Krischek Jan-Ole, Mannherz Hans Georg, Napirei Markus
Department of Anatomy and Molecular Embryology, Medical Faculty, Ruhr-University Bochum, Bochum, Germany.
Department of Cellular and Translational Physiology, Medical Faculty, Ruhr-University Bochum, Bochum, Germany.
PLoS One. 2025 Apr 29;20(4):e0321094. doi: 10.1371/journal.pone.0321094. eCollection 2025.
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 Pichia pastoris by employing the α-mating factor prepro signal peptide (αMF-SP) a method, which we now applied to express recombinant human DNASE1 (rhDNASE1). In addition to an impaired cleavage of the αMF pro-peptide, which we also detected previously for mDNase1, expression of hDNASE1 resulted in a 70-80 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 α-subunit of the Oligosaccharyltransferase complex (Ost1) from Saccharomyces cerevisiae. These improvements resulted in the purification of ~ 8 mg pure mature rmDNase1 and ~ 0.4 mg rhDNASE1 per Liter expression medium of a culture with a cell density of OD600 = 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 P. pastoris is comparable to Pulmozyme®, 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 Ca2 + /Mg2 + and the lower inhibition by monomeric actin.
细胞外染色质和DNA的长期存在是囊性纤维化、系统性红斑狼疮和新冠病毒相关微血管病等疾病的一个显著特征。由于脱氧核糖核酸酶I（DNase1）是参与DNA相关废物处理的主要内切酶，重组DNase1是一种重要的治疗性生物制品。最近，我们描述了通过使用α-交配因子前体信号肽（αMF-SP）在毕赤酵母中生产重组鼠DNase1（rmDNase1）的方法，我们现在将该方法应用于表达重组人DNASE1（rhDNASE1）。除了αMF前肽的切割受损（我们之前在mDNase1中也检测到这种情况）外，hDNASE1的表达导致产量降低了70 - 80倍，尽管这两种直系同源物具有高度的结构和功能同源性。以mDNase1的表达为指导，我们通过优化营养物质、培养温度、甲醇供应和密码子使用等参数，使hDNASE1的产量提高了四倍。此外，通过使用来自酿酒酵母的寡糖基转移酶复合物（Ost1）α亚基的信号肽（SP），将翻译后导入粗面内质网（rER）改为共翻译导入。这些改进使得在细胞密度为OD600 = 40的培养物中，每升表达培养基在24小时内可纯化得到约8毫克纯成熟rmDNase1和约0.4毫克rhDNASE1。作为表达差异的主要原因，我们认为折叠能力不同以达到天然构象，这在hDNASE1表达过程中会在rER内引发升高的非生产性未折叠蛋白反应。在功能方面，在毕赤酵母中表达的rhDNASE1与Pulmozyme®相当，即罗氏 - 基因泰克公司在中国仓鼠卵巢（CHO）细胞中生产的rhDNASE1。在生化有效性方面，rmDNase1由于在Ca2 + /Mg2 +存在下具有更高的比活性且受单体肌动蛋白的抑制较低，优于rhDNASE1。
