Laboratoire de Biotechnologie Cellulaire (CBUE), Ecole Polytechnique Fédérale de Lausanne, 1015, Ecublens, Switzerland.
Cytotechnology. 1999 Jul;30(1-3):159-68. doi: 10.1023/A:1008091200262.
The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown to be capable of separating plasmid DNA from typical impurities.
大规模制备质粒 DNA 是生物分离中的一个紧迫问题。本文首次研究了置换色谱法作为一种分离质粒 DNA（4.7kb）与大肠杆菌脂多糖和蛋白质（全转铁蛋白）的方法。在这方面，置换色谱法具有优势，因为物质混合物被分离成各个组分的矩形区域，而不是峰。因此，可以在合并的产物馏分中维持更高的总浓度。实验中包括羟磷灰石（I 型和 II 型）和阴离子交换固定相。除了填充有多孔颗粒的常规阴离子交换柱外，还研究了最近推出的连续床 UNOTM 阴离子交换柱。羟磷灰石材料均不能用于 DNA 纯化。一般来说，常规的基于颗粒的柱子不适合分离分子量差异很大的任何两种物质，例如质粒 DNA 和标准蛋白质。据推测，在置换色谱中至关重要的结合位点的直接竞争受到了这种情况下可及固定相表面积的尺寸依赖性的限制。连续床柱的结果更好，其中吸附表面与流经孔的壁重合。因此，与常规固定相材料相比，与相互作用分子的大小相关的可及表面变化不大。从 UNOTM 柱中回收的物质区之间也观察到更明显的过渡。在阴离子交换方法中，使用立体质量作用模型来辅助方法开发。虽然显然需要进一步研究，但已经表明连续床柱上的置换色谱能够分离质粒 DNA 与典型杂质。
