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en Heterologous Expression, Purification, and Characterization of the HspX, Ppe44, and EsxV Proteins of Mycobacterium tuberculosis بیوشیمی Biochemistry مقالات اصلی Original Article <div style="text-align: justify;">Background: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe44, and EsxV as a new construction, were expressed alone and as a fusion protein to develop a new vaccine candidate against tuberculosis infection. Methods: To make the fusion protein, the three genes were linked together by AEAAAKEAAAKA linkers and inserted into pET21b and pET32b vectors. Escherichia coli (E. coli) Top10 cells were transformed with the plasmid, and the purified plasmid was used to transform E. coli BL21 cells. Protein expression was induced with IPTG. After optimizing protein expression, the recombinant proteins were purified by Ni-NTA chromatography. Protein purification was confirmed by SDS-PAGE and Western blotting with an anti-poly histidine-peroxidase monoclonal antibody against the 6His–tags at the proteins’ C termini. Results: Directional cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing. The highest expression of the tri-fusion protein and HspX were obtained by the addition of 0.2 mM of IPTG to E. coli BL-21 cells at 37 ˚C and 18 h of incubation. For Ppe44 and EsxV, the optimum expression conditions were 18 ˚C and 16 h of incubation. SDS-PAGE and Western blots confirmed that the desired proteins were produced. Conclusions: The three desired proteins and the fusion protein were successfully expressed and the conditions for optimum expression determined. These recombinant proteins will be evaluated as vaccine candidates against tuberculosis. Further studies are needed to evaluate the abilities of these proteins to induce strong immunological responses.</div> EsxV, Expression, HspX, Mycobacterium tuberculosis, Ppe44, Purification 125 130 http://rbmb.net/browse.php?a_code=A-10-31-1&slc_lang=en&sid=1 Yousef Amini aminiy901@mums.ac.ir 10031947532846002870 10031947532846002870 No Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran. Mohsen Tafaghodi tafaghodim@mums.ac.ir 10031947532846002871 10031947532846002871 No Nanotechnology Research Center, Pharmaceutical Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Saied Amel Jamehdar amelJS@mums.ac.ir 10031947532846002872 10031947532846002872 No Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran. zahra Meshkat meshkatz@mums.ac.ir 10031947532846002873 10031947532846002873 No Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran. Bagher Moradi moradib@gmail.com 10031947532846002874 10031947532846002874 No Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran. Mojtaba Sankian sankianm@mums.ac.ir 10031947532846002875 10031947532846002875 Yes Immunology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.