Voss Carsten, Lindau Dennis, Flaschel Erwin
Chair of Fermentation Engineering, Faculty of Technology, Bielefeld University, PO 100 131, D-33501 Bielefeld, Germany.
Biotechnol Prog. 2006 May-Jun;22(3):737-44. doi: 10.1021/bp050417e.
The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.
由于核酸作为基因治疗和基因疫苗中的活性药物成分（API）的重要性，对生物制药质粒DNA下游加工新策略的需求有所增加。受临床应用试剂生产中动物源蛋白使用问题的影响，我们提出了一种机会，即在色谱步骤之前使用重组核糖核酸酶Ba（解淀粉芽孢杆菌的芽孢杆菌核糖核酸酶）替代牛核糖核酸酶A来去除RNA。构建了一个用于生产核糖核酸酶Ba的表达载体，使重组蛋白定位于周质。产核糖核酸酶克隆的培养在整个培养过程中显示出稳定的活性（稳定期为3.6 kU mL(-1)）。纯化后测试了核糖核酸酶活性，并与市售核糖核酸酶A进行了比较。即使与质粒DNA长时间孵育，核糖核酸酶Ba也没有显示出脱氧核糖核酸酶活性。因此，它是药物纯化过程中牛核糖核酸酶A的合适替代品。
