Characterization of the major membrane protein of virulent Mycobacterium tuberculosis

Abstract

A protein with a molecular mass of 19 kDa was isolated and purified from enriched membrane fractions of the virulent Erdman strain of Mycobacterium tuberculosis. The protein is different from another 19-kDa protein, a lipoprotein, that was recently described (D. B. Young and T. R. Garbe, Res. Microbiol. 142:55-65, 1991). The sequencing strategy applied to this major membrane protein employed four different endoproteinases and resulted in sufficient overlapping peptide sequences for assignment of the entire protein sequence. Electron spray ionization mass spectrometry demonstrated a measured mass of 16,100, deviating from the predicted mass by only 2.86 atomic mass units. The sequence of this protein is unique. However, some similarities with other low-molecular-weight heat shock proteins were observed. Immunoblotting indicated that this protein is highly expressed in the virulent strains of M. tuberculosis. Its application to sera from patients with pulmonary tuberculosis showed promise as a serodiagnostic tool.

Serological distinction of integral plasma membrane proteins as a class of mycobacterial antigens and their relevance for human T cell activation

“…The biphasic Triton X-l 14 extraction (38] has been used widely and successfully as a standard procedure for isolationcharacterization of integral and peripheral membrane proteins from a variety of pathogenic species including mycobacteria [20][21][22]26,28]. In the present case of Myco.…”

“…tuherculosis protein, on the other hand, was characterized as a novel antigen capable of eliciting antibody responses in most pulmonary tuberculosis patients [28], Serological specificity of the detergent-soluble integral membrane proteins of Myco. fortuitum qualified them by and large as a group of antigens quite distinct from the watersoluble membrane proteins or cytosolic proteins, Antigenic characterization of detergent-soluble proteins was not attempted in the studies by Hunter et al [26] and Lee et al [28]. Nevertheless, a study by Young & Garbe [19] comes closest to characterizing such proteins from Myco.…”

“…UK) was grown in Sauton's medium at 37 C on an orbital shaker and harvested in the late-log phase of growth (day 4). Subcellular fractionation was carried out according to the standard protocol [26,28,35]. Brietly.…”

“…The available reports describe isolation and characterization of water-soluble CperipheraD proteins of 22 and 35 kD from Myco, leprae [26,27] and 19 kD from Myco. tuberculosis [28]. In both these 'slow growing' mycobaeterial species, co-partitioning of the immunosuppressive moiety lipoarabinomannan (LAM) [6][7][8] in the detergent phase posed a major obstacle for further resolution and immunological evaluation of the integral membrane proteins.…”

T cell clones from a non-leprosy exposed subject recognize theMycobacterium leprae18-kD protein

“…The antigen was purified from the bacteria by solubilization in 8 M urea, precipitation with ammonium sulphate and purification with high performance liquid chromatography (HPLC) [15]. The 16-kD membrane protein of A/, tuberculosis [16] was also expressed in E. coli DH5a from plasmid pML3tb. Lysates containing the 16-kD protein and control E. coli lysates were lyophilized, reconstituted in distilled water and filtered prior to use.…”