Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli

Authors

  • Arshid Yousefi-Avarvand 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 Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
  • Farzad Khademi Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
  • Kiarash Ghazvini 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 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 Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
  • Mojtaba Sankian Immunobiochemistry Lab, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
  • Zahra Meshkat Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, 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. 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.

Upgrade to premium to download articles

Sign up to access the full text

Already have an account?login

similar resources

mycobacterium tuberculosis hspx/esxs fusion protein: gene cloning, protein expression, and purification in escherichia coli

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 wit...

full text

Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

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 wit...

full text

Molecular Cloning, Expression and Purification of Protein TB10.4 Secreted by Mycobacterium Tuberculosis

Objective(s) Tuberculosis (TB) is the leading cause of mortality among the infectious diseases, especially in developing countries. One of the main goals in tuberculosis research is to identify antigens which have the ability of inducing cellular and/or humoral immunity in order to use them in diagnostic reagents or vaccine design. The aim of this study was to clone and express the TB'0.4 prot...

full text

cloning and expression of mycobacterium tuberculosis major secreted protein antigen 85b (ag85b) in escherichia coli

background the 30 kda major secretory protein of mycobacterium tuberculosis (antigen 85b) is a primary vaccine candidate. this secreted antigen induces a protective immune response and stimulates the production of ifn-γ in animal models. objectives the aim of this study was cloning and expression of ag 85b of m. tuberculosis in escherichia coli. materials and methods to produce recombinant ag85...

full text

Cloning and evaluation of gene expression and purification of gene encoding recombinant protein containing binding subunit of coli surface antigens CS1 and CS2 from Enterotoxigenic Escherichia coli

Background & Objective: Enterotoxigenic Escherichia coli (ETEC) is a major causative agent of diarrhea. Enterotoxins and the colonization factors (CFs) are major virulence factors in ETEC infections. The bacterium binds to the intestinal epithelial cell surface through colonization factors and produces enterotoxins that cause excessive fluid and electrolyte secretion in the lumen of the intesti...

full text

Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli

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 rv1...

full text

My Resources

Save resource for easier access later

Save to my library Already added to my library

{@ msg_add @}


Journal title

volume 6  issue 1

pages  15- 21

publication date 2017-10

By following a journal you will be notified via email when a new issue of this journal is published.

Keywords

Hosted on Doprax cloud platform doprax.com

copyright © 2015-2023