IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisboa, Portugal.
J Chromatogr A. 2011 Dec 2;1218(48):8629-37. doi: 10.1016/j.chroma.2011.10.004. Epub 2011 Oct 6.
Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl(2) solutions since it maximised pDNA yield (96.2±4.9%) and protein removal (61.3±3.0%), while providing for a substantial removal of RNA (65.5±3.5%) and gDNA (44.7±14.1%). Although the use of pH 3.5 maximised removal of impurities (75%), the best compromise between plasmid yield (96%) and RNA clearance (~60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3' termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.
质粒 DNA（pDNA）可直接从碱裂解衍生的大肠杆菌（E. coli）裂解物中通过苯硼酸（PB）层析进行纯化。该方法利用 PB 配体与 RNA 和脂多糖（LPS）等含顺式二醇的杂质共价结合，但可逆转的特性，使 pDNA 留在溶液中。尽管具有这种特异性，但也会去除不含顺式二醇的物质，如蛋白质和基因组 DNA（gDNA）。这是一个主要的优势，因为该过程旨在防止目标 pDNA 结合。本文的重点是研究含有 pDNA 的裂解物中杂质（RNA、gDNA、蛋白质、LPS）与 3-氨基 PB 控制孔玻璃（CPG）基质之间的次级相互作用。设计运行以评估吸附缓冲液组成、进料类型（pH 值、盐含量）、CPG 基质和样品预处理（RNase A、异丙醇沉淀）的作用。选择水作为吸附缓冲液而不是 MgCl2 溶液，因为它可以使 pDNA 产量最大化（96.2±4.9%）并去除蛋白质（61.3±3.0%），同时提供大量去除 RNA（65.5±3.5%）和 gDNA（44.7±14.1%）。尽管使用 pH 值 3.5 可以最大程度地去除杂质（75%），但在质粒产量（96%）和 RNA 清除率（~60-70%）之间的最佳折衷是 pH 值 5.2。当进入裂解物进料中的醋酸盐和钾离子浓度分别为 1.7 M 和 1.0 M 时，质粒产量达到最大值（>96%）。用 RNase A 预处理裂解物会降低性能，因为由此产生的寡核糖核苷酸在其 3'末端缺乏顺式二醇基团。总的来说，这些结果支持这样一种观点，即在酸性 pH 值下硼原子与核酸的芳香碱基和蛋白质的侧残基中的电子供体基团之间的电荷转移相互作用负责非特异性去除 gDNA、RNA 和蛋白质。
