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--d-thiogalactopyranoside at 37C 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.