High Resolution Electroelution of Polyacrylamide Gels for the Purification of Single Proteins from <i>Mycobacterium tuberculosis</i> Culture Filtrate

Weldingh, Karin; Hansen, Anker; Jacobsen, Susanne; Andersen, Peter

doi:10.1046/j.1365-3083.2000.00655.x

Cited by 15 publications

(12 citation statements)

References 34 publications

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“…Moreover, recombinant M. tuberculosis GS was abundantly exported by M. smegmatis while very little of the endogenous M. smegmatis GS was released (30). Others have also identified M. tuberculosis GS in culture filtrates (55,56,62,69). In this study, we found a somewhat lower percentage of total GS (8 to 14%) in M. tuberculosis culture filtrates compared with our previous work (33%) and that of Raynaud et al (17%) (55).…”

Section: Discussioncontrasting

confidence: 56%

“…In contrast, although there was a readily detectable quantity of MDH in the culture filtrate at all time points (10 to 70 mU per ml of original culture VOL. 69,2001 EXPORT OF GS AND SOD BY MYCOBACTERIA 6355 volume), this extracellular MDH activity represented less than 0.5% of the total MDH activity. The SDS-PAGE results were consistent, showing no accumulation of MDH in the culture filtrate.…”

Section: Resultsmentioning

confidence: 92%

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High Extracellular Levels of Mycobacterium tuberculosis Glutamine Synthetase and Superoxide Dismutase in Actively Growing Cultures Are Due to High Expression and Extracellular Stability Rather than to a Protein-Specific Export Mechanism

2001

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Glutamine synthetase (GS) and superoxide dismutase (SOD), large multimeric enzymes that are thought to play important roles in the pathogenicity of Mycobacterium tuberculosis, are among the bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leader peptide, their presence in the extracellular medium during early stages of growth suggested that they might be actively secreted. To understand their mechanism of export, we cloned the homologous genes (glnA1 and sodA) from the rapid-growing, nonpathogenic Mycobacterium smegmatis, generated glnA1 and sodA mutants of M. smegmatis by allelic exchange, and quantitated expression and export of both mycobacterial and nonmycobacterial GSs and SODs in these mutants. We also quantitated expression and export of homologous and heterologous SODs from M. tuberculosis. When each of the genes was expressed from a multicopy plasmid, M. smegmatis exported comparable proportions of both the M. tuberculosis and M. smegmatis GSs (in the glnA1 strain) or SODs (in the sodA strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinant M. smegmatis and M. tuberculosis strains even exported nonmycobacterial SODs. To determine the extent to which export of these large, leaderless proteins is expression dependent, we constructed a recombinant M. tuberculosis strain expressing green fluorescent protein (GFP) at high levels and a recombinant M. smegmatis strain coexpressing the M. smegmatis GS, M. smegmatis SOD, and M. tuberculosis BfrB (bacterioferritin) at high levels. The recombinant M. tuberculosis strain exported GFP even in early stages of growth and at proportions very similar to those of the endogenous M. tuberculosis GS and SOD. Similarly, the recombinant M. smegmatis strain exported bacterioferritin, a large (ϳ500-kDa), leaderless, multimeric protein, in proportions comparable to GS and SOD. In contrast, high-level expression of the large, leaderless, multimeric protein malate dehydrogenase did not lead to extracellular accumulation because the protein was highly unstable extracellularly. These findings indicate that, contrary to expectations, export of M. tuberculosis GS and SOD in actively growing cultures is not due to a protein-specific export mechanism, but rather to bacterial leakage or autolysis, and that the extracellular abundance of these enzymes is simply due to their high level of expression and extracellular stability. The same determinants likely explain the presence of other leaderless proteins in the extracellular medium of actively growing M. tuberculosis cultures.Mycobacterium tuberculosis, the primary etiologic agent of tuberculosis, is one of the world's leading causes of death, killing 2 million persons annually worldwide (23). New modalities to combat M. tuberculosis and a greater understanding of the biology and immunology of this pathogen are high priorities of tuberculosis research.The extracellular proteins of M. tuberculosis have been the focus of many studies investigating ...

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Section: Discussioncontrasting

confidence: 56%

Section: Resultsmentioning

confidence: 92%

High Extracellular Levels of Mycobacterium tuberculosis Glutamine Synthetase and Superoxide Dismutase in Actively Growing Cultures Are Due to High Expression and Extracellular Stability Rather than to a Protein-Specific Export Mechanism

2001

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“…Although their functions have not been established, these proteins have been considered as antigens, able to distinguish between tuberculosis clinical states, or attractive targets for the development of new drugs, vaccines and diagnostic strategies for TB [58-60]. …”

Section: Discussionmentioning

confidence: 99%

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

et al. 2011

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BackgroundBacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.ResultsThe 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern.ConclusionsHere we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

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“…The second fractionation by gel filtration was unsatisfied for narrow separation (data not shown). Weldingh et al [32,33] and Bhasker et al [34] used a preparative two‐dimensional gel for fractionation, while another study [29] used a combination of classical chromatographic methods and a multi‐elution method using PAGE gels to search for reliable antigens. Similarly, we used a combination of DEAE ion‐exchange chromatography and narrow molecular mass fractionation from SDS–PAGE gels, which proved more effective for identifying valuable proteins from the complex antigen system.…”

Section: Discussionmentioning

confidence: 99%

Identification of the new T-cell-stimulating antigens fromMycobacterium tuberculosisculture filtrate

Lim

Kim

Lee

et al. 2004

FEMS Microbiology Letters

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The proteins secreted by Mycobacterium tuberculosis are an important target for vaccine development. To identify the antigens from M. tuberculosis culture filtrate (CF) that strongly stimulate T-cells, the CF was fractionated by ion-exchange chromatography and then nonreducing sodium dodecyl sulfate^polyacrylamide gel electrophoresis with mini-whole gel elution. Each fraction was screened for its ability to induce interferon-gamma (IFN-Q) production in peripheral blood mononuclear cells isolated from healthy tuberculin reactors. The protein bands that strongly induced IFN-Q production were subjected to N-terminal sequencing. Two new proteins, a 17-kDa protein (Rv0164, MTSP17) and an 11-kDa (Rv3204, MTSP11) protein, were identified. The recombinant MTSP17 (rMTSP17) and rMTSP11 induced significant production of IFN-Q and interleukin (IL)-12p40 in peripheral blood mononuclear cells from healthy tuberculin reactors. Interestingly, IL-12p40 production in response to rMTSP11 was significantly higher than that in response to rMTSP17 or the three components of the antigen 85 complex. These results suggest that MTSP11 antigen should be further evaluated as a component of a subunit vaccine.

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High Resolution Electroelution of Polyacrylamide Gels for the Purification of Single Proteins from Mycobacterium tuberculosis Culture Filtrate

Cited by 15 publications

References 34 publications

High Extracellular Levels of Mycobacterium tuberculosis Glutamine Synthetase and Superoxide Dismutase in Actively Growing Cultures Are Due to High Expression and Extracellular Stability Rather than to a Protein-Specific Export Mechanism

High Extracellular Levels of Mycobacterium tuberculosis Glutamine Synthetase and Superoxide Dismutase in Actively Growing Cultures Are Due to High Expression and Extracellular Stability Rather than to a Protein-Specific Export Mechanism

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

Identification of the new T-cell-stimulating antigens fromMycobacterium tuberculosisculture filtrate

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