Investigation of the use of self-cleaving intein tag including chitin binding domain in the recombinant production of MPT64, the immunogenic protein of M. tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a global public health challenge, largely due to diagnostic limitations. Current diagnostic methods, including the Tuberculin Skin Test (TST), often yield false positives due to cross-reactivity with the Bacillus Calmette-Guérin (BCG) vaccine. MPT64 protein, a component of the Purified Protein Derivative (PPD) used in TST and can be used to improve the specificity of TB diagnosis. In this study, it was investigated the feasibility of using self-associated intein protein and chitin binding tag in the recombinant production of MPT64 protein. For this purpose, MPT64 coding DNA sequences were cloned into two different plasmid vectors, pTXB1 and pTYB21, and expression conditions were investigated. In the conditions of enriched medium, the expression was more successful in the pTYB21vector. Both E. coli ER2566 and E. coli BL21(DE3) Rare were used as hosts. Since the recombinant protein was obtained as inclusion body, urea was used for solubilization. Since urea was incompatible with the chitin column, refolding was achieved by dialysis. These additional steps resulted in a decrease in the protein yield.