1. High-yield production of major T-cell ESAT6-CFP10 fusion antigen of M. tuberculosis complex employing codon-optimized synthetic gene.
- Author
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Gutiérrez-Ortega A, Moreno DA, Ferrari SA, Espinosa-Andrews H, Ortíz EP, Milián-Suazo F, and Alvarez AH
- Subjects
- Amino Acid Sequence, Animals, Antigens, Bacterial immunology, Bacterial Proteins immunology, Cattle, Cloning, Molecular, Codon, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Histidine genetics, Histidine metabolism, Immunogenicity, Vaccine, Interferon-gamma biosynthesis, Mycobacterium bovis chemistry, Mycobacterium bovis genetics, Mycobacterium tuberculosis chemistry, Mycobacterium tuberculosis genetics, Mycobacterium tuberculosis immunology, Oligopeptides genetics, Oligopeptides metabolism, Peptide Fragments immunology, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Sequence Alignment, Tuberculosis Vaccines genetics, Tuberculosis Vaccines immunology, Tuberculosis, Bovine immunology, Tuberculosis, Bovine microbiology, Vaccination methods, Antigens, Bacterial genetics, Bacterial Proteins genetics, Mycobacterium bovis immunology, Peptide Fragments genetics, Recombinant Fusion Proteins administration & dosage, Tuberculosis Vaccines administration & dosage, Tuberculosis, Bovine prevention & control
- Abstract
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., Competing Interests: Declaration of competing interest The authors confirm that this article content has no conflicts of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2021
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