Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 9 2 2017 08 12 64 73 EN Mitra Ashayeri-Panah Department of Microbiology, Faculty of Biological Sciences and Technology, Shahid Beheshti University, Tehran, Iran Fereshteh Eftekhar Department of Microbiology, Faculty of Biological Sciences and Technology, Shahid Beheshti University, Tehran, Iran Bahram Kazemi Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Joan Joseph Hospital Clinic/HIVACAT, School of Medicine, University of Barcelona, Barcelona, Spain 2017 08 12 2017 08 12 Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E. coli BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting. Results: Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the E. coli cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4 and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient’s serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in E. coli which was successfuly purified. Conclusion: We believe that the purified Rv1733c recombinant protein from M. tuberculosis might be a good candidate for vaccine production against tuberculosis. https://ijm.tums.ac.ir/index.php/ijm/article/view/1458 https://ijm.tums.ac.ir/index.php/ijm/article/download/1458/706