Otimização da expressão e purificação de proteínas recombinantes miméticas a antígenos de Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), a serious global public health problem. TB is primarily transmitted via the respiratory route, and disease establishment involves the formation of an immune cell granuloma. Its progression can lead to symptoms such as persistent cough, fever, weight loss, general malaise, and in severe cases, even death. Despite the availability of treatment, diagnostic methods still present limitations, highlighting the need for the development of optimized diagnostic tools. In this context, research involving recombinant proteins that mimic MTB antigens has shown promise, as these biomarkers can make diagnostic methods faster and more accessible. This study aimed to optimize the production of two recombinant proteins, MTG3F3 and PMT64, mimetic to the MPT64 antigen of M. tuberculosis, with potential application in serological diagnostic tests. The genes of interest were cloned into pET32a expression plasmids and expressed in E. coli BL21(DE3). Different conditions were evaluated to maximize protein expression, including variations in temperature (30ºC and 37ºC), induction time (from 1 hour to overnight), and presence or absence of IPTG. The most favorable condition was incubation at 30ºC overnight with IPTG induction. After expression, the recombinant proteins were purified by affinity chromatography and evaluated by SDS-PAGE, with bands consistent with the expected molecular weights: 31.7 kDa for MTG3F3 and 29.9 kDa for PMT64. The purified samples were tested in Dot blot assays with sera from individuals with and without tuberculosis, showing reactivity of TB-positive sera against the proteins. The results demonstrated the feasibility of obtaining immunoreactive recombinant proteins from E. coli, reinforcing the potential of these biomolecules as candidates for use in rapid immunodiagnostic tests. Despite the positive results, future research aiming to improve the standardization of large-scale protein production is essential, along with additional studies such as ELISA assay standardization.