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

Abstract

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.

Graphical abstract

Keywords

Arenin / Amphibian / Kunitz-type peptide / Wound healing / Anticancer activity

References

[1] Ahangar P, Strudwick XL, Cowin AJ. Wound healing from an actin cytoskeletal perspective. Cold Spring Harb Perspect Biol, 2022, 14(8): a041235

[2] Alonso-Castro AJ, Carranza-Álvarez C, Maldonado-Miranda JJ, Del Rosario Jacobo-Salcedo M, Quezada-Rivera DA, Lorenzo-Márquez H, Figueroa-Zúñiga LA, Fernández-Galicia C, Ríos-Reyes NA, De León-Rubio MÁ, Rodríguez-Gallegos V, Medellín-Milán P. Zootherapeutic practices in Aquismón, San Luis Potosí, México. J Ethnopharmacol, 2011, 138(1): 233-237.

[3] Alvarado D, Cardoso-Arenas S, Corrales-García LL, Clement H, Arenas I, Montero-Dominguez PA, Olamendi-Portugal T, Zamudio F, Csoti A, Borrego J, Panyi G, Papp F, Corzo G. A novel insecticidal spider peptide that affects the mammalian voltage-gated ion channel hKv1.5. Front Pharmacol, 2021, 11: 563858.

[4] Bhatwa A, Wang W, Hassan YI, Abraham N, Li XZ, Zhou T. Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications. Front Bioeng Biotechnol, 2021, 9: 630551.

[5] Bonturi CR, Teixeira ABS, Rocha VM, Valente PF, Oliveira JR, Filho CMB, Batista IdeFC, Oliva MLV. Plant Kunitz inhibitors and their interaction with proteases: current and potential pharmacological targets. Int J Mol Sci, 2022, 23(94742

[6] Cao X, Wang Y, Wu C, Li X, Fu Z, Yang M, Bian W, Wang S, Song Y, Tang J, Yang X. Cathelicidin-OA1, a novel antioxidant peptide identified from an amphibian, accelerates skin wound healing. Sci Rep, 2018, 8(1): 1-15.

[7] Chan HS, Chang SJ, Wang TY, Ko HJ, Lin YC, Lin KT, Chang KM, Chuang YJ. Serine protease PRSS23 is upregulated by estrogen receptor α and associated with proliferation of breast cancer cells. PLoS ONE, 2012, 7(1): e30397

[8] Chen X, Wang H, Shen Y, Wang L, Zhou M, Chen T, Shaw C. Kunitzins: prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs. Biochem Biophys Res Commun, 2016, 4772): 302-309.

[9] Chen Y, Gong Y, Shi M, Zhu H, Tang Y, Huang D, Wang W, Shi C, Xia X, Zhang Y, Liu J, Huang J, Liu M, Chen H, Ma Y, Wang Z, Wang L, Tu W, Zhao Y, Shi X. miR-3606-3p alleviates skin fibrosis by integratively suppressing the integrin/FAK, p-AKT/p-ERK, and TGF-β signaling cascades. J Adv Res, 2024, 75: 271-290.

[10] Chhillar A, Jaiswal A. Hyaluronic acid-based self-healing hydrogels for diabetic wound healing. Adv Healthc Mater, 2025, 14(4): 2404255

[11] Cialdai F, Risaliti C, Monici M. Role of fibroblasts in wound healing and tissue remodeling on Earth and in space. Front Bioeng Biotechnol, 2022, 10: 958381.

[12] Dayer C, Stamenkovic I. Recruitment of matrix metalloproteinase-9 (MMP-9) to the fibroblast cell surface by lysyl hydroxylase 3 (LH3) triggers transforming growth factor-β (TGF-β) activation and fibroblast differentiation. J Biol Chem, 2015, 290(22): 13763

[13] de Marco A. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb Cell Fact, 2009, 8(1): 1-18.

[14] Estévez N, Fuciños P, Sobrosa AC, Pastrana L, Pérez N, Luisa Rúa M. Modeling the angiotensin-converting enzyme inhibitory activity of peptide mixtures obtained from cheese whey hydrolysates using concentration–response curves. Biotechnol Prog, 2012, 28(5): 1197-1206.

[15] Francis DM, Page R. Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci, 2010, 5(1): 5.24.1-5.24.29.

[16] Fratini E, Rossi MN, Spagoni L, Riccieri A, Mancini E, Polticelli F, Bologna MA, Mariottini P, Cervelli M. Molecular characterization of Kunitz-type protease inhibitors from blister beetles (Coleoptera, Meloidae). Biomolecules, 2022, 12(7988

[17] Hancock REW, Haney EF, Gill EE. The immunology of host defence peptides: beyond antimicrobial activity. Nat Rev Immunol, 2016, 16(5): 321-334.

[18] Hernández-Pérez J, Serra A, Sze SK, Conway PL, Schlundt J, Benavides J. Identification of Arenin, a novel Kunitz-like polypeptide from the skin secretions of Dryophytes arenicolor. Int J Mol Sci, 2018, 19(11): 3644.

[19] Huang C, Fu X, Liu J, Qi Y, Li S, Wang H. The involvement of integrin β1 signaling in the migration and myofibroblastic differentiation of skin fibroblasts on anisotropic collagen-containing nanofibers. Biomaterials, 2011, 336): 1791.

[20] Huang L, Chen D, Wang L, Lin C, Ma C, Xi X, Chen T, Shaw C, Zhou M. Dermaseptin-PH: a novel peptide with antimicrobial and anticancer activities from the skin secretion of the South American orange-legged leaf frog, Pithecopus (Phyllomedusa) hypochondrialis. Molecules, 2017, 22(10): 1805.

[21] Ibraheem A, Yap H, Ding Y, Campbell RE. A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors. BMC Biotechnol, 2011, 11: 105

[22] Kobayashi H, Shigetomi H, Imanaka S. Reassessing the role of tissue factor pathway inhibitor 2 in neoplastic and non-neoplastic lesions. Cancers, 2025, 17(9): 1447

[23] König E, Bininda-Emonds ORP, Shaw C. The diversity and evolution of anuran skin peptides. Peptides, 2015, 63: 96-117.

[24] Lavergne M, Guillon-Munos A, Lenga Ma Bonda W, Attucci S, Kryza T, Barascu A, Moreau T, Petit-Courty A, Sizaret D, Courty Y, Iochmann S, Reverdiau P. Tissue factor pathway inhibitor 2 is a potent kallikrein-related protease 12 inhibitor. Biol Chem, 2021, 402(10): 1257-1268.

[25] Li C, Fu Z, Jin T, Liu Y, Liu N, Yin S, Wang Z, Huang Y, Wang Y, Zhang Y, Li J, Wu Y, He L, Tang J, Wang Y, Yang X. A frog peptide provides new strategies for the intervention against skin wound healing. Cell Mol Biol Lett, 2023, 28(1): 61

[26] Liu C, Liu Y, Yu Y, Huang S, Sun C, Zhang D, Yu A. High glucose-induced senescent fibroblasts-derived exosomal miR-497 inhibits wound healing by regulating endothelial cellular autophagy via ATG13. Anal Cell Pathol, 2025, 2025: 8890200

[27] Liu Y, Liu Y, He W, Mu X, Wu X, Deng J, Nie X. Fibroblasts: immunomodulatory factors in refractory diabetic wound healing. Front Immunol, 2022, 13: 918223

[28] Lu J, Yang H, Yu H, Gao W, Lai R, Liu J, Liang X. A novel serine protease inhibitor from Bungarus fasciatus venom. Peptides, 2008, 29(3): 369-374.

[29] Lu Z, Tan K, Xiang S, Zhang Y, Luo F, Liu X, Zhao X, Ouyang L. Peptide loaded self-healing hydrogel promotes diabetic skin wound healing through macrophage orchestration and inflammation inhibition. Mater Today Bio, 2025, 32: 101690

[30] Ludewig S, Kossner M, Schiller M, Baumann K, Schirmeister T. Enzyme kinetics and hit validation in fluorimetric protease assays. Curr Top Med Chem, 2010, 10(3): 368-382.

[31] Maria DA, Will SEAL, Bosch RV, Souza JG, Sciani JM, Goldfeder MB, Rondon GG, Chudzinski-Tavassi AM. Preclinical evaluation of Amblyomin-X, a Kunitz-type protease inhibitor with antitumor activity. Toxicol Rep, 2018, 6: 51-63.

[32] Martin CE, List K. Cell-surface anchored serine proteases in cancer progression and metastasis. Cancer Metastasis Rev, 2019, 38(3): 357.

[33] Mishra V. Dot-blotting: a quick method for expression analysis of recombinant proteins. Curr Protoc, 2022, 2(9): e546

[34] Mookherjee N, Anderson MA, Haagsman HP, Davidson DJ. Antimicrobial host defence peptides: functions and clinical potential. Nat Rev Drug Discov, 2020, 19(5): 311-332.

[35] Mukherjee AK, Mackessy SP, Dutta S. Characterization of a Kunitz-type protease inhibitor peptide (Rusvikunin) purified from Daboia russelii russelii venom. Int J Biol Macromol, 2014, 67: 154-162.

[36] Nishikai-Yan Shen T, Kado M, Hagiwara H, Fujimura S, Mizuno H, Tanaka R. MMP9 secreted from mononuclear cell quality and quantity culture mediates STAT3 phosphorylation and fibroblast migration in wounds. Regen Ther, 2021, 18: 464-471.

[37] Pang L, Dunterman M, Guo S, Khan F, Liu Y, Taefi E, Bahrami A, Geula C, Hsu WH, Horbinski C, James CD, Chen P. Kunitz-type protease inhibitor TFPI2 remodels stemness and immunosuppressive tumor microenvironment in glioblastoma. Nat Immunol, 2023, 24(10): 1654

[38] Pouresmaeil M, Azizi-Dargahlou S. Factors involved in heterologous expression of proteins in E. coli host. Arch Microbiol, 2023, 205(5): 1-18.

[39] Ranasinghe SL, Fischer K, Zhang W, Gobert GN, McManus DP. Cloning and characterization of two potent Kunitz type protease inhibitors from Echinococcus granulosus. PLoS Negl Trop Dis, 2015, 9(12): e0004268.

[40] Ranasinghe S, McManus DP. Structure and function of invertebrate Kunitz serine protease inhibitors. Dev Comp Immunol, 2013, 39(3): 219-227.

[41] Rashno Z, Rismani E, Ghasemi JB, Mansouri M, Shabani M, Afgar A, Dabiri S, Rezaei Makhouri F, Hatami A, Harandi MF. Design of ion channel blocking, toxin-like Kunitz inhibitor peptides from the tapeworm, Echinococcus granulosus, with potential anti-cancer activity. Sci Rep, 2023, 13(1): 1-18.

[42] Rath A, Glibowicka M, Nadeau VG, Chen G, Deber CM. Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proc Natl Acad Sci U S A, 2009, 106(6): 1760

[43] Rigi G, Rostami A, Ghomi H, Ahmadian G, Mirbagheri VS, Jeiranikhameneh M, Vahed M, Rahimi S. Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide. BMC Biotechnol, 2021, 21(1): 21-51.

[44] Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol, 2014, 5(4): 79503.

[45] Schmidt MCB, Morais KLP, de Almeida MES, Iqbal A, Goldfeder MB, Chudzinski-Tavassi AM. Amblyomin-X, a recombinant Kunitz-type inhibitor, regulates cell adhesion and migration of human tumor cells. Cell Adhes Migr, 2020, 14(1): 129-138.

[46] Schnittert J, Bansal R, Storm G, Prakash J. Integrins in wound healing, fibrosis and tumor stroma: high potential targets for therapeutics and drug delivery. Adv Drug Deliv Rev, 2018, 129: 37-53.

[47] Shabestani Monfared G, Ertl P, Rothbauer M. An on-chip wound healing assay fabricated by xurography for evaluation of dermal fibroblast cell migration and wound closure. Sci Rep, 2020, 10(1): 1-14.

[48] Shelake RM, Ito Y, Masumoto J, Morita EH, Hayashi H. A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1. PLoS ONE, 2017, 12(2e0172182

[49] Shigetomi H, Onogi A, Kajiwara H, Yoshida S, Furukawa N, Haruta S, Tanase Y, Kanayama S, Noguchi T, Yamada Y, Oi H, Kobayashi H. Anti-inflammatory actions of serine protease inhibitors containing the Kunitz domain. Inflamm Res, 2010, 59(9): 679-687.

[50] Shimomura T, Denda K, Kitamura A, Kawaguchi T, Kito M, Kondo J, Kagaya S, Qin L, Takata H, Miyazawa K, Kitamura N. Hepatocyte growth factor activator inhibitor, a novel Kunitz-type serine protease inhibitor. J Biol Chem, 1997, 272(10): 6370-6376.

[51] Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Fact, 2015, 14(1): 1-10.

[52] Song YY, Zhang Y, Ren HN, Sun GG, Qi X, Yang F, Jiang P, Zhang X, Cui J, Wang ZQ. Characterization of a serine protease inhibitor from Trichinella spiralis and its participation in larval invasion of host’s intestinal epithelial cells. Parasit Vectors, 2018, 11(1): 1-12.

[53] Sørensen HP, Mortensen KK. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact, 2005, 4(1): 1-8.

[54] Spriestersbach A, Kubicek J, Schäfer F, Block H, Maertens B. Purification of His-tagged proteins. Methods Enzymol, 2015, 559: 1-15.

[55] Sun F, Deng X, Gao H, Ding L, Zhu W, Luo H, Ye X, Luo X, Chen Z, Qin C. Characterization of Kunitz-domain anticoagulation peptides derived from Acinetobacter baumannii exotoxin protein F6W77. Toxins, 2024, 450(10): 450.

[56] Vázquez-Iglesias L, Barcia-Castro L, Rodríguez-Quiroga M, De La Cadena MP, Rodríguez-Berrocal J, Cordero OJ. Surface expression marker profile in colon cancer cell lines and sphere-derived cells suggests complexity in CD26+ cancer stem cells subsets. Biol Open, 2019, 87): bio041673.

[57] Wan H, Lee KS, Kim BY, Zou FM, Yoon HJ, Je YH, Li J, Jin BR. A spider-derived Kunitz-type serine protease inhibitor that acts as a plasmin inhibitor and an elastase inhibitor. PLoS ONE, 2013, 8(1): e53343

[58] Wang L, Yang Y, Han W, Ding H. Novel design and development of Centella Asiatica extract—loaded poloxamer/ZnO nanocomposite wound closure material to improve anti-bacterial action and enhanced wound healing efficacy in diabetic foot ulcer. Regen Ther, 2024, 27: 92-103.

[59] Xu X, Lai R. The chemistry and biological activities of peptides from amphibian skin secretions. Chem Rev, 2015, 115(4): 1760-1846.

[60] Yin S, Wang Y, Yang X. Amphibian-derived wound healing peptides: chemical molecular treasure trove for skin wound treatment. Front Pharmacol, 2023, 14: 1120228

[61] Zhu MJ, Zhang GQ, Wang HX, Ng TB. Isolation and characterization of a Kunitz-type trypsin inhibitor with antiproliferative activity from Gymnocladus chinensis (Yunnan bean) seeds. Protein J, 2011, 30(4): 240-246.

Funding

SECIHTI (previously CONAHCyT)(Scholarship CVU: 1095127)

Tecnologico de Monterrey(Challenge-Based Research Funding Program 2022 (Project ID: IJXT070–22EG57001))

RIGHTS & PERMISSIONS

The Author(s)