Expression, purification and characterization of the membrane-associated HrcA repressor protein of Helicobacter pylori.
Helicobacter pylori, a microaerophilic, gram-negative bacterium is a human pathogen that colonizes the gastric niche and is associated with several acute and chronic stomach diseases. In order to survive in the gastric environment and become pathogenic, the bacterium relies on a plethora of virulence factors, which also include heat shock proteins. We previously showed that two out of the three operons encoding the major cellular chaperone machineries are transcriptionally repressed by two regulators, HrcA and HspR. Till now, molecular studies aimed at the understanding of the role of each protein in controlling transcription was hampered by toxicity and insolubility of HrcA in heterologous expression systems. Similar problems were encountered by many other groups studying HrcA from different bacteria. In this study, we analyzed the amino acid sequence of HrcA that predicted association of this protein to the inner membrane, which was experimentally verified. Subsequently, we implemented a dedicated induction protocol which enabled the overexpression of the recombinant His-HrcA protein in the soluble fraction of Escherichia coli cells. Moreover, we developed a purification procedure for His-HrcA that allowed us to obtain highly pure preparation of the protein. The functionality of the purified protein was then confirmed with an in vitro DNA-binding assay.
Roncarati
Davide
D
Department of Biology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy.
Spohn
Gunther
G
Tango
Nunzio
N
Danielli
Alberto
A
Delany
Isabel
I
Scarlato
Vincenzo
V
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D016480:Helicobacter pylori / Q000737:chemistry*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D012097:Repressor Proteins