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It is also an entry point for various pathogens, such as South American hemorrhagic fever caused by arenaviruses and the malaria parasite\n                    <jats:italic>Plasmodium vivax<\/jats:italic>\n                    , which utilize TfR to gain access to cells. The receptor is additionally upregulated in many aggressive cancers and at the blood\u2013brain barrier. Altogether, the TfR is a highly relevant target for many medical applications, and novel protein\u2010interacting partners are sought after. A protein design strategy was explored here to develop a small protein that can be used for drug delivery across cell membranes, to investigate blood\u2013brain barrier crossing, study endocytosis, or to block pathogen access to the apical domain. A computationally docked library of small protein scaffolds to the TfR apical domain, the native binding site of the\n                    <jats:italic>Machupo<\/jats:italic>\n                    arenavirus, was a starting point for the design and optimization. The best variants were expressed in a yeast surface display system and assessed for interaction with TfR by flow cytometry. One protein variant, which initially showed a low binding signal, was further optimized by directed evolution to bind to the target receptor at nanomolar concentration. The evolved construct, tagged with the enhanced green fluorescent protein (eGFP) and bacterially expressed, showed uptake similar\u00a0to that of FITC\u2010coupled transferrin\u00a0in a cell\u2010based assay. The designed protein can be utilized as a tool to target cell entry via TfR for drug delivery applications or as a foundation for developing antiviral therapeutics against arenaviruses.\n                  <\/jats:p>","DOI":"10.1002\/pro.70384","type":"journal-article","created":{"date-parts":[[2025,11,19]],"date-time":"2025-11-19T04:30:36Z","timestamp":1763526636000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Targeting the apical domain of the transferrin receptor: Development of a new protein scaffold for cellular delivery"],"prefix":"10.1002","volume":"34","author":[{"given":"Anuthariq Alikkam","family":"Veetil","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biomedical Sciences Linnaeus University  Kalmar Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dick J.","family":"Sj\u00f6str\u00f6m","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biomedical Sciences Linnaeus University  Kalmar Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cristian","family":"Iribarren","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biomedical Sciences Linnaeus University  Kalmar Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Camilla","family":"Mohlin","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biomedical Sciences Linnaeus University  Kalmar Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena","family":"Ambrosetti","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry and Biophysics Karolinska Institutet  Stockholm Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9300-614X","authenticated-orcid":false,"given":"Sinisa","family":"Bjelic","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biomedical Sciences Linnaeus University  Kalmar Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,11,18]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.1772"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.physiol.69.031905.164337"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/gzq002"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/47417"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2006.94"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0022477"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-13924-6"},{"key":"e_1_2_8_9_1","doi-asserted-by":"crossref","unstructured":"CuffM EvdokimovaE KudritskaM EdwardsA JoachimiakA SavchenkoA.The structure of uncharacterized protein TM1266 from Thermotoga maritima. 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