Mapping and identification ofMycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection

Rosenkrands, Ida; Weldingh, Karin; Jacobsen, Susanne; Hansen, Christina Veggerby; Florio, Walter; Gianetri, Isabella; Andersen, Peter

doi:10.1002/(sici)1522-2683(20000301)21:5<935::aid-elps935>3.0.co;2-p

Cited by 153 publications

(24 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thirty-five of the identified proteins have no known function, but are now shown to be synthesized in C. glutamicum under the employed culture conditions. In a few cases, homologues of the proteins of unknown function were also identified on 2-D gels of M. tuberculosis (Rv0046c, Rv1636, Rv2140c, Rv2145c) and M. bovis [13,14]. With the exception of AmyE and PS1, which have also been detected in the culture supernatant [9], and of AtpA and Mqo, which are associated with the inner face of the cytoplasmic membrane, all soluble identified proteins with known function are cytoplasmic proteins.…”

Section: Figurementioning

confidence: 97%

“…In E. coli, proteins with N-terminal Arg, Lys, Phe, Tyr, Trp and Leu residues are highly unstable [17] and none of the N-termini of 223 cytoplasmic E. coli proteins starts with one of these amino acids [15]. In contrast, mature proteins starting with Arg, Lys, Tyr and Leu residues were detected in C. glutamicum, M. tuberculosis [14], Streptococcus pneumoniae [18], B. subtilis [19] and Sinorhizobium meliloti [20]. …”

Section: Determination Of N-terminimentioning

confidence: 99%

See 1 more Smart Citation

A high-resolution reference map for cytoplasmic and membrane-associated proteins ofCorynebacterium glutamicum

Schaffer¹,

Weil²,

Nguyen

et al. 2001

Electrophoresis

121

View full text Add to dashboard Cite

We present a high-resolution reference map for soluble proteins obtained from Corynebacterium glutamicum cells grown in glucose minimal medium. The analysis window covers the pl range from 4-6 and the molecular mass range from 5-100 kDa. Using overlapping narrow immobilized pH gradients for isoelectric focusing, 970 protein spots were detected after second-dimensional separation on SDS-polyacrylamide gels and colloidal Coomassie-staining. By tryptic peptide mass fingerprinting 169 protein spots were identified, representing 152 different proteins including many enzymes involved in central metabolism (18), amino acid biosynthesis (24) and nucleotide biosynthesis (11). Thirty-five of the identified proteins have no known function. A comparison of the observed and the expected physicochemical properties of the identified proteins indicated that nine proteins were covalently modified, since variants with apparently identical molecular mass, but differing pl were detected. The N-termini of eight proteins were determined by post-source decay (PSD) analysis of selected peptides. In addition to the soluble proteins, a map of the membrane-bound proteins within the pl range 4-7 is presented, which contains 660 protein spots, 22 of which were identified, representing 13 different proteins.

show abstract

Section: Figurementioning

confidence: 97%

Section: Determination Of N-terminimentioning

confidence: 99%

A high-resolution reference map for cytoplasmic and membrane-associated proteins ofCorynebacterium glutamicum

Schaffer¹,

Weil²,

Nguyen

et al. 2001

Electrophoresis

121

View full text Add to dashboard Cite

show abstract

“…protein A or fibronectin binding protein of S. aureus) [18,41,42]. An especially great effort has been made to exlore the extracellular proteomes of pathogenic bacteria as for instance for S. aureus [43], group A streptococci [44], Mycobacterium tuberculosis [45][46][47] and Helicobacter pylori [48]. Strikingly, global cell surface proteome analyses using the proteomic approach are still lacking for most of the pathogenic bacteria.…”

Section: Cell Wall Binding Proteins and Cell Wall Retention Signalsmentioning

confidence: 99%

Stabilization of cell wall proteins in Bacillus subtilis: A proteomic approach

et al. 2002

View full text Add to dashboard Cite

Even though cell wall proteins of Bacillus subtilis are characterized by specific cell wall retention signals, some of these are also components of the extracellular proteome. In contrast to the majority of extracellular proteins, wall binding proteins disappeared from the extracellular proteome during the stationary phase and are subjected to proteolysis. Thus, the extracellular proteome of the multiple protease-deficient strain WB700 was analyzed which showed an increased stability of secreted WapA processing products during the stationary phase. In addition, stabilization of the WapA processing products was observed also in a sigD mutant strain which is impaired in motility and cell wall turnover. Next, we analyzed if proteins that can be extracted from B. subtilis cell walls are stabilized in the WB700 strain as well as in the sigD mutant. Thus, the cell wall proteome of B. subtilis wild type was defined showing most abundantly cell wall binding proteins (CWBPs) resulting from the WapA and WprA precursor processing. The inactivation of extracellular proteases as well as SigmaD caused an increase of CWBP105 and a decrease of CWBP62 in the cell wall proteome. We conclude that WapA processing products are substrates for the extracellular proteases which are stabilized in the absence of sigD due to an impaired cell wall turnover.

show abstract

“…Today most laboratories use non-heated M. bovis culture filtrates rather than tuberculin for the identification of specific M. bovis antigens for use as diagnostic and vaccine candidates [10,12,13,15-17]. Separation and characterization of non-heated M. bovis CFPs using molecular techniques such as two dimensional polyacrylamide gel electrophoresis (2-DE), mass spectronomy (MASS-SPEC) analysis and in vitro antigenicity assays has lead to the identification of several, highly antigenic M. bovis proteins.…”

Section: Introductionmentioning

confidence: 99%

Antibody response to a sterile filtered PPD tuberculin in M. bovis infected and M. bovis sensitized cattle

2010

View full text Add to dashboard Cite

BackgroundBovine tuberculosis, caused by Mycobacterium bovis, afflicts approximately 50 million cattle worldwide and is detected by the tuberculin skin test (TST). While it has long been recognized that purified protein derivative (PPD) tuberculin is composed of a mixture of M. bovis derived protein components, little is known about the quality, relative quantity and identity of the proteins that make up PPD tuberculin. We manufactured a sterile filtered PPD tuberculin (SF-PPD) from a nine-week-old M. bovis culture supernatant in order to characterise the culture filtrate proteins (CFP) which make up M. bovis PPD tuberculin and to compare the antibody response of M. bovis infected versus M. bovis sensitized cattle.ResultsSF-PPD resolved into approximately 200 discrete spots using two-dimensional polyacrylamide gel electrophoresis (2-DE) while fewer than 65 spots could be discerned from 2-DE gels of tuberculin derived from autoclaved culture supernatant. Two dimensional Western blot analyses indicated that sera from M. bovis sensitized cattle recognized additional SF-PPD antigens as compared to M. bovis infected cattle at seven weeks post infection/sensitization. However, application of a comparative tuberculin skin test resulted in an antibody boosting response to the same set of M. bovis CFPs in both the M. bovis infected and M. bovis sensitized cattle.ConclusionsWe concluded that it is the heat sterilization of the M. bovis CFPs that causes severe structural changes to the M. bovis proteins. This work suggests that M. bovis infected cattle and cattle artificially sensitized to M. bovis with an injection of heat killed cells exhibit similar antibody responses to M. bovis antigens.

show abstract

Mapping and identification ofMycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection

Cited by 153 publications

References 56 publications

A high-resolution reference map for cytoplasmic and membrane-associated proteins ofCorynebacterium glutamicum

A high-resolution reference map for cytoplasmic and membrane-associated proteins ofCorynebacterium glutamicum

Stabilization of cell wall proteins in Bacillus subtilis: A proteomic approach

Antibody response to a sterile filtered PPD tuberculin in M. bovis infected and M. bovis sensitized cattle

Contact Info

Product

Resources

About