# Gene expression, purification, and functional characterization of recombinant conotoxin μ-TIIIA and TIIIAlaMut in Escherichia coli with clinical evaluation of antiwrinkle efficacy

## TL;DR

This study shows that recombinant conotoxins, produced in bacteria, can inhibit nerve channels and reduce wrinkles when applied as a cream.

## Contribution

The novel contribution is the successful bacterial expression and functional validation of conotoxins for potential cosmeceutical use.

## Key findings

- Recombinant conotoxins TIIIA and TIIIAlaMut inhibited Nav1.4 channel activity effectively.
- A cream containing the conotoxins reduced wrinkles in 47% of participants and improved skin tone and sebum balance.
- The bioactivity of the conotoxins from the cream was comparable to a synthetic standard.

## Abstract

Conotoxins are small peptides known for their potent and selective activity on ion channels, offering potential applications in both medicine and cosmetology. This study aimed to design and validate recombinant conotoxin TIIIA and its mutant TIIIAlaMut, assess their biological activity on the voltage-gated Na+ (Nav) channel Nav1.4, and evaluate the antiwrinkle efficacy of a topical cream containing the recombinant peptide in a group of volunteers.

Fusion genes encoding TRX::TIIIA and TRX::TIIIAlaMut were cloned into the pDM vector and expressed in Escherichia coli S4B cells. The proteins were purified using Ni-NTA chromatography, cleaved with CNBr under optimized acidic conditions, and analyzed. Biological activity was assessed using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing the human Nav1.4 channel. Additionally, a conotoxin-containing cream was applied to 55 human volunteers in an application study assessing its antiaging effects.

Both recombinant genes were successfully expressed, purified, and activated. Electrophysiological measurements demonstrated their ability to inhibit Nav1.4 channel activity, including the version extracted directly from the cream. In the human study, 47% of participants reported a visible reduction in wrinkles. Additional benefits included evening of skin tone, reduced erythema, and balanced sebum production in oily skin types.

This study describes the design, bacterial expression, and functional analysis of recombinant conotoxins TIIIA and TIIIAlaMut. Their bioactivity was confirmed on human Nav1.4 channels. The recombinant toxins, including the form extracted from the cream, showed effects comparable to a synthetic standard. Application tests demonstrated the conotoxin’s potential in cosmeceuticals, particularly in reducing periocular wrinkles and improving skin texture and tone.