Abstract
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.
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Acknowledgements
This project was funded by a grant from the Canadian Institutes of Health Research to Alain Stintzi and Jean-Francois Couture. Dr. Couture acknowledges grants from Canada Foundation for Innovation and a Canada Research Chair in Structural biology and Epigenetics. The authors thank Dr. James Butcher for helpful comments on the manuscript.
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Sarvan, S., Yeung, A., Charih, F. et al. Purification and characterization of Campylobacter jejuni ferric uptake regulator. Biometals 32, 491–500 (2019). https://doi.org/10.1007/s10534-019-00177-5
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DOI: https://doi.org/10.1007/s10534-019-00177-5