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

## TL;DR

This study explores the production and activity of ProTx-I, a toxin from tarantula venom, which could help develop new painkillers by targeting specific ion channels.

## Contribution

The study introduces efficient methods for producing ProTx-I and reveals species-specific activity and the impact of an N-terminal methionine residue.

## Key findings

- ProTx-I was successfully produced using cytoplasmic inclusion bodies and periplasmic secretion methods.
- ProTx-I showed higher activity on rat TRPA1 channels compared to human TRPA1 channels.
- An extra N-terminal methionine residue reduced the toxin's activity.

## 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.