The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method.
Keywords: MPT64; electroelution; intracellular; non-tagged; passive elution.