High-yield production of major T-cell ESAT6-CFP10 fusion antigen of M. tuberculosis complex employing codon-optimized synthetic gene

Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 μg/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-γ release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes. [Display omitted] •Differences in codon usage of Mycobacterium genes in E. coli may impede the translation of heterologous proteins.•A soluble rESAT6-CFP10 protein was produced in E. coli with a codon-optimized synthetic gene.•The theoretical topology of the target rESAT6-CFP10 antigen was consistent with the natural complex molecule.•A multi-epitope rESAT6-CFP10 protein was achieved with featured antigenic properties.•Translation engineering is a feasible and cost-effective strategy for the rational design of multi-epitope antigens.