Purification of plasmid DNA with aqueous two phase systems of PEG 600 and sodium citrate/ammonium sulfate
Purification of plasmid DNA with aqueous two phase systems of PEG 600 and sodium citrate/ammonium sulfate
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DOI：
10.1016/j.seppur.2008.01.026
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发表时间：
2009-02-02
影响因子：
8.6
通讯作者：
Prazeres, D. Miguel F.
中科院分区：
文献类型：
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作者：
Gomes, Gabriela A.;Azevedo, Ana M.;Prazeres, D. Miguel F.
采用以聚乙二醇（PEG）600、柠檬酸钠和硫酸铵为基础的聚合物/盐双水相系统（ATPS）从大肠杆菌碱性裂解液中部分纯化质粒DNA（pDNA）。分析了pH和裂解物上样量对双结点曲线的影响，并确定了连接线，以建立ATPS形成的最佳条件。使用20%（w/w）裂解物上样量和含16.5%（w/w）盐、19.0%（w/w）PEG 600和37.1%（w/w）连接线长度的系统，在pH 6.9下进行了一系列浸提实验。在这些条件下，在富盐底相中回收质粒DNA。然而，尽管使用基于柠檬酸钠的系统获得了接近100%的回收率，但使用硫酸铵能够实现更高的纯化，尽管回收率较低。因此，还评价了通过组合柠檬酸钠和硫酸铵制备的ATPS的性能。25%（w/w）硫酸铵和75%（w/w）柠檬酸钠的混合物提供了质粒回收率（91.1%）和纯度（17.2%）之间的良好折衷。为了提高工艺性能，对多步提取程序进行了评价。虽然在第一步中除去了大部分杂质，但通过加入额外的步骤，纯度得到了逐步提高。为了增加底相中的质粒浓度，增加了顶相与底相的体积比。虽然这是使用4的相比率实现的，但是相对于起始裂解物不可能浓缩质粒。总之，结果表明，硫酸铵，一种具有高环境影响的盐，可以在ATPS中被柠檬酸钠部分替代，而不会显著降低质粒纯化的性能。(C)2008 Elsevier B. V.保留所有权利。
Polymer/salt aqueous two phase systems (ATPS) based on polyethylene glycol (PEG) 600, sodium citrate and ammonium sulfate were used to partially purify plasmid DNA (pDNA) from Escherichia coli alkaline lysates. The effect of pH and lysate load on the binodal curve was analyzed and tie-lines were determined in order to establish the optimal conditions for ATPS formation. A series of extraction experiments were performed at pH 6.9 using a 20% (w/w) lysate load and systems with 16.5% (w/w) salt, 19.0% (w/w) PEG 600, and a tie-line length of 37.1% (w/w). Under these conditions, plasmid DNA was recovered in the salt-rich bottom phase. However, whereas with sodium citrate-based systems recovery yields closer to 100% were obtained, the use of ammonium sulfate enabled higher purification although with lower recoveries. Thus, the performance of ATPS prepared by combining sodium citrate and ammonium sulfate was also evaluated. A mixture of 25% (w/w) ammonium sulfate and 75% (w/w) sodium citrate offered a good compromise between plasmid recovery (91.1%) and purity (17.2%). Multi-step extraction procedures were evaluated in order to improve the process performance. Although the majority of the impurities were removed in the first step, incremental increases in the purity were obtained with the inclusion of extra steps. The top to bottom phase volume ratio was increased in order to increase plasmid concentration in the bottom phase. Although this was achieved using a phase ratio of 4, it was not possible to concentrate plasmid relatively to the starting lysate. Overall, the results show that ammonium sulfate, a salt which has a high environmental impact, can be partially replaced in ATPS by sodium citrate, without significant decrease in the performance of plasmid purification. (C) 2008 Elsevier B.V. All rights reserved.