Cloning, Optimized Expression and Purification of Mycobacterium tuberculosis RpfE Protein in Escherichia coli

Background and Objective: One of the five resuscitation promoting factors (Rpf) encoded by Mycobacterium tuberculosis is RpfE which is antigenic, immunogenic, and probably secretory. This indicates RpfE holds promise as a potential vaccine candidate against tuberculosis. The aim of this study was to clone RfpE and subsequently optimize the expression of mature RpfE protein in Escherichia coli for purification. Materials and Methods: The coding sequence of RpfE was chemically synthesized. Full-length RpfE and a fragment of the gene lacking the signal peptide sequence (encoding mature RpfE) were respectively cloned in pET-23a (+) and pET-28a (+) plasmids. The polyhistidine (His)-tagged recombinant proteins were expressed in E. coli BL21 (DE3) and their cellular localization was determined. The optimal induction conditions were determined for mature RpfE. The protein was purified using a His-tag purification kit, was resolved on SDS-PAGE gel, and detected by western blot using anti-His antibody and serum from a consented tuberculosis patient. Results: Full-length RpfE (~26 kDa) was secreted into broth medium while mature RpfE (multimerized as a band of ~35 kDa) was accumulated in the cytoplasm of E. coli. The optimal expression of the mature RpfE was obtained in Luria-Bertani broth containing 1 mM isopropyl-β-d-thiogalactopyranoside at 37°C after 6 hrs (0.8 g/L of culture medium). The His-tag affinity chromatography followed by anti-His western blot analysis confirmed the purification of the mature His-tagged protein and the same-sized band was detected by the tuberculosis patient’s serum. Conclusion: The recombinant mature RpfE from M. tuberculosis lays the basis for immunogenicity studies as a vaccine candidate against tuberculosis.