In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR.
AUTOR(ES)
Grob, P
RESUMO
The spv regulon of Salmonella dublin is essential for virulence in mice. SpvR, a LysR-type regulator, induces the expression of the spvABCD operon and its own expression in the stationary phase of bacterial growth and in macrophages. We constructed fusion proteins to the maltose-binding protein (MBP) and a His tag peptide (His) to overcome the insolubility and to facilitate purification of SpvR. We demonstrated that both fusion proteins, MBP-SpvR and His-SpvR, were able to induce spvA expression in vivo. MBP-SpvR was produced as soluble protein, whereas His-SpvR was only marginally present in the soluble cell fraction. Affinity chromatography resulted in at least 95% pure MBP-SpvR protein and in an enrichment of His-SpvR. Gel mobility shift assay revealed that the SpvR fusion proteins were able to bind to 125-and 147-bp DNA fragments of the spvA and spvR promoter regions, respectively. DNase I footprint experiments showed that the fusion proteins protected DNA regions of 54 and 50 bp within the spvA and spvR promoter regions, respectively.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=177874Documentos Relacionados
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