Work

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

Year: 2025

Type: article

Abstract: Background 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 conotoxins (TIIIA and its mutant TIIIAlaMut), assess their biological activity on Nav1.4 sodium channels, and evaluate the anti-wrinkle efficacy of a topical cream containing the recombinant peptide in a group of volunteers. Material and methods 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 tested using patch-clamp electrophysiology in Xenopus laevis oocytes expressing Nav1.4 channels. Additionally, the conotoxin-containing cream was applied by 55 human volunteers in an application study assessing anti-aging effects. Results Both recombinant peptides were expressed, purified, and activated. Electrophysiological measurements demonstrated their ability to modulate Nav1.4 channel activity, including the version extracted from the cream. In the human study, 47% of participants reported visible reduction of wrinkles. Additional benefits included skin tone evening, erythema reduction, and balanced sebum production in oily skin types. Conclusions This study describes the design, bacterial expression, and functional analysis of recombinant conotoxins TIIIA and TIIIAlaMut. Their bioactivity was confirmed on Xenopus laevis oocytes expressing human Nav 1.4 channels. The recombinant toxins, including one extracted from a cream, showed effects comparable to a synthetic standard. Application tests demonstrated the conotoxin’s potential in cosmeceuticals, especially in reducing periocular wrinkles and improving skin texture and tone.

Source: Biotechnologia

DOI: 10.5114/bta/214377