Volume 6, Number 1 (Vol.6 No.1 Oct 2017)                   rbmb.net 2017, 6(1): 15-21 | Back to browse issues page


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Khademi F, Yousefi-Avarvand A, Derakhshan M, Meshkat Z, Tafaghodi M, Ghazvini K, et al . Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli. rbmb.net. 2017; 6 (1) :15-21
URL: http://rbmb.net/article-1-134-en.html

Immunobiochemistry Lab, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Abstract:   (2365 Views)

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.

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Type of Article: Original Article | Subject: Special
Received: 2017/01/16 | Accepted: 2017/01/16 | Published: 2017/01/16

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