Biochemical and structural characterization of the Macrophage Migration Inhibitory Factor (MIF) protein as a potential target for drugs against Paracoccidioidomycosis
Name: PAULO ARTHUR COUTINHO
Type: MSc dissertation
Publication date: 14/12/2020
Advisor:
Name |
Role |
|---|---|
| JULIANA BARBOSA COITINHO GONCALVES | Advisor * |
Examining board:
| Name |
Role |
|---|---|
| JULIANA BARBOSA COITINHO GONCALVES | Advisor * |
| LUCAS CUNHA DIAS DE REZENDE | Internal Examiner * |
| RENATO GRACIANO DE PAULA | External Examiner * |
Summary: Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America, with a high incidence in Brazil and with cases reported in Espírito Santo. This disease, caused by fungi of the genus Paracoccidioides, affects the lungs and other organs and can be lethal if the correct treatment is not used. The treatment of PCM is carried out with traditional antifungals, and its cost is extremely high and prolonged. The MIF protein (macrophage migration inhibiting factor), which, in helminths, seems to facilitate the establishment of infection, does not have a three-dimensional structure resolved in Paracoccidioides and its primary sequence differs from other MIF proteins. Initially, to choose MIF as the object of study, the servers XtalPred, Expasy and BlastP were used and demonstrated that MIF is a good target for biochemical-structural characterization. In addition, the secondary and tertiary structure of the MIF was predicted by the servers ESPrit, PsiPred and i-TASSER, demonstrating that its predicted global structure agrees with the MIF of other organisms, but presents important differences for the FIM of humans that can be explored in a context treatment of PCM. Thus, the coding region for MIF was cloned into the vector pET-28aTEV. The recombinant vector was inserted in the strains of Escherichia coli BL21, ArcticExpress, Rosetta, Lemo21 and C43. Several expression tests varying inducer concentration, temperature and induction time and use of different additives were carried out, in addition to different lysis protocols in order to obtain the MIF protein in the soluble fraction. Thus, the recombinant protein was expressed in the soluble fraction only of the E. coli Rosetta strain with induction at 18 ° C for 72 hours and 1 mmol.L -1 of IPTG with addition of 1% ethanol (v.v -1 ) and was purified by cobalt affinity chromatography. All efforts to purify MIF from the soluble fraction are justified because this step is essential for its biochemical-structural characterization, which will certainly contribute to the development of more effective drugs for treatment, as well as a more efficient diagnostic alternative.
KEYWORDS: Paracoccidioides, Paracoccidioidomycosis, PCM, Macrophage Migration Inhibitory Factor, MIF.
