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en Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli تخصصي Special مقالات اصلی Original Article Background: The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. Methods: An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. Results: The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. Conclusions: An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies. Mycobacterium tuberculosis, HspX/ EsxS fusion protein, Gene cloning, Protein expression, Protein purification 15 21 http://rbmb.net/browse.php?a_code=A-10-1-79&slc_lang=en&sid=1 Farzad Khademi 10031947532846002202 10031947532846002202 No Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Arshid Yousefi-Avarvand 10031947532846002203 10031947532846002203 No Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Mohammad Derakhshan 10031947532846002204 10031947532846002204 No Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Zahra Meshkat 10031947532846002205 10031947532846002205 No Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Mohsen Tafaghodi 10031947532846002206 10031947532846002206 No Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Kiarash Ghazvini 10031947532846002207 10031947532846002207 No Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Ehsan Aryan 10031947532846002208 10031947532846002208 No Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Mojtaba Sankian sankianm@mums.ac.ir 10031947532846002209 10031947532846002209 Yes Immunobiochemistry Lab, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran