Dual affinity method for plasmid DNA purification in aqueous two-phase systems
Dual affinity method for plasmid DNA purification in aqueous two-phase systems
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DOI：
10.1016/j.chroma.2009.12.059
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发表时间：
2010-02-26
影响因子：
4.1
通讯作者：
Marcos, J. C.
中科院分区：
文献类型：
--
作者：
Barbosa, H. S. C.;Hine, A. V.;Marcos, J. C.
DNA 结合融合蛋白 Lacl-His(6)-GFP 与缀合物 PEG-IDA-Cu(II) (10 kDa) 一起作为双亲和系统进行评估，用于从聚乙二醇 (PEG)/葡聚糖 (DEX) 水性两相系统 (ATPS) 中的碱性细菌细胞裂解液中提取 pUC19 质粒。在含有 0.273 nmol Lacl 融合蛋白和 0.14% (w/w) 功能化 PEG-IDA-Cu(II) 的 PEG 600-DEX 40 ATPS 中，超过 72% 的质粒 DNA 分配到 PEG 相，通过琼脂糖凝胶电泳评估，没有 RNA 或基因组 DNA 污染。在第二个提取阶段，使用葡聚糖或磷酸盐缓冲液作为第二相，从 Lacl 结合复合物中洗脱 pDNA 被证明是困难的，尽管在两个系统中都去除了超过 75% 的总蛋白质。使用 PEG-葡聚糖系统作为第二提取系统，pCU19 质粒的最大回收率达到约 27%，其中 80-90% 的 pDNA 分配到底部相。这代表每 1 mL pUC19 脱盐裂解液提取约 7.4 μg pDNA。 (C) 2009 Elsevier B.V. 保留所有权利。
The DNA binding fusion protein, Lacl-His(6)-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of Lacl fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the Lacl binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 mu g of pDNA extracted per 1 mL of pUC19 desalted lysate. (C) 2009 Elsevier B.V. All rights reserved.