tuberculosis, the plasmid construct pTBOBGE was made to overexpress

Obg in E. coli. Log phase E. coli cells (strain BL21) bearing the plasmid pTBOBGE were induced by IPTG to overexpress a protein that migrates at around 55 kDa in SDS-PAGE gels. This overexpressed protein, purified as detailed in the Methods section, showed a single protein in SDS-PAGE (Figure 1A). This was designated as His10-Obg, to distinguish it from the native, Selleckchem GDC-0994 normally expressed Obg protein in M. tuberculosis. Figure 1 Analysis of overexpressed Obg and its GTP BX-795 supplier binding and hydrolysis activities. A. SDS-PAGE protein profile showing overexpression and purification of M. tuberculosis Obg. E. coli was grown in LB broth at 37°C, and lysates were prepared by sonication. Lane 1, Molecular markers; Lanes 2 and 3, extracts of E. coli strain BL21 carrying the overexpression plasmid pTBOBGE in the absence (Lane 2) and presence (Lane 3) of 1 mM IPTG; Lane 4, supernatant of E. coli lysate after 10,000 g centrifugation; Lane 5, His10-Obg after Ni-NTA affinity chromatography. The arrow points to the His10-Obg band. B. Autoradiogram of SDS-PAGE-separated M. tuberculosis His10-Obg after UV-crosslinking with [α32P]GTP. UV-cross-linking was performed by incubating 5 μg of His10-Obg click here with 10 μCi of [α32P]GTP

in the binding buffer as described in the Methods section I. Crosslinking of His10-Obg with [α32P]GTP after 0, 30 and 60 minutes of exposure to UV

light (256 nm). II. Crosslinking of His10-Obg with [α32P]GTP for 30 min Metalloexopeptidase without any additional GTP or ATP in the reaction mixture (Lane 1) or with 5 mM of unlabeled GTP (Lane 2), or with 500 mM of unlabeled ATP (Lane 3). C. GTPase activity of His10-Obg. GTP hydrolysis of His10-Obg was performed using [γ-32P] GTP at 37°C. The GTPase activity is expressed as 32Pi released (cpm)/μg protein/hour. Columns indicate GTPase activity in the absence of [γ-32P]GTP and His10-Obg (Column 1), in the presence of His10-Obg alone (Column 2), in the presence of both [γ-32P]GTP and His10-Obg (Column 3), in the presence of [γ -32P]GTP, His10-Obg and 5 mM unlabeled GTP (Column 4), in the presence of [γ -32P]GTP, His10-Obg and 5 mM unlabeled GDP (Column 5) and in the presence of [γ-32P]GTP, His10-Obg and 5 mM unlabeled ATP (Column 6). * indicates value significant from column 3 (paired t-test P = 0.0163). To verify whether the overexpressed Obg of M. tuberculosis can interact with GTP, we performed GTP-UV-crosslinking experiments [31]. The autoradiogram in Figure 1B shows that His10-Obg binds physically to [α32P]-GTP. Exposure of the reaction mixtures to UV irradiation for 0, 30 and 60 min revealed that binding of GTP with His10-Obg is increased between 0 and 30 min of exposure, but not after 30 min (Figure 1B).