HPLC Purification of TRPM8 and Experimental Confirmation of Its Cholesterol Affinity on Synthetic Lipid Raft-like Models

first_pagesettingsOrder Article Reprints Open AccessArticle HPLC Purification of TRPM8 and Experimental Confirmation of Its Cholesterol Affinity on Synthetic Lipid Raft-like Models by Clotilde Beatrice Angelucci 1,†,Annalaura Sabatucci 2,†ORCID,Alexandrine Kurtz 2,Davide Laurenti 3ORCID,Beatrice Dufrusine 2ORCID,Enrico Dainese 2,*ORCID andAntonio Francioso 2,*ORCID 1 Department of Veterinary Medicine, University of Teramo, 64100 Teramo, Italy 2 Department of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo, Italy 3 Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, 00185 Rome, Italy * Authors to whom correspondence should be addressed. † These authors contributed equally to this work. Life 2026, 16(3), 392; https://doi.org/10.3390/life16030392 Submission received: 18 December 2025 / Revised: 13 February 2026 / Accepted: 13 February 2026 / Published: 28 February 2026 (This article belongs to the Special Issue Channel Proteins and Transporters in Human Health and Disease) Downloadkeyboard_arrow_down Browse Figures Versions Notes Abstract This study presents the successful expression, purification, and functional characterization of the human TRPM8 ion channel, a key player in temperature sensing and pain modulation. Using a modified bacterial expression protocol and DDM-based solubilization, TRPM8 was purified via HPLC-SEC and analyzed for its membrane-binding properties. FRET-based assays with synthetic lipid rafts revealed a strong and selective affinity of TRPM8 for cholesterol-containing membranes, suggesting cholesterol’s role in modulating TRPM8 localization and activity. These findings provide quantitative in vitro evidence of TRPM8–cholesterol interactions and establish a robust model system for future structural and functional studies of membrane-associated proteins.