tuberculosis His 10 -Obg after autophosphorylation. Autophosphorylation reactions were set up by selleck chemicals incubating 5 μg of His10-Obg with 10 μCi of [γ-32P] GTP in autophosphorylation buffer, as detailed in the Methods section. A. Autophosphorylation of His10-Obg by [γ-32P] GTP or [γ-32P]ATP after 0, 15, 30 and 60 minutes of incubation at 37°C. B. Autophosphorylation of His10-Obg by [γ-32P]GTP in the presence (+ lane) and absence of (- lane) 1.5
mM MgCl2 . C. Autophosphorylation of His10-Obg by [γ-32P]GTP in the presence of 5 mM (Lane 1), 50 mM (Lane 2) and 500 mM (Lane 3) ATP; 5 mM (Lane 1), 50 mM (Lane 2) and 500 mM (Lane 3) of GTP; 5 mM (Lane 1), 50 mM (Lane 2) and 500 mM (Lane 3) of GDP. Expression of M. tuberculosis Obg is growth-dependent, and Obg is associated with the membrane fraction In the sporulating bacterium S. coelicolor, the expression of Obg is regulated developmentally and is linked to the onset of sporulation [9]. By contrast, no such change in expression of
Obg occurs in C. crescentus, although it also has a clear developmental cycle involving sporulation [10]. M. tuberculosis is a slow growing bacterium which exhibits neither sporulation nor a developmental cell cycle during its growth in culture. To determine learn more whether the expression of Obg changes during the growth of M. tuberculosis in culture, we developed a rabbit anti-Obg antiserum against M. tuberculosis His10-Obg, and used it in Western blots of M. tuberculosis protein extracts. This antiserum detects multiple bands in immunoblotted extracts of M. tuberculosis, particularly at 55 kDa and 75 kDa. To confirm that the 55 kDa protein reacting with anti-Obg antiserum is in fact Obg, we cloned the coding region of Obg downstream of the hsp60 promoter in the plasmid INCB018424 price pMV261, and transformed the resulting construct (pMVOBG) into M. tuberculosis
to overproduce Obg. Figure 3A shows that protein extracts of M. tuberculosis strains harboring plasmid pMVOBG, but not strains bearing the vector plasmid pMV261, reveal strong 55 kDa protein bands, indicating that the protein at 55 kDa is Obg. Further analysis revealed that the 75 kDa band was a false reactivity due to the second antibody, and that it is not an Obg protein. Methane monooxygenase Figure 3 Immunoblot analysis of Obg of M. tuberculosis. A. Immunoblot analysis of Obg from M. tuberculosis strains harboring plasmids. M. tuberculosis strains were grown in 7H9-OADC-TW broth at 37°C to early log phase and lysates prepared using a bead beater and separated (100 μg protein for each lane) on SDS-PAGE. The immunoblots were probed with anti-Obg antiserum (1:500 dilution) followed by alkaline phosphatase labeled anti-rabbit IgG (1:1000 dilution, Zymed). The antibody-incubated blots were then developed with NBT/BCIP substrates. Lane 1, M. tuberculosis carrying the plasmid pMV261(empty vector control); Lane 2, M. tuberculosis carrying the plasmid pMVOBG (plasmid overexpressing Obg). B. Immunoblot analysis of Obg at different growth points in M.