Farzad Khademi1, Arshid Yousefi-Avarvand1, Mohammad Derakhshan1, Zahra Meshkat1, Mohsen Tafaghodi2, Kiarash Ghazvini1, Ehsan Aryan1, Mojtaba Sankian3. 1. Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. 2. Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. 3. Immunobiochemistry Lab, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Abstract
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. CONCLUSION: 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.
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. CONCLUSION: 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.
Entities:
Keywords:
Gene cloning; HspX/ EsxS fusion protein; Mycobacterium tuberculosis; Protein expression; Protein purification
Authors: Annemieke Geluk; May Young Lin; Krista E van Meijgaarden; Eliane M S Leyten; Kees L M C Franken; Tom H M Ottenhoff; Michèl R Klein Journal: Infect Immun Date: 2007-03-26 Impact factor: 3.441
Authors: Abebech Demissie; Eliane M S Leyten; Markos Abebe; Liya Wassie; Abraham Aseffa; Getahun Abate; Helen Fletcher; Patrick Owiafe; Philip C Hill; Roger Brookes; Graham Rook; Alimuddin Zumla; Sandra M Arend; Michel Klein; Tom H M Ottenhoff; Peter Andersen; T Mark Doherty Journal: Clin Vaccine Immunol Date: 2006-02
Authors: Michala V Rose; Godfather Kimaro; Thomas N Nissen; Inge Kroidl; Michael Hoelscher; Ib C Bygbjerg; Sayoki G Mfinanga; Pernille Ravn Journal: PLoS One Date: 2012-07-12 Impact factor: 3.240