Heterologous expression and functional characterization of recombinant arenin to assess its anticancer and wound-healing potential.

Arenin is a cystine-rich Kunitz-type protease inhibitor originally isolated from the skin secretion of the tree frog Dryophytes arenicolor, whose limited natural yield has hindered comprehensive functional studies. In this work, we established an efficient recombinant production system in Escherichia coli and evaluated its anticancer and wound-healing properties. A codon-optimized arenin gene, fused to an N-terminal 6 × His-TEV tag, was cloned into a T7-lac expression cassette. IPTG induction at 30 °C and 37 °C revealed distinct temperature-dependent partitioning: at 30 °C, arenin predominantly accumulated in the soluble fraction, whereas at 37 °C, it was confined to inclusion bodies. Both strategies yielded ≥ 90% pure peptide, producing 7.8 ± 0.6 mg and 12.4 ± 1.0 mg per 250 mL culture, respectively. Bioassays showed that HDFa fibroblasts tolerated 31.25-500 µg mL-1 arenin and had increased viability at 1000 µg mL-1. ER⁺ MCF-7 cells showed mild inhibition at low doses but growth stimulation at the highest, suggesting hormetic protease-receptor effects. Caco-2 cells were sensitive, with viability at 60.2 ± 3.4% at 31.25 µg mL-1 and below 80% up to 250 µg mL-1. Scratch-wound assays under serum deprivation or high glucose showed complete closure within 72 h at all arenin concentrations, comparable to Centella asiatica (10 µg mL-1), consistent with other Kunitz-family peptide activities in diverse protease environments. Overall, our results demonstrate the feasibility of a heterologous expression strategy for large-scale arenin production and highlight its dual functional potential, pro-regenerative effects in fibroblasts, and selective cytotoxicity toward hormone-independent cancer cells.