-Terminal Residue of ProTx-I Toxin from Thrixopelma Pruriens Tarantula Venom

M. Shulepko,Mendi Zhang,Eugenii A. Zhivov,D. Kulbatskii,A. S. Paramonov,Yuqi Che,A. Kuznetsov,A. Popov,M. Kirpichnikov,Z. Shenkarev,E. Lyukmanova

Published 2025 in Acta Naturae

ABSTRACT

The ProTx-I toxin from Thrixopelma pruriens tarantula venom inhibits voltage-gated sodium (NaV), potassium, and calcium channels, as well as the chemosensitive TRPA1 ion channel, affecting the activating processes of these channels. Due to its action at the NaV1.7, NaV1.8, and TRPA1 channels involved in pain perception and propagation, ProTx-I may be used as a model for the development of next-generation analgesics. ProTx-I consists of 35 amino acid residues, with three disulfide bonds forming an inhibitor cystine knot (ICK) motif, which challenges its heterologous production. An efficient ProTx-I production system is necessary to study, at the molecular level, the mechanism by which the toxin acts. In this study, we tested several approaches for bacterial production of disulfide-containing toxins. Cytoplasmic expression of ProTx-I fused with either thioredoxin or glutathione-S-transferase failed to yield a correctly folded toxin. However, the natively folded ProTx-I was successfully obtained by “direct” expression in the form of cytoplasmic inclusion bodies, followed by renaturation, as well as by secretion into the periplasmic space via fusion with maltose-binding protein. The activity of the recombinant ProTx-I was studied by electrophysiology in X. laevis oocytes expressing rat and human TRPA1 channels. The toxin proved to be more active on the rat channel than on the human channel (IC50 = 250 ± 85 and 840 ± 190 nM, respectively). The presence of an additional N-terminal methionine residue in the toxin obtained through “direct” expression significantly attenuated its activity.

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