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

• Published in: International journal of biological macromolecules Vol. 171; pp. 82 - 88
• Main Authors: Gutiérrez-Ortega, A., Moreno, D.A., Ferrari, S.A., Espinosa-Andrews, H., Ortíz, E.P., Milián-Suazo, F., Alvarez, A.H.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 28.02.2021
• Subjects: Amino Acid Sequence; Animals; Antigenicity; Antigens, Bacterial - genetics; Antigens, Bacterial - immunology; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Cattle; Cloning, Molecular; Codon; Codon optimization; Escherichia coli - genetics; Escherichia coli - metabolism; Fusion protein; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Histidine - genetics; Histidine - metabolism; Immunogenicity, Vaccine; Interferon-gamma - biosynthesis; Mycobacterium bovis - chemistry; Mycobacterium bovis - genetics; Mycobacterium bovis - immunology; Mycobacterium tuberculosis - chemistry; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - immunology; Oligopeptides - genetics; Oligopeptides - metabolism; Peptide Fragments - genetics; Peptide Fragments - immunology; Recombinant Fusion Proteins - administration & dosage; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Sequence Alignment; Soluble ESAT6-CFP10; Synthetic gene; Tuberculosis Vaccines - administration & dosage; Tuberculosis Vaccines - genetics; Tuberculosis Vaccines - immunology; Tuberculosis, Bovine - immunology; Tuberculosis, Bovine - microbiology; Tuberculosis, Bovine - prevention & control; Vaccination - methods; Soluble ESAT6-CFP10; Synthetic gene; Antigenicity; Fusion protein; Codon optimization
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2020.12.179

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.