Over-expression, purification, and characterisation of Mycobacterium tuberculosis MPT64 from inclusion bodies.

Tuberculosis remains among the top ten deadliest infectious diseases worldwide, primarily due to delayed diagnosis, co-infection with HIV, and multidrug resistance. Targeting early secreted mycobacterial antigens, including MPT64, ESAT6, and CFP10, may be a viable strategy for detecting the onset of tuberculosis. Although recombinant MPT64 has been produced previously, limited information is available regarding its structural integrity and thermodynamic unfolding behaviour following recovery from inclusion bodies. In this study, MPT64 protein was overexpressed, affinity-purified, and characterised using biophysical and biochemical methods. The purified recombinant MPT64 had a purity greater than 95%, yielding 2 mg from 1 L of bacterial culture. Western blot analysis confirmed MPT64 identity, while ELISA demonstrated antibody recognition of refolded MPT64, with markedly reduced binding following heat denaturation, indicating that refolding preserved conformational epitopes. Far-UV circular dichroism (CD) and intrinsic fluorescence spectroscopy spectral profiles confirmed that the purified MPT64 protein retained its folded secondary and tertiary structural integrity. Thermodynamic analysis by fluorescence and Far-UV CD yielded stability values (ΔG°) of 19.9 and 26.2 kJ·mol-1, with unfolding midpoints of 5.3 and 5.5 M urea, respectively. Differential scanning calorimetry and thermal circular dichroism indicated melting transitions between 62.5 and 58 °C, supporting moderate thermal stability. Collectively, these complementary measurements provide a detailed biophysical assessment of the conformational and thermal stability of recombinant MPT64 following inclusion-body recovery and refolding. The findings provide a biophysical basis for further investigation of MPT64 as a recombinant antigen for tuberculosis diagnostics.