The 34kDa putative serine protease of M.a.paratuberculosis shows conserved features of the HtrA family of bacterial proteases, implicated in the virulence of several intracellular pathogens. However its role in virulence of M.a.paratuberculosis remained to be determined. To this end the 34kDa putative serine protease ORF was cloned into a mycobacterial shuttle vector under the transcriptional control of the M. tuberculosis hsp60 promoter. The expression and subsequent secretion of the recombinant protein into the extracellular milieu by M. smegmatis mc2 155 was shown. M. smegmatis mc2 155 that expressed the 34kDa putative serine protease demonstrated enhanced intracellular survival in ex-vivo sheep alveolar macrophages in the 24 hours following macrophage ingestion in comparison to M. smegmatis mc2 155 expressing E-GFP and wild-type M. smegmatis mc2 155. The presence of the protein had no affect on the ability of M. smegmatis mc2 155 to enter macrophages or on the long-term intracellular survival. The upstream region of the ORF in M.a.paratuberculosis was examined for transcriptional activity and shown to be capable of expressing the 34kDa putative serine protease in M. smegmatis mc2 155. A similar level of increased intracellular survival was noted when the 34kDa putative serine protease was expressed from the putative upstream promoter as observed when the 34kDa putative serine protease was expressed by the hsp60 promoter. A putative IdeR regulatory sequence was identified in the upstream region. However, further characterisation of the upstream region failed to demonstrate a role for IdeR or the cellular concentration of iron in the regulation of the 34kDa putative serine protease. Homologues of the 34kDa putative serine protease were observed in the genomes of several pathogenic mycobacterial species, including members of the TB complex and M. leprae. Significantly, only weak homology noted in the genome of the non-pathogenic M. smegmatis. Therefore, the possibility exists that the 34kDa putative serine protease represents a conserved feature of mycobacterial pathogenesis.