Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Road, Roger Guindon Hall, Ottawa, ON, K1H 8M5, Canada.
Biometals. 2019 Jun;32(3):491-500. doi: 10.1007/s10534-019-00177-5. Epub 2019 Jan 31.
The ferric uptake regulator (Fur) is a superfamily of transcription factors found in bacteria which control the expression of a myriad of genes. In this study, we report a simple protocol for the purification of recombinant untagged Campylobacter jejuni Fur (CjFur). CjFur was isolated using a combination of three ion exchange chromatography steps followed by size exclusion chromatography on a Superdex 75. ESI-MS analysis shows that our method yields pure CjFur and that this tag-free version incorporates metal more efficiently than recombinant CjFur harboring a tag or tag remnants. Finally, electrophoretic mobility shift assays show that this new purification method yields a CjFur preparation that binds DNA more efficiently. These results suggest that adding a N-terminus tag onto CjFur is detrimental to its activity. Overall, the approaches detailed in this study offer an alternative strategy for the purification of CjFur, and likely other metalloregulators, for future biochemical and biophysical studies.
铁摄取调节因子（Fur）是一类在细菌中发现的转录因子超家族，它控制着众多基因的表达。在本研究中，我们报告了一种简单的方案，用于纯化重组无标签空肠弯曲菌 Fur（CjFur）。CjFur 是通过三种离子交换色谱步骤的组合分离出来的，然后在 Superdex 75 上进行大小排阻色谱。ESI-MS 分析表明，我们的方法得到了纯的 CjFur，并且这种无标签版本比含有标签或标签残基的重组 CjFur 更有效地结合金属。最后，电泳迁移率变动分析表明，这种新的纯化方法得到了一种更有效地结合 DNA 的 CjFur 制剂。这些结果表明，在 CjFur 上添加 N 端标签对其活性有害。总的来说，本研究中详述的方法为未来的生化和生物物理研究提供了一种替代的 CjFur 及其它金属调节因子的纯化策略。
