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.

中文翻译：

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空肠弯曲杆菌铁摄取调节剂的纯化和鉴定。

铁摄取调节剂（Fur）是细菌中发现的转录因子的超家族，可控制多种基因的表达。在这项研究中，我们报告了一个简单的协议，用于纯化未标记的空肠弯曲菌毛皮（CjFur）。CjFur的分离是通过三个离子交换色谱步骤的结合，然后在Superdex 75上进行尺寸排阻色谱法进行的。ESI-MS分析表明，我们的方法可产生纯净的CjFur，并且这种无标签版本比带有标签的重组CjFur更有效地掺入金属或标记残留物。最后，电泳迁移率变动分析表明，这种新的纯化方法可产生CjFur制剂，该制剂可更有效地结合DNA。这些结果表明，在CjFur上添加一个N末端标签对它的活性是有害的。总体，