MPB70 and MPB83 as indicators of protein localization in mycobacterial cells

Morten Harboe, Harald G. Wiker, Gunni Ulvund, Bent Lund-Pedersen, Åse Bengård Andersen, R. Glyn Hewinson, Sadamu Nagai*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

48 Citations (SciVal)

Abstract

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

Original languageEnglish
Pages (from-to)289-296
Number of pages8
JournalInfection and Immunity
Volume66
Issue number1
DOIs
Publication statusPublished - 13 Jan 1998

Keywords

  • Antibodies, Monoclonal/immunology
  • Antigens, Bacterial/isolation & purification
  • Antigens, Surface/immunology
  • Bacterial Proteins/immunology
  • Blotting, Western
  • Cell Membrane/chemistry
  • Chaperonin 10/isolation & purification
  • Chaperonin 60/isolation & purification
  • Chromatography, Gel
  • Culture Media, Conditioned/chemistry
  • Cytosol/chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli Proteins
  • Flow Cytometry
  • HSP70 Heat-Shock Proteins/isolation & purification
  • Lipopolysaccharides/isolation & purification
  • Lipoproteins/isolation & purification
  • Membrane Proteins
  • Mycobacterium avium subsp. paratuberculosis/chemistry
  • Mycobacterium bovis/chemistry
  • Mycobacterium tuberculosis/chemistry
  • Octoxynol
  • Polyethylene Glycols/pharmacology

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