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Bioprocess."],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Arenin is a cystine-rich Kunitz-type protease inhibitor originally isolated from the skin secretion of the tree frog\n                    <jats:italic>Dryophytes arenicolor<\/jats:italic>\n                    , whose limited natural yield has hindered comprehensive functional studies. In this work, we established an efficient recombinant production system in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    and evaluated its anticancer and wound-healing properties. A codon-optimized arenin gene, fused to an N-terminal 6\u2009\u00d7\u2009His-TEV tag, was cloned into a T7-lac expression cassette. IPTG induction at 30\u00a0\u00b0C and 37\u00a0\u00b0C revealed distinct temperature-dependent partitioning: at 30\u00a0\u00b0C, arenin predominantly accumulated in the soluble fraction, whereas at 37\u00a0\u00b0C, it was confined to inclusion bodies. Both strategies yielded\u2009\u2265\u200990% pure peptide, producing 7.8\u2009\u00b1\u20090.6\u00a0mg and 12.4\u2009\u00b1\u20091.0\u00a0mg per 250\u00a0mL culture, respectively. Bioassays showed that HDFa fibroblasts tolerated 31.25\u2013500\u00a0\u00b5g\u00a0mL\n                    <jats:sup>\u22121<\/jats:sup>\n                    arenin and had increased viability at 1000\u00a0\u00b5g\u00a0mL\n                    <jats:sup>\u22121<\/jats:sup>\n                    . ER\u207a MCF-7 cells showed mild inhibition at low doses but growth stimulation at the highest, suggesting hormetic protease\u2013receptor effects. Caco-2 cells were sensitive, with viability at 60.2\u2009\u00b1\u20093.4% at 31.25\u00a0\u00b5g\u00a0mL\n                    <jats:sup>\u22121<\/jats:sup>\n                    and below 80% up to 250\u00a0\u00b5g\u00a0mL\n                    <jats:sup>\u22121<\/jats:sup>\n                    . Scratch-wound assays under serum deprivation or high glucose showed complete closure within 72\u00a0h at all arenin concentrations, comparable to\n                    <jats:italic>Centella asiatica<\/jats:italic>\n                    (10\u00a0\u00b5g\u00a0mL\n                    <jats:sup>\u22121<\/jats:sup>\n                    ), 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.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Graphical abstract<\/jats:bold>\n                  <\/jats:p>","DOI":"10.1186\/s40643-025-00986-2","type":"journal-article","created":{"date-parts":[[2025,12,6]],"date-time":"2025-12-06T08:25:39Z","timestamp":1765009539000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Heterologous expression and functional characterization of recombinant arenin to assess its anticancer and wound-healing potential"],"prefix":"10.1186","volume":"12","author":[{"given":"Enrique","family":"Hidalgo-V\u00e1zquez","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jes\u00fas","family":"Hern\u00e1ndez-P\u00e9rez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marilena","family":"Antunes-Ricardo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Calef","family":"S\u00e1nchez-Trasvi\u00f1a","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mario E.","family":"Barocio","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mar\u00eda Isabela","family":"Avila Rodr\u00edguez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jorge","family":"Benavides","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,12,6]]},"reference":[{"issue":"8","key":"986_CR1","doi-asserted-by":"publisher","first-page":"a041235","DOI":"10.1101\/CSHPERSPECT.A041235","volume":"14","author":"P Ahangar","year":"2022","unstructured":"Ahangar P, Strudwick XL, Cowin AJ (2022) Wound healing from an actin cytoskeletal perspective. 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