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Title: Characterisation of a putative serine protease expressed in vivo by Mycobacterium avium subsp. paratuberculosis
Author: Cameron, Rona Mary
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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As part of a larger study to identify genes and gene products expressed in vivo by Mycobacterium avium subsp. paratuberculosis, two immunogenic clones designated S4 (1.6 kb) and S8 (3 kb) were extracted from a λgt11 genomic expression library of M.a. paratubeculosis 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 M.a. paratuberculosis. 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 Escherichia coli, Salmonella typhimurium, Bruncella abortus, Rochalimea henselae, and Campylobacter jejuni, 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 E. coli 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 M. avium complex (M. avium, M.a. paratuberculosis, M.a. silvaticum, M. intracellulare and M. scrofulaceum were found to contain the gene, although M. malmoense and M. marinum 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 M. avium complex of mycobacteria. However, no proteolytic activity has been demonstrated by either native or recombinant proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available