Purification of ArcR, an oxidation-sensitive regulatory protein from Bacillus licheniformis.
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, C178 and C205, 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.
Laboratoire de Microbiologie, Université Libre de Bruxelles, Brussels, Belgium.
Alexandre
A
Wohlkönig
Victor
V
Stalon
Corinne
C
Vander Wauven
D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012492:Salts / Q000737:chemistry; D016415:Sequence Alignment
C416526:ArcR protein, bacteria; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004220:Disulfides; D011994:Recombinant Proteins; D012492:Salts; D003545:Cysteine