Sousa A, Almeida A M, Černigoj U, Sousa F, Queiroz J A
CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
J Chromatogr A. 2014 Aug 15;1355:125-33. doi: 10.1016/j.chroma.2014.06.003. Epub 2014 Jun 6.
Preparation of high quantities of supercoiled plasmid DNA of pharmaceutical grade purity is a research area where intensive investigation is being performed. From this standpoint, several downstream methods have been proposed, among them the monolithic chromatographic strategies owing to excellent mass transfer properties of monolithic supports and their high binding capacity for large biomolecules. The present study explores the physicochemical properties of histamine ligand in a supercoiled plasmid DNA purification process from an Escherichia coli clarified lysate, where the emphasis is given to the elution strategy that allows higher selectivity and efficient removal of other impurities besides the open circular isoform. The combination of high NaCl concentration and acidic pH allowed the elimination of 89% of RNA during the preparative loading of the lysate sample. The results of the purification strategy with ascending sodium chloride gradient revealed that 97% of supercoiled plasmid DNA was recovered with a purity degree of 99%. In addition, using a combined purification strategy with ascending sodium chloride (capture step) and then descending ammonium sulfate (polishing step) gradient, it was achieved a lower supercoiled plasmid DNA recovery yield of 79% with a purity degree of 92%, although the dynamic binding capacity under these conditions was higher than in the previous strategy. A significant reduction of host contents, such as proteins, RNA and genomic DNA, was obtained in both purification strategies. Accordingly, histamine is a useful and versatile ligand that allows the desirable supercoiled plasmid purification with high yield and purity level.
制备大量具有药用级纯度的超螺旋质粒DNA是一个正在进行深入研究的领域。从这个角度来看，已经提出了几种下游方法，其中整体色谱策略因其整体载体优异的传质性能及其对大生物分子的高结合能力而备受关注。本研究从大肠杆菌澄清裂解物中探索了超螺旋质粒DNA纯化过程中组胺配体的物理化学性质，重点是洗脱策略，该策略除了能有效去除开环异构体之外，还能实现更高的选择性和有效去除其他杂质。高NaCl浓度和酸性pH的组合使得在裂解物样品的制备加载过程中能够去除89%的RNA。氯化钠梯度上升的纯化策略结果表明，97%的超螺旋质粒DNA被回收，纯度为99%。此外，使用氯化钠梯度上升（捕获步骤）然后硫酸铵梯度下降（精制步骤）的组合纯化策略，虽然在此条件下的动态结合能力高于前一种策略，但超螺旋质粒DNA的回收率较低，为79%，纯度为92%。在两种纯化策略中均显著降低了宿主成分，如蛋白质、RNA和基因组DNA的含量。因此，组胺是一种有用且通用的配体，能够以高产量和纯度水平实现理想的超螺旋质粒纯化。
