Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography.
Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 microM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp-His-His-His), suggested that UreE may act as a Ni2+ chelator for the urease operon. To exploit this potential metal-binding motif, we attempted to purify UreE from cytoplasmic extracts of E. coli containing cloned urease genes. Soluble protein was loaded onto a nickel-nitrilotriacetic acid column, a metal chelate resin with high affinity for polyhistidine tails, and bound protein was eluted with a 0 to 0.5 M imidazole gradient. A single polypeptide of 20-kDa apparent molecular size, as shown by sodium dodecyl sulfate-10 to 20% polyacrylamide gel electrophoresis, was eluted between 0.25 and 0.4 M imidazole. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated on a Superdex 75 column to be 36 kDa, suggesting that the protein is a dimer. These data suggest that UreE is a Ni(2)+-binding protein that is necessary for synthesis of a catalytically active urease at low Ni(2+) concentrations.
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201.
B
B
Sriwanthana
M D
MD
Island
D
D
Maneval
H L
HL
Mobley
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification*; D002614:Chelating Agents; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006639:Histidine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009942:Organometallic Compounds; D011513:Proteus mirabilis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017421:Sequence Analysis; D014510:Urease / Q000096:biosynthesis*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002614:Chelating Agents; D009942:Organometallic Compounds; D011994:Recombinant Proteins; C090404:UreE protein, Bacteria; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D009532:Nickel; D014510:Urease; D009571:Nitrilotriacetic Acid