PlasmidFactory GmbH & Co. KG, Meisenstrasse 96, 33607, Bielefeld, Germany.
Fermentation Engineering, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany.
Microb Cell Fact. 2021 Aug 28;20(1):171. doi: 10.1186/s12934-021-01642-y.
The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar.
The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production.
The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.
牛源核糖核酸酶的使用是质粒 DNA 纯化标准方案的一部分。随着基因治疗领域现在进入临床阶段，需要逐步淘汰此类酶或开发替代的纯化方案，以确保产品安全和符合法规要求。重组表达细菌核糖核酸酶存在毒性问题，使其成为一种具有挑战性的酶表达。本研究描述了一种质粒构建体，允许 barnase 在大肠杆菌中表达，同时表达其天然抑制剂 barstar。
没有抑制剂 barstar 的纯酶通过周质被运送到细胞外空间，然后从无细胞上清液中纯化。离子交换色谱法被用作主要的纯化步骤。然后进行疏水性相互作用色谱法，得到活性酶的浓缩部分。尽管目前达到的比体积活性水平相当低（4 个 Kunitz 单位/mL），但原则上可以证明其在质粒 DNA 纯化中的应用。目前，它能够处理用于质粒生产的少量（13g）细菌生物质。
目前的工作集中在重组核糖核酸酶的下游纯化策略上，如果通过优化宿主和/或重新设计质粒来提高特定生产力，则为大规模生产奠定了框架。同样重要的是要减少阳离子交换色谱纯化过程中大量酶的损失。即使应用相对少量的重组核糖核酸酶，也有助于大大降低碱性裂解物中的初始 RNA 水平，从而增强进一步的下游质粒纯化步骤。
