Use of plasmids for expression of proteins from the genus Leishmania in Escherichia coli: current state and perspectives.

Leishmaniosis is caused by the protozoa of the genus Leishmania with a wide spectrum of clinical and epidemiological manifestations which are characterized into four clinical groups: cutaneous, mucocutaneous, diffuse cutaneous, and visceral. American visceral leishmaniosis (AVL) or visceral leishmaniosis (VL) has been known as the most severe form of the disease. However, despite the growing number of people exposed to the infection risk and the great effort done by the scientific community worldwide to significantly increase the knowledge about these diseases, there is no vaccine capable of preventing VL in humans. In this short review, we present some of the plasmids used for the expression of recombinant protein by Escherichia coli strains used mainly for the second generation of vaccines for leishmaniosis. It can be emphasized that currently, these vectors and hosts play an important role in developing vaccine strategies against the disease. Indeed, use of the E. coli BL21 (DE) strain is remarkable mainly due to its characteristics for being a stable protein producer as well as the use of histidine tags for antigen purification. KEY POINTS: • Plasmid vectors and E. coli will continue being important for studies about leishmaniosis. • Protein purification exploiting histidine tags is a key technique.

Evaluation of induction conditions for plasmid pQE-30 stability and 503 antigen of Leishmania i. chagasi expression in E. coli M15.

The present study aimed to evaluate the influence of induction conditions (IPTG concentration, temperature, and induction time) on the plasmid pQE-30 stability and 503 antigen expression of Leishmania i. chagasi in Escherichia coli M15. Batch cultures were performed at 37 °C and induced by the addition of different IPTG concentrations (0.01 to 1.5 mM). Subsequently, experiments were carried out at different temperatures (27 to 42 °C), evaluating the influence of induction time (0.5 to 6 h after the start of the culture). The results showed that IPTG toxicity caused a metabolic stress in the cells and, consequently, the microorganism growth reduced. The induction with IPTG may also be associated with the plasmid pQE-30 instability, due to metabolic burden imposed by the recombinant protein expression. The optimal conditions for 503 antigen expression of Leishmania i. chagasi in Escherichia coli M15 were an IPTG concentration of 1.0 mM, temperature of 37 °C, and induction time of 2 h. The maximum antigen concentration obtained was 0.119 ± 0.009 g/L, about seven times higher than the lowest concentration. Therefore, the results showed that 503 antigen can be produced in laboratory; however, it requires more studies to minimize the plasmid instability and improve to industrial scale.