{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,28]],"date-time":"2025-10-28T05:24:09Z","timestamp":1761629049106,"version":"build-2065373602"},"reference-count":34,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,10,25]],"date-time":"2025-10-25T00:00:00Z","timestamp":1761350400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["NRF-2022R1F1A1064161","NRF-2021R1I1A3046499","RS-2023-00217471"],"award-info":[{"award-number":["NRF-2022R1F1A1064161","NRF-2021R1I1A3046499","RS-2023-00217471"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: Osteoporosis is caused by excessive osteoclast activation via the receptor activator nuclear factor kappa B ligand (RANKL), which is released from osteoblasts or osteocytes. RANKL regulates osteoclast activity by binding to the receptor activator of nuclear factor kappa B (RANK) in the canonical pathway or leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) in the non-canonical pathway. In this study, we attempted to develop an intact small-fragment protein based on RANKL by removing the RANK-binding site and transforming the amino acid residues at crucial sites to inhibit osteoclast activity and treat osteoporosis. Methods: We expressed a small-fragment variant of RANKL as a soluble glutathione S-transferase (GST) or 6x histidine (His)-tagged fusion protein using a GST- or His-binding domain tag expression vector system. To generate an intact form of small-fragment RANKL, ribosome-inactivating protein\u2013His-fusion RANKL was purified using HisTrap affinity chromatography and treated with tobacco etch virus nuclear inclusion endopeptidase to remove the His-tag fusion protein. Tartrate-resistant acid phosphatase (TRAP) and bone resorption pit formation assays were performed to analyze the inhibitory effects on osteoclast differentiation and activation. Results: The intact forms of 225RANKL295P and 225RANKL295A showed the strongest inhibitory effects on TRAP activity and bone resorption pit formation. Conclusions: Using an optimal construct design, a large and diverse range of small RANKL fragments could be generated. This suggests that the generation of small-fragment RANKL provides a promising avenue for the advancement of novel therapeutic approaches to osteoporosis.<\/jats:p>","DOI":"10.3390\/pharmaceutics17111385","type":"journal-article","created":{"date-parts":[[2025,10,27]],"date-time":"2025-10-27T02:50:48Z","timestamp":1761533448000},"page":"1385","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Generation and Purification of RANKL-Derived Small-Fragment Variants for Osteoclast Inhibition"],"prefix":"10.3390","volume":"17","author":[{"given":"Hyungjun","family":"Lee","sequence":"first","affiliation":[{"name":"Department of Premedical Science, College of Medicine, Chosun University, Gwangju 61452, Republic of Korea"},{"name":"Laboratory of Orthopedic Research, Chosun University Hospital, Gwangju 61452, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4781-7539","authenticated-orcid":false,"given":"Hyungseok","family":"Park","sequence":"additional","affiliation":[{"name":"Laboratory of Orthopedic Research, Chosun University Hospital, Gwangju 61452, Republic of Korea"},{"name":"Department of Orthopaedic Surgery, College of Medicine, Chosun University, Gwangju 61452, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8320-5030","authenticated-orcid":false,"given":"Kabsun","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Premedical Science, College of Medicine, Chosun University, Gwangju 61452, Republic of Korea"},{"name":"Laboratory of Orthopedic Research, Chosun University Hospital, Gwangju 61452, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Youngjong","family":"Ko","sequence":"additional","affiliation":[{"name":"Department of Premedical Science, College of Medicine, Chosun University, Gwangju 61452, Republic of Korea"},{"name":"Laboratory of Orthopedic Research, Chosun University Hospital, Gwangju 61452, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9119-6154","authenticated-orcid":false,"given":"Chang-Moon","family":"Lee","sequence":"additional","affiliation":[{"name":"Regional Leading Research Center, Chonnam National University, Yeosu 59626, Republic of Korea"},{"name":"School of Healthcare and Biomedical Engineering, Chonnam National University, Yeosu 59626, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6337-6599","authenticated-orcid":false,"given":"Wonbong","family":"Lim","sequence":"additional","affiliation":[{"name":"Department of Premedical Science, College of Medicine, Chosun University, Gwangju 61452, Republic of Korea"},{"name":"Laboratory of Orthopedic Research, Chosun University Hospital, Gwangju 61452, Republic of Korea"},{"name":"Department of Orthopaedic Surgery, College of Medicine, Chosun University, Gwangju 61452, Republic of Korea"},{"name":"Regional Leading Research Center, Chonnam National University, Yeosu 59626, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1186\/s41232-019-0111-3","article-title":"RANKL biology: Bone metabolism, the immune system, and beyond","volume":"40","author":"Ono","year":"2020","journal-title":"Inflamm. 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