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首页> 外文期刊>Biotechnology Progress >Production of Recombinant RNase Ba and Its Application in Downstream Processing of Plasmid DNA for Pharmaceutical Use
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Production of Recombinant RNase Ba and Its Application in Downstream Processing of Plasmid DNA for Pharmaceutical Use

机译：重组RNase Ba的制备及其在质粒DNA下游加工中的应用

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摘要

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.
机译：由于核酸在基因治疗和基因疫苗接种中作为活性药物成分（API）的重要性，对生物制药质粒DNA下游加工的新策略的需求增加了。在临床应用中，我们提供了通过使用重组RNase Ba（解淀粉芽孢杆菌的barnase）作为牛RNase A的替代品在色谱步骤之前去除RNA的机会。构建了用于RNase Ba产生的表达载体，可实现重组蛋白的周质定位在整个培养过程中，产生RNase的克隆的培养显示稳定的活性（在固定相期间为3.6 kU mL〜（-1））。纯化后，对RNase活性进行了测试，并与市售RNase A进行了比较。长时间与质粒DNA孵育后仍具有DNase活性。因此，它是b的合适替代品药物纯化过程中的绵羊RNaseA。

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