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Expression, purification, and characterization of iron-sulfur cluster assembly regulator IscR from Acidithiobacillus
Jia Zeng1, Ke Zhang, Jianshe Liu
1Department of Bioengineering, School of Resources Processing and Bioengineering, Central South University, Changsha 410083, P R China.
Abstract:
IscR (iron-sulfur cluster regulator) has been reported to be a repressor of the iscRSUA operon, and in vitro transcription reactions have revealed that IscR has a repressive effect on the iscR promoter in the case of [Fe(2)S(2)] cluster loading. In the present study, the iscR gene from A. ferrooxidans ATCC 23270 was cloned and successfully expressed in Escherichia coli, and then purified by one-step affinity chromatography to homogeneity. The molecular mass of the IscR was 18 kDa by SDS-PAGE. The optical and EPR spectra results for the recombinant IscR confirmed that an iron-sulfur cluster was correctly inserted into the active site of the protein. However, no [Fe(2)S(2)] cluster was assembled in apoIscR with ferrous iron and sulfide in vitro. Therefore, the [Fe(2)S(2)] cluster assembly in IscR in vivo would appear to require scaffold proteins and follow the Isc "AUS" pathway.
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