Mycobacterium tuberculosis has evolved mechanisms to circumvent the hostile environment of the macrophage and it has been suggested that tyrosine phosphatases (PTPs) may play an important role in the pathogenicity of mycobacteria by interfering with phosphotyrosine-mediated signals in macrophages. M. tuberculosis has two Tyr phosphatases (PtpA and PtpB) and there appears to be no counterpart Tyr kinase, as shown by genome analysis. The absence of Tyr kinase suggests that the substrates of PtpA and PtpB are proteins of the host cells (macrophages). Recently, "substrate-trapping" mutants have been proposed as a tool for identifying potential substrates of PTPs. In this work, the substrate trapping mutant PtpAD126A was generated by site direct mutagenesis using the pET28 expression vector containing the wild type PtpA. The capture of the PTP substrate complex becomes possible because this mutant retains the ability to bind substrates, but is severely impaired in carrying out substrate dephosphorylation. The constructs containing the wild type and the "substrate-trapping" variants of PtpA were expressed in E. coli strain BL21(DE3). Transformed E. coli cells were grown at 37ºC in LB medium with 50 mg/ml kanamycin to the end of exponential phase of growth. Then a final concentration of 0.5 mM of IPTG was added to start protein induction at 15ºC. Bacterial pellets were resuspended in a lysis buffer and sonicated. The recombinant proteins were purified from the supernatant by immobilized Cu-affinity chromatography using Chelating Sepharose Fast Flow and eluted by an imidazol gradient. Then, proteins were subsequently dialyzed to eliminate the imidazol, concentrated and the purity of samples was checked by SDS-PAGE. All proteins were produced pure and in soluble form (2-5mg recombinant protein/L culture) At present, the determination of the kinetic parameters of the wild type PtpA and PtpAD126A using paranitrophenol phosphate as a substrate is in progress. In order to find the physiological substrates of PtpA in vitro tests of substrate trapping ability of PtpA using proteins extracts from infected macrophages will be perform.