Infection with the bacterium Mycobacterium tuberculosis (MTB) causes tuberculosis, a pulmonary infection that may be fatal if left untreated. Misuse or mismanagement of tuberculosis drugs may lead to drug-resistant pathogen forms that are difficult to treat and contribute to a global health problem. The MTB SenX3/RegX3 signal transduction system allows bacteria to externally sense the environment and mediate an appropriate internal response; SenX3 is also associated with MTB virulence, suggesting that this protein may provide a potential therapeutic target. To investigate the role of SenX3 and MTB drug resistance, SenX3 was cloned, expressed and purified in Escherichia coli. SenX3 was cloned from the genome of the MTB strain H37Rv by polymerase chain reaction and an internal NcoI restriction site was destroyed by site-directed mutagenesis to allow cloning into the pET-28b prokaryotic expression vector. SenX3 expression from the resulting pET‑28b-mSenX3 plasmid was induced with isopropyl β-D-thiogalactoside and the protein was purified using Ni-NTA agarose affinity chromatography. A pure protein of the expected size was identified. The examination of purified SenX3 protein is considered to enable the in‑depth investigation of SenX3-mediated drug resistance.