Figure 1

Analysis of overexpressed Obg and its GTP 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 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 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 ³²P_i 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).