Vlatakis G, Skarpelis G, Stratidaki I, Bouriotis V, Clonis Y D
Research Center of Crete, Institute of Molecular Biology and Biotechnology, Iraklio, Greece.
Appl Biochem Biotechnol. 1987 Oct;15(3):201-12. doi: 10.1007/BF02798449.
The resolution of restriction endonucleases from the same microorganism is conventionally achieved by lengthy fractionation protocols. We now report effective single-step procedures that exploit dye-ligand chromatography for the resolution and purification of restriction enzymes. After suitable initial screening, we demonstrated that resolution of two restriction activities can be achieved in one chromatographic step, and further purification can subsequently be effected using selected dye-adsorbents. Accordingly, we resolved in one step, Hpa I from Hpa II, Hind II from Hind III, and Sac I from Sac II. Furthermore, a three-step chromatographic procedure has been developed to purify EcoRV suitable for commercial exploitation, as judged by the "overdigestion" and "cut-ligate-recut" quality control tests.
传统上，从同一微生物中分离限制性内切酶需要通过冗长的分级分离方案来实现。我们现在报告了一些有效的单步程序，这些程序利用染料配体色谱法来分离和纯化限制性酶。经过适当的初步筛选，我们证明了在一个色谱步骤中可以实现两种限制性活性的分离，随后可以使用选定的染料吸附剂进行进一步纯化。因此，我们一步就分离出了Hpa II中的Hpa I、Hind III中的Hind II和Sac II中的Sac I。此外，还开发了一种三步色谱程序来纯化适用于商业用途的EcoRV，通过“过度消化”和“切割-连接-再切割”质量控制测试可以判断其适用性。
