{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T22:01:22Z","timestamp":1780696882048,"version":"3.54.1"},"reference-count":130,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2025,7,18]],"date-time":"2025-07-18T00:00:00Z","timestamp":1752796800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"state of S\u00e3o Paulo Research Foundation and Universidad de La Frontera, FAPESP-UFRO","award":["2020\/06982-3"],"award-info":[{"award-number":["2020\/06982-3"]}]},{"name":"state of S\u00e3o Paulo Research Foundation and Universidad de La Frontera, FAPESP-UFRO","award":["21231090"],"award-info":[{"award-number":["21231090"]}]},{"name":"ANID Scholarship File","award":["2020\/06982-3"],"award-info":[{"award-number":["2020\/06982-3"]}]},{"name":"ANID Scholarship File","award":["21231090"],"award-info":[{"award-number":["21231090"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Acute lymphoblastic leukemia (ALL) is the most common cancer affecting children, making up about 80% of all acute leukemia cases in the pediatric population. While treatment with L-asparaginase (ASNase) has greatly improved survival rates, its bacterial origin often causes immune reactions in some patients, which can reduce how well the therapy works. To overcome this challenge, previous in silico studies designed a humanized chimeric ASNase by swapping out the predicted immunogenic parts of the bacterial enzyme with similar, less immunogenic segments from the human version\u2014while keeping the enzyme\u2019s active site intact. In this study, the chimeric L-asparaginase designed was successfully cloned, expressed, and purified using the Escherichia coli Rosetta strain. The production conditions (37 \u00b0C, 0.01 mM IPTG, 2\u20134 h) were optimized, and we purified the enzyme in a single step with nickel-affinity chromatography. The enzyme\u2019s activity was confirmed in vitro, showing that it is possible to produce a functional humanized variant in a bacterial system. These results lay important groundwork for future research to assess the immune response and therapeutic potential of this novel chimeric enzyme.<\/jats:p>","DOI":"10.3390\/ijms26146919","type":"journal-article","created":{"date-parts":[[2025,7,18]],"date-time":"2025-07-18T13:33:27Z","timestamp":1752845607000},"page":"6919","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Toward Safer Biotherapeutics: Expression and Characterization of a Humanized Chimeric L-Asparaginase in E. coli"],"prefix":"10.3390","volume":"26","author":[{"given":"Alejandro","family":"Pedroso","sequence":"first","affiliation":[{"name":"Department of Chemical Engineering, Faculty of Engineering and Science, Universidad de La Frontera, Temuco 4811230, Chile"},{"name":"Departamento de Ciencias B\u00e1sicas, Facultad de Ciencias, Universidad Santo Tomas, Temuco 4780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Javiera","family":"Miranda","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Faculty of Engineering and Science, Universidad de La Frontera, Temuco 4811230, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-8901-0358","authenticated-orcid":false,"given":"Nicol\u00e1s","family":"Lefin","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Faculty of Engineering and Science, Universidad de La Frontera, Temuco 4811230, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2339-2049","authenticated-orcid":false,"given":"Brian","family":"Effer","sequence":"additional","affiliation":[{"name":"Center of Excellence in Translational Medicine (CEMT) and Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco 4811230, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Enrique Pedroso","family":"Reyanldo","sequence":"additional","affiliation":[{"name":"Comit\u00e9 Cient\u00edfico Araucania Sur, Colegio M\u00e9dico de Chile, Temuco 4780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1467-5178","authenticated-orcid":false,"given":"Yolanda","family":"Calle","sequence":"additional","affiliation":[{"name":"School of Life and Health Sciences, University of Roehampton, London SW15 4JD, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3385-047X","authenticated-orcid":false,"given":"Gisele","family":"Monteiro","sequence":"additional","affiliation":[{"name":"Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of S\u00e3o Paulo, S\u00e3o Paulo 05508-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5268-8690","authenticated-orcid":false,"suffix":"Jr.","given":"Adalberto","family":"Pessoa","sequence":"additional","affiliation":[{"name":"Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of S\u00e3o Paulo, S\u00e3o Paulo 05508-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7660-9603","authenticated-orcid":false,"given":"Jorge G.","family":"Farias","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Faculty of Engineering and Science, Universidad de La Frontera, Temuco 4811230, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,18]]},"reference":[{"key":"ref_1","unstructured":"SEPEAP (2025, June 03). 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