coli and purified to homogeneity by metal chelating chromatography using Ni(II)-NTA-resin. 200 ml LB-broth medium (Gibco BRL, Gaithersburg, Md) containing ampicillin (100 μg/ml) was inoculated with 20 ml of overnight culture of the respective
E. coli BL 21-Lys clone for 1 h at 37°C with vigorous shaking until an OD600 nm of 0.6 to 0.9 was reached. Protein expression was induced by isopropylthio-β-D- galactoside (0.2 mM). After 3 h of shaking at 37°C the cells were harvested by centrifugation (15,000 × g, 20 min, 4°C) and frozen at -20°C. After thawing on ice the cells were resuspended in 17 ml buffer A [20 mM Tris/HCl pH 8.0, 500 mM KCl, 10 mM imidazole, 10 mM β-mercaptoethanol, 10% [v/v] glycerol, 5% [w/v] N-lauroylsarcosine, 1 tablet protease inhibitor (Roche, Grenzach-Wyhlen, Germany)] and incubated for 2 h on a rotating wheel followed by one burst of sonication on ice (5 min at 95 W). The lysate was centrifuged (15,000 × g, 20 min, 4°C) and find more the supernatant transferred to 0.5
ml 50% slurry of Ni-NTA- sepharose (Qiagen, Hilden, Germany) selleck chemicals llc and incubated for 4 h at RT on a rotating wheel. The sepharose was loaded into a 1 cm diameter column and check details washed with 20 ml washing buffer [20 mM Tris/HCl pH 8.0, 500 mM KCl, 10 mM imidazole, 10 mM β-mercaptoethanol, 10% [v/v] glycerol, 0.5% [w/v] N-lauroylsarcosine]. The bound proteins were eluted from the Ni-NTA resin by using wash buffer supplemented with 150 mM imidazole. 10 fractions of 0.5 ml were collected and 20 μl of each fraction analyzed on 9.5% polyacrylamide gels [42]. Adhesion assays The adhesion assays with wild type proteins of M.
hominis (OppA, P50, the P60/P80 membrane complex) and the recombinant OppA mutants were performed as a cell ELISA according to the description of Henrich et al., 1993 [6] with the following modifications: HeLa cells (1 × 105 cells/well) were immobilized Liothyronine Sodium with 1.25% (v/v) glutaraldehyde to lysine- coated 96-well micro-plates (Greiner Bio-one GmbH, Frickenhausen, Germany) as described formerly [45] and incubated in DMEMFCS [DMEM 10% (v/v) fetal bovine serum] (Lonza Cologne GmbH, Cologne, Germany) for 30 min at 37°C. The proteins were serial diluted 1:5 in DMEMFCS, using a starting concentration of 1 μg protein/well for the wild type proteins and 5 μg protein/well for the OppA mutants, and incubated with the immobilized HeLa cells for 2 h at 37°C. To analyze the influence of ATPase inhibitors the OppA protein or M. hominis cells were preincubated for 20 min with DIDS, Suramin, Ouabain, Oligomycin, FSBA or MgATP (Sigma) in concentrations as written in the figure legends before incubating with the HeLa cells. After removal of unbound protein by washing twice with DMEMFCS adherent wild type proteins were detected by protein-specific antibodies as described formerly [6]. For the detection of His-tagged OppA mutants monoclonal tetra-His antibody (Qiagen, Hilden, Germany) was used.
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