Mycobacterium Tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia Coli

Overview

Journal Rep Biochem Mol Biol

Specialty Biochemistry

Date 2017 Nov 2

PMID 29090225

Citations 6

Authors

Farzad Khademi

Arshid Yousefi-Avarvand

Mohammad Derakhshan

Zahra Meshkat

Mohsen Tafaghodi

Kiarash Ghazvini

Ehsan Aryan

Mojtaba Sankian

Affiliations

Soon will be listed here.

Abstract

Background: The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of in a prokaryotic system.

Methods: An gene construct was synthesized and ligated into a pGH plasmid, 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. 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 gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in 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 fusion protein was expressed in 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.

Citing Articles

Enhancement of the immunogenicity of a fusion protein using ISCOMATRIX and PLUSCOM nano-adjuvants as prophylactic vaccine after nasal administration in mice.

Yousefi Avarvand A, Meshkat Z, Khademi F, Aryan E, Sankian M, Tafaghodi M Iran J Basic Med Sci. 2024; 27(1):24-30.

PMID: 38164481 PMC: 10722485. DOI: 10.22038/IJBMS.2023.69295.15100.

Evaluation of Immunogenicity, Protective Immunity on Aquaculture Pathogenic and Fermentation of Flagellin FlaC Protein.

Chen C, Kang C, Rong N, Wu N, Chen C, Wu S Iran J Biotechnol. 2020; 17(3):e2628.

PMID: 32195288 PMC: 7080974. DOI: 10.29252/ijb.2628.

Enhancing immunogenicity of novel multistage subunit vaccine of using PLGA:DDA hybrid nanoparticles and MPLA: Subcutaneous administration.

Khademi F, Yousefi A, Derakhshan M, Najafi A, Tafaghodi M Iran J Basic Med Sci. 2019; 22(8):893-900.

PMID: 31579445 PMC: 6760476. DOI: 10.22038/ijbms.2019.33962.8079.

Formulation and Optimization of a New Cationic Lipid-Modified PLGA Nanoparticle as Delivery System for HspX/EsxS Fusion Protein: An Experimental Design.

Khademi F, Yousefi-Avarvand A, Derakhshan M, Abbaspour M, Sadri K, Tafaghodi M Iran J Pharm Res. 2019; 18(1):446-458.

PMID: 31089379 PMC: 6487422.

Cloning and expression of MPT83 gene from in BL21 as vaccine candidate of tuberculosis: A preliminary study.

Ahmad A, Agus R, Massi M, Natzir R, Madhyastha R, Madhyastha H J Genet Eng Biotechnol. 2019; 16(2):335-340.

PMID: 30733743 PMC: 6353755. DOI: 10.1016/j.jgeb.2018.04.001.

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