Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli.
The genes encoding Helicobacter pylori urease, a nickel metalloenzyme, have been cloned and expressed in Escherichia coli. Enzymatic activity, however, has been very weak compared with that in clinical isolates of H. pylori. Conditions under which near wild-type urease activity was achieved were developed. E. coli. SE5000 containing recombinant H. pylori urease genes was grown in minimal medium containing no amino acids, NiCl2 was added to 0.75 microM, and structural genes ureA and ureB (pHP902) were overexpressed in trans to the complete urease gene cluster (pHP808). Under these conditions, E. coli SE5000 pHP808/pHP902) expressed a urease activity up to 87 mumol of urea per min per mg of protein (87 U/mg of protein), a level approaching that of wild-type H. pylori UMAB41 (100 U/mg of protein), from which the genes were cloned. Poor catalytic activity of recombinant clones grown in Luria broth or M9 medium containing 0.5% Casamino Acids was due to chelation of nickel ions by medium components, particularly histidine and cysteine. In cultures containing these amino acids, 63Ni2+ was prevented from being transported into cells and was not incorporated into urease protein. As a consequence, M9 minimal medium cultures containing histidine or cysteine produced only 0.05 and 0.9%, respectively, of active urease produced by control cultures containing no amino acids. We conclude that recombinant H. pylori urease is optimally expressed when Ni2+ transport is not inhibited and when sufficient synthesis of urease subunits UreA and UreB is provided.
Hu
L T
LT
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201.
Mobley
H L
HL
D000596:Amino Acids / Q000494:pharmacology; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D009532:Nickel / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014510:Urease / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D011994:Recombinant Proteins; C022838:nickel chloride; D009532:Nickel; D014510:Urease