Purification of ArcR, an oxidation-sensitive regulatory protein from Bacillus licheniformis.

Abstract In Bacillus licheniformis , ArcR, a transcriptional activator of the Crp/Fnr family, is required for expression of the anaerobic pathway of arginine catabolism, the arginine deiminase pathway. The method described here allows the purification of milligram quantities of functional ArcR from a recombinant Escherichia coli strain. The solubility properties of ArcR were much exploited during the purification process. The protein appeared highly sensitive to oxidation. Oxidation-induced precipitation of the protein was attributed to the formation of intermolecular disulfide bridges. Alkylation of mutant proteins with single substitutions showed that both cysteine residues of the protein, C 178 and C 205 , are involved in formation of the disulfide bridges. Substitution of both cysteines yielded a functional protein insensitive to oxidation and able to form a complex with its cognate target on the DNA.