Development of a large-scale HPLC-based purification for the urease from Staphylococcus leei and determination of subunit structure.
Coagulase-positive Staphylococcus species, related to but distinct from the genetic homology group containing Staphylococcus cohnii, Staphylococcus xylosus, and Staphylococcus saphrophyticus, were isolated from biopsy material obtained from a cluster of patients in Korea suffering from gastritis. The prototype isolate, Staphylococcus leei, has high urease activity that is similar with respect to a low K(m) value and acid resistance of the urease found in the stomach adapted pathogen, Helicobacter pylori. S. leei is remarkably resistant to lysis and only a small fraction of the cells are broken using sonication, a French press, Niro homogenizer, or a Gaulin mill. In the present report, we describe an efficient cell lysis procedure for S. leei using three passes through a Dynomill with 0.5mm glass beads that results in lysis of more than 95% of the cells. We also developed an efficient and large-scale purification procedure for the S. leei urease using a BioCAD HPLC Workstation using Q-Sepharose, Poros HP2, Sephacryl S-300, and hydroxyapatite chromatography. The urease of S. leei was purified 98-fold to a specific activity of 731U/mg. The urease protein is composed of three subunits, alpha (65kDa), beta (21kDa), and gamma (12kDa), and in situ enzyme assay and molecular sieve chromatography indicate that multiple high molecular weight forms are present, including an apparent pentamer of 1:1:1 alphabetagamma-heterotrimers of 480kDa. This purification procedure was used to purify 16mg of enzyme from 120-liters of cell culture. This improved lysis and purification procedure will make it possible to obtain sufficient quantities of urease for use as an antigen in ELISA assays to carry out studies to determine the incidence and demographic prevalence of gastritis due to S. leei.
Jin
Ming
M
Department of Chemistry, City College of New York, 138th Street and Convent Avenue, New York, NY 10031, USA.
Rosario
Wildys
W
Watler
Elsie
E
Calhoun
David H
DH
D000180:Acrylic Resins / Q000737:chemistry; D000641:Ammonia / Q000032:analysis / Q000378:metabolism; D001433:Bacteriolysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017886:Durapatite / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013210:Staphylococcus / Q000201:enzymology*; D014508:Urea / Q000378:metabolism; D014510:Urease / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000180:Acrylic Resins; D021122:Protein Subunits; C037239:Sephacryl Superfine; D000641:Ammonia; D014508:Urea; D017886:Durapatite; D014510:Urease