Search for a command to run...
Type I ribosome-inactivating proteins, such as gelonin, are enzymatically potent toxins but are effectively non-toxic under physiological conditions because they are unable to cross the plasma membrane[1]. Pulsed electric fields (PEF) overcome this delivery barrier by transiently permeabilizing the plasma membrane, allowing direct cytosolic entry. This enhances gelonin cytotoxicity by several thousand-fold, highlighting gelonin’s potential as an electrochemotherapy candidate[2]. Here, we present a recombinant gelonin (rGel) expression platform in E. coli with HPLC-based purification that enables reproducible protein production and structure-guided engineering. The mature gelonin sequence (residues 47–297) was cloned into pET-32a(+) with an N-terminally fused Trx-6×His tag and TEV cleavage site. The protein was expressed in E. coli, captured from the cell lysate using affinity chromatography, and the fusion part cleaved with TEV. Untagged rGel was recovered in the flow-through in a second affinity round. To remove any residual impurities, rGel underwent cation-exchange chromatography and endotoxin removal. Our purification workflow yields 10 ± 1 mg rGel per liter of culture at >99% purity with endotoxin levels below 2 EU/mg. rGel exhibits translation-inhibiting activity comparable to native gelonin (nGel) in a cell-free assay (EC₅₀: 4.9 vs 3.2 pM). Electroporation-enhanced cytotoxicity assessed in CT26 cells (8 × 100 µs, 1.1 kV/cm), revealed similar potency between rGel and nGel (EC₅₀: 0.8 nM vs 0.7 nM). Given its comparable activity and cytotoxicity, rGel will replace nGel in future work. This platform enables engineering of size- and charge-optimized rGel variants to improve tissue penetration. Funding: Pam and Rich Nuccitelli Foundation (to OP), Graduate Summer Award Program 2025, Graduate School and Office of Research, Old Dominion University (to EZ). References: [1] Pizzo E, Di Maro A. J Biomed Sci 2016;23:54. [2] Pakhomova ON, Zivla E, Silkuniene G, Silkunas M, Pakhomov AG. Int J Mol Sci 2025;26:458.