| SapM is a 28-kDa acid phosphatase specific to the pathogenic mycobacteria. It is secreted and its expression is selectively induced by the mildly acidic phagosomal pH in macrophages. These properties suggest that SapM may play a role in the pathogenesis of Mycobacterium tuberculosis and that it may also serve as a marker in diagnostic assays. This thesis focuses on the development of optimized protein expression and purification methods in order to obtain sufficient amounts of SapM for future functional studies. Factors affecting the expression and purification of SapM such as promoters, host strains, temperature, fusion tags and chaperones were tested and an optimal system has been developed. The construction of a sapM knock-out mutant was also investigated. Homologous recombination approaches to construct sapM mutants proved to be inefficient. Enzyme-Linked Immunosorbent Assay (ELISA) using denatured SapM as antigen revealed that SapM could be potentially used in clinical diagnosis of tuberculosis. |
