{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T03:02:47Z","timestamp":1787022167613,"version":"build-2736575974"},"reference-count":124,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2025,4,29]],"date-time":"2025-04-29T00:00:00Z","timestamp":1745884800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Kommission f\u00fcr Finanzautonomie, Erg\u00e4nzungs- und Reservemittel (KOFFER) of the Ruhr-Universit\u00e4t Bochum, Medical Faculty","award":["26-04-2023"],"award-info":[{"award-number":["26-04-2023"]}]},{"name":"Funding by the Open-access Publikationsfonds, University library, Ruhr-University Bochum","award":["30-10-2024"],"award-info":[{"award-number":["30-10-2024"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    The prolonged persistence of extracellular chromatin and DNA is a salient feature of diseases like cystic fibrosis, systemic lupus erythematosus and COVID-19 associated microangiopathy. Since deoxyribonuclease I (DNase1) is a major endonuclease involved in DNA-related waste disposal, recombinant DNase1 is an important therapeutic biologic. Recently we described the production of recombinant murine DNase1 (rmDNase1) in\n                    <jats:italic>Pichia pastoris<\/jats:italic>\n                    by employing the \u03b1-mating factor prepro signal peptide (\u03b1MF-SP) a method, which we now applied to express recombinant human DNASE1 (rhDNASE1). In addition to an impaired cleavage of the \u03b1MF pro-peptide, which we also detected previously for mDNase1, expression of hDNASE1 resulted in a 70\u201380 times lower yield although both orthologues share a high structural and functional homology. Using mDNase1 expression as a guideline, we were able to increase the yield of hDNASE1 fourfold by optimizing parameters like nutrients, cultivation temperature, methanol supply, and codon usage. In addition, post-translational import into the rough endoplasmic reticulum (rER) was changed to co-translational import by employing the signal peptide (SP) of the \u03b1-subunit of the Oligosaccharyltransferase complex (Ost1) from\n                    <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n                    . These improvements resulted in the purification of ~\u20098\u2009mg pure mature rmDNase1 and ~\u20090.4\u2009mg rhDNASE1 per Liter expression medium of a culture with a cell density of OD\n                    <jats:sub>600<\/jats:sub>\n                    =\u2009 40 in 24 hours. As a main cause for the expression difference, we assume varying folding abilities to reach a native conformation, which induce an elevated unproductive unfolded protein response within the rER during hDNASE1 expression. Concerning functionality, rhDNASE1 expressed in\n                    <jats:italic>P. pastoris<\/jats:italic>\n                    is comparable to Pulmozyme\u00ae, i.e. rhDNASE1 produced in Chinese hamster ovary (CHO) cells by Roche - Genentech. With respect to the biochemical effectivity, rmDNase1 is superior to rhDNASE1 due to its higher specific activity in the presence of Ca\n                    <jats:sup>2\u2009+\u2009<\/jats:sup>\n                    \/Mg\n                    <jats:sup>2\u2009+<\/jats:sup>\n                    and the lower inhibition by monomeric actin.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0321094","type":"journal-article","created":{"date-parts":[[2025,4,29]],"date-time":"2025-04-29T13:54:47Z","timestamp":1745934887000},"page":"e0321094","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":3,"title":["Different results despite high homology: Comparative expression of human and murine DNase1 in Pichia pastoris"],"prefix":"10.1371","volume":"20","author":[{"given":"Jan-Ole","family":"Krischek","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hans Georg","family":"Mannherz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1300-3246","authenticated-orcid":true,"given":"Markus","family":"Napirei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,4,29]]},"reference":[{"issue":"6","key":"pone.0321094.ref001","doi-asserted-by":"crossref","first-page":"1873","DOI":"10.1002\/art.20267","article-title":"Chromatin breakdown during necrosis by serum Dnase1 and the plasminogen system","volume":"50","author":"M Napirei","year":"2004","journal-title":"Arthritis Rheum"},{"issue":"6367","key":"pone.0321094.ref002","doi-asserted-by":"crossref","first-page":"1202","DOI":"10.1126\/science.aam8897","article-title":"Host DNases prevent vascular occlusion by neutrophil extracellular traps","volume":"358","author":"M 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