As part of a larger study to identify genes and gene products expressed <I>in vivo</I> by <I>Mycobacterium avium </I>subsp. <I>paratuberculosis, </I>two immunogenic clones designated S4 (1.6 kb) and S8 (3 kb) were extracted from a λgt11 genomic expression library of <I>M.a. paratubeculosis </I>by screening with serum from a sheep with clinical Paratuberculosis (Johne's disease). The clones were shown by antibody elution and DNA sequencing to contain fragments of the same 1.1 kb open reading frame (ORF), which encoded a native protein of approximately 34 kDa in <I>M.a. paratuberculosis. </I>The ORF also encoded a possible signal peptide from amino acids 1-39, suggesting the native protein is secretory. Database searches using the deduced amino acid sequence of the ORF identified a motif 'GDSGG' which displayed 100% homology to the residues surrounding the active serine in a trypsin-like serine protease. An overall homology of approximately 30% was detected with the HtrA proteins of <I>Escherichia coli, Salmonella typhimurium, Bruncella abortus, Rochalimea henselae, </I>and <I>Campylobacter jejuni, </I>which are also thought to be serine proteases. The S8 DNA insert contained the entire 1.1 kb ORF, but due to the presence of the signal peptide, the recombinant could not be expressed as a fusion protein. However the insert was successfully expressed as a free protein by translational coupling, and was found to be secreted into the <I>E. coli</I> periplasm, confirming the presence of a signal sequence. When several species of mycobacteria were screened with the 1.1. kb ORF only members of the <I>M. avium </I>complex (<I>M. avium, M.a. paratuberculosis, M.a. silvaticum, M. intracellulare</I> and <I>M. scrofulaceum </I>were found to contain the gene, although <I>M. malmoense </I>and <I>M. marinum </I>reacted weakly and may therefore contain homologous genes. The native 34 kDa protein encoded by these clones therefore appears to be a novel secretory serine protease specific to the <I>M. avium </I>complex of mycobacteria. However, no proteolytic activity has been demonstrated by either native or recombinant proteins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642340 |
| Date | January 1996 |
| Creators | Cameron, Rona Mary |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13292 |
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