{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,14]],"date-time":"2025-11-14T13:07:17Z","timestamp":1763125637073,"version":"3.45.0"},"reference-count":41,"publisher":"Wiley","issue":"11","license":[{"start":{"date-parts":[[2025,10,28]],"date-time":"2025-10-28T00:00:00Z","timestamp":1761609600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002784","name":"Canada Excellence Research Chairs, Government of Canada","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100002784","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000196","name":"Canada Foundation for Innovation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000196","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2025,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Following the SARS\u2010CoV\u20102 pandemic, many direct\u2010acting antivirals targeting viral cysteine protease were developed. SARS\u2010CoV\u20102, as well as other viruses, rely on cysteine proteases for their replication, suggesting future generations of antivirals targeting cysteine proteases will emerge. A major concern for these first\u2010generation drugs is the off\u2010target effects on host cysteine proteases. Therefore, screening for inhibitor specificity is a crucial step in antiviral drug development. Cathepsins are one of the most abundant human proteases, which have roles in maintaining cell health and are key to many physiological processes. Here we describe a general expression and purification protocol for cathepsins B, S, and L using the Expi293\u2122 mammalian expression system. We characterized the glycosylation pattern and kinetic parameters of purified enzymes with commercially available cathepsin\u2010specific fluorogenic substrates and also established cathepsin inhibition screen assays. We tested specificity indices of peptidic inhibitors of SARS\u2010CoV\u20102 M\n                    <jats:sup>pro<\/jats:sup>\n                    synthesized by our team and nirmatrelvir as a benchmark for all three cathepsins. Establishing a reliable cathepsin inhibition assay would assist with screening of newly developed cysteine protease inhibitors for off\u2010target activity within the scope of pandemic preparedness.\n                  <\/jats:p>","DOI":"10.1002\/pro.70361","type":"journal-article","created":{"date-parts":[[2025,10,28]],"date-time":"2025-10-28T19:47:22Z","timestamp":1761680842000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Production of human cathepsins using\n                    <scp>Expi293\u2122<\/scp>\n                    mammalian cell expression system for off\u2010target activity of cysteine protease inhibitor screening"],"prefix":"10.1002","volume":"34","author":[{"given":"Zoe","family":"Turner","sequence":"first","affiliation":[{"name":"Department of Biochemistry University of Alberta  Edmonton Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4119-3622","authenticated-orcid":false,"given":"Elena","family":"Arutyunova","sequence":"additional","affiliation":[{"name":"Department of Biochemistry University of Alberta  Edmonton Alberta Canada"},{"name":"Li Ka Shing Institute of Virology University of Alberta  Edmonton Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9274-0759","authenticated-orcid":false,"given":"Duong T.","family":"Bui","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Alberta  Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3088-0374","authenticated-orcid":false,"given":"Ling","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Alberta  Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Appan Srinivas","family":"Kandadai","sequence":"additional","affiliation":[{"name":"Li Ka Shing Applied Virology Institute University of Alberta  Edmonton Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bing","family":"Bai","sequence":"additional","affiliation":[{"name":"Li Ka Shing Applied Virology Institute University of Alberta  Edmonton Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Howard S.","family":"Young","sequence":"additional","affiliation":[{"name":"Department of Biochemistry University of Alberta  Edmonton Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4791-8524","authenticated-orcid":false,"given":"Lara K.","family":"Mahal","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Alberta  Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3389-7112","authenticated-orcid":false,"given":"John S.","family":"Klassen","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Alberta  Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M. Joanne","family":"Lemieux","sequence":"additional","affiliation":[{"name":"Department of Biochemistry University of Alberta  Edmonton Alberta Canada"},{"name":"Li Ka Shing Institute of Virology University of Alberta  Edmonton Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,10,28]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsbiomedchemau.3c00039"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1039\/D1MD00247C"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.1c00616"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1046-5928(02)00646-0"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbamcr.2008.10.016"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biochi.2007.07.024"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1046-2023(03)00212-3"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.1c04779"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/ed040pA386.1"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1996.tb00934.x"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms17121969"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M508381200"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/0014-5793(94)00924-4"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-021-00418-x"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2019.02037"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1432-1033.1996.00558.x"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pharmthera.2020.107587"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)99979-5"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1042\/bj2820577"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.0c00875"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1515\/bchm.1998.379.2.219"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1006\/bbrc.1996.5905"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.abl4784"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biopha.2018.05.148"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1039\/D1SC00785H"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1742-4658.2008.06815.x"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.57.070188.004033"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI42918"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1358\/dnp.2005.18.10.949485"},{"key":"e_1_2_9_31_1","first-page":"99","volume-title":"Essentials 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