Vlatakis G, Bouriotis V
Enzyme Technology Division, Institute of Molecular Biology and Biotechnology, Crete, Greece.
J Chromatogr. 1991 Feb 1;538(2):311-21. doi: 10.1016/s0021-9673(01)88852-x.
Partitioning of restriction endonucleases between two liquid aqueous phases can be strongly influenced by group-specific ligands included in the two-phase system. Three restriction endonucleases, namely EcoR I, EcoR V and BamH I, were partitioned within an aqueous dextran-polyethylene glycol (PEG) system. The enzymes could be extracted into the upper PEG phase by using either triazine dyes or herring DNA as affinity ligands. The influence of the endogenous bacterial nucleic acids, concentration of polymerbound dye and concentration of sodium chloride on the system were examined. A partial purification of EcoR I (up to 52-fold) and EcoR V (up to 37-fold) was achieved using a combination of affinity partitioning and ion-exchange chromatography, providing an extremely fast and economical method for the isolation of restriction endonucleases free from contaminating nuclease activities.
两相体系中特定基团配体可强烈影响限制性内切酶在两个液相水相之间的分配。三种限制性内切酶，即EcoR I、EcoR V和BamH I，在葡聚糖-聚乙二醇（PEG）水相体系中进行分配。通过使用三嗪染料或鲱鱼DNA作为亲和配体，这些酶可以被萃取到上层PEG相中。研究了内源性细菌核酸、聚合物结合染料浓度和氯化钠浓度对该体系的影响。结合亲和分配和离子交换色谱法，实现了EcoR I（高达52倍）和EcoR V（高达37倍）的部分纯化，为分离无核酸酶污染活性的限制性内切酶提供了一种极其快速且经济的方法。
