1887

Abstract

The gene encoding an immunodominant secreted antigen, MPB70, of was cloned into the plasmid vector pBluescript II KS along with its native ribosome-binding site. In this construct translation of the protein in was from the native AUG initiation codon and was directed by the mycobacterial ribosome-binding site. Two different molecular mass forms (26 kDa and 22 kDa) of MPB70 were observed in whole-cell pellets of recombinant . The difference in size indicates cleavage of the signal peptide of MPB70 by an endopeptidase of . MPB70 was secreted into the periplasm of recombinant , where the 22 kDa form of the protein was predominant. The culture filtrate contained only the 22 kDa form of the protein, which was soluble. The passage of MPB70 from the periplasm into the growth medium was found to be due, at least in part, to non-specific leakage of periplasmic proteins across the outer membrane associated with the expression of recombinant MPB70.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-6-1253
1993-06-01
2021-04-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/6/mic-139-6-1253.html?itemId=/content/journal/micro/10.1099/00221287-139-6-1253&mimeType=html&fmt=ahah

References

  1. Abou-Zeid C., Harboe M., Rook G. A. W. 1987; Characterization of the secreted antigens of Mycobacterium bovis BCG: comparison of the 46-kilodalton dimeric protein with proteins MPB64 and MPB70. Infection and Immunity 55:3213–3214
    [Google Scholar]
  2. Angus B. L., Carey A. M., Canon D. A., Kropinski A. M. B., Hancock R. E. W. 1982; Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptible mutant. Antimicrobial agents and Chemotherapy 21:299–309
    [Google Scholar]
  3. Ashbridge K. R., Backstrom B. T., Liu H.-X., Vikereors T., Englebretsen D. R., Harding D. R. K., Watson J. D. 1992; Mapping of T helper cell epitopes by using peptides spanning the 19-kDa protein of Mycobacterium tuberculosis . Journal of Immunology 148:2248–2255
    [Google Scholar]
  4. Auer L. A. 1987; Assessment of an enzyme linked immunosorbent assay for the detection of cattle infected with Mycobacterium bovis . Australian Veterinary Journal 64:172–176
    [Google Scholar]
  5. Blake M. S., Johnston K. H., Russel-Jones G. J., Gatschlich E. C. 1984; A rapid, sensitive method for detection of alkaline phosphatase conjugated anti-antibody on western blots. Analytical Biochemistry 136:175–179
    [Google Scholar]
  6. Daniel T. M., Janicki B. W. 1978; Mycobacterial antigens: a review of their isolation, chemistry, and immunological properties. Microbiological Reviews 42:84–113
    [Google Scholar]
  7. Fifis T., Plackett P., Corner L. A., Wood P. R. 1989; Purification of a major Mycobacterium bovis antigen for the diagnosis of bovine tuberculosis. Scandanavian Journal of Immunology 29:91–101
    [Google Scholar]
  8. Fifis T., Costopoulos C., Radford A. J., Bacic A., Wood P. R. 1991; Purification and characterization of major antigens froma Mycobacterium bovis culture filtrate. Infection and Immunity 59:800–807
    [Google Scholar]
  9. Fifis T., Costopoulos C., Corner L. A., Wood P. R. 1992; Serological reactivity to Mycobacterium bovis protein antigens in cattle. Veterinary Microbiology 30:343–354
    [Google Scholar]
  10. Griffin J. F. T., Nagai S., Buchan G. S. 1991; Tuberculosis in domesticated red deer: comparison of purified protein derivative and the specific protein MPB70 for in vitro diagnosis. Research in Veterinary Science 50:279–285
    [Google Scholar]
  11. Harboe M., Nagai S., Patarroyo M. E., Torres M. L., Ramirez C., Cruz N. 1986; Properties of proteins MPB64, MPB70 and MPB80 of Mycobacterium bovis BCG . Infection and Immunity 52:293–302
    [Google Scholar]
  12. Harboe M., Wiker H. G., Duncan J. R., Garcia M. M., Dukes T. W., Brooks B. W., Turcotte C., Nagai S. 1990; Protein G-based enzyme-linked immunosorbent assay for anti-MPB70 antibodies in bovine tuberculosis. Journal of Clinical Microbiology 28:913–921
    [Google Scholar]
  13. Hasl𝜙v K., Andersen A. B., Bentzon M. W. 1987; Biological activity in sensitized guinea pigs of MPB70, a protein specific for some strains of Mycobacterium bovis BCG. Scandanavian Journal of Immunology 26:445–454
    [Google Scholar]
  14. Koshland D., Botstein D. 1980; Secretion of beta-lactamase requires the carboxy end of the protein. Cell 20:749–760
    [Google Scholar]
  15. Laemmli U. K. 1970; Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  16. Matsudaira P. 1987; Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. Journal of Biological Chemistry 262:10035–10038
    [Google Scholar]
  17. Miura K., Nagai S., Kinomoto M., Haga S., Tokunaga T. 1983; Comparative studies with various substrains of Mycobacterium bovis BCG on the production of an antigenic protein, MPB70. Infection and Immunity 39:540–545
    [Google Scholar]
  18. Morris J. A., Thorns C. J., Woolley J. 1985; The identification of antigenic determinants on Mycobacterium bovis using monoclonal antibodies. Journal of General Microbiology 131:1825–1831
    [Google Scholar]
  19. Nagai S., Matsumoto J., Nagasuga T. 1981; Specific skin- reactive protein from culture filtrate of Mycobacterium bovis BCG. Infection and Immunity 31:1152–1160
    [Google Scholar]
  20. Nagai S., Miura K., Tokunaga T., Harboe M. 1986; MPB70, a unique antigenic protein isolated from the culture filtrate of BCG substrain Tokyo. Developments in Biological Standardization 58:511–516
    [Google Scholar]
  21. O‘Callaghan C. H., Morris A., Kirby S. M., Singler A. H. 1972; Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy 1:283–288
    [Google Scholar]
  22. Pugsley A. 1989; Later stages in the prokaryotic secretory pathway. In Protein Targeting pp. 112–125 Pugsley A. Edited by London: Academic Press;
    [Google Scholar]
  23. Radford A. J., Wood P. R., Billman-Jacobe H., Geyson H. M., Mason T. J., Tribbick G. 1990; Epitope mapping of the Mycobacterium bovis secretory protein MPB70 using overlapping peptide analysis. Journal of General Microbiology 136:265–272
    [Google Scholar]
  24. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.;
    [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74:5463–5467
    [Google Scholar]
  26. Schoner B. E., Hsiung H. M., Belagaje R. M., Mayne N. G., Schoner R. G. 1984; Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli . Proceedings of the National Academy of Sciences of the United States of America 81:5403–5407
    [Google Scholar]
  27. Sutcliffe J. G. 1978; Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proceedings of the National Academy of Sciences of the United States of America 75:3737–3741
    [Google Scholar]
  28. Terasaka K., Yamaguchi R., Matsuo K., Yamazuki A., Nagai S., Yamada T. 1989; Complete nucleotide sequence of immunogenic protein MPB70 from Mycobacterium bovis BCG. FEMS Microbiology Letters 58:273–276
    [Google Scholar]
  29. Von Heijne G. 1988; Trancending the impenetrable: how proteins come to terms with membranes. Biochimica et Biophvsica Acta 947:307–333
    [Google Scholar]
  30. Vordermeier H. M., Harris D. P., Roman E., Lathigra R., Moreno C., Ivanyi J. 1991; Identification of T cell stimulatory peptides from the 38-kDa protein of Mycobacterium tuberculosis . Journal of Immunology 147:1023–1029
    [Google Scholar]
  31. Waley S. G. 1981; An easy method for the determination of initial rates. Biochemical Journal 205:631–633
    [Google Scholar]
  32. Whipple D. L., Le Febure R. B., Andrews R. E. JR Thiernann A. B. 1987; Isolation and analysis of restriction endonuclease digestive patterns of chromosomal DNA from Mycobacterium paratuberculosis and other mycobacterium species . Journal of Clinical Microbiology 25:1511–1515
    [Google Scholar]
  33. Wood P. R., Ripper J., Radford A. J., Bundesen P. G., Rylatt D. B., Cottis L. E., John M., Plackett P. 1988; Production and characterization of monoclonal antibodies specific for Mycobacterium bovis . Journal of General Microbiology 134:2599–2604
    [Google Scholar]
  34. Wood P. R., Corner L. A., Rothel J. S., Ripper J. L., Fifis T., Mccormick B. S., Francis B., Melville L., Small K., De Witte K., Tolson J., Ryan T. J., De Lisle G. W., Cox J. C., Jones S. L. 1992; A field evaluation of serological and cellular diagnostic tests for bovine tuberculosis. Veterinary Microbiology. 31:71–79
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-6-1253
Loading
/content/journal/micro/10.1099/00221287-139-6-1253
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error