Banca de DEFESA: BRUNO GILDO DALLA VECCHIA MORALES

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : BRUNO GILDO DALLA VECCHIA MORALES
DATE: 09/11/2023
TIME: 13:00
LOCAL: Sala Virtual Remora https://bit.ly/Def_Doc_BrunoGildo_PGBIOEXP
TITLE:

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KEY WORDS:

Malaria. Plasmodium falciparum. Protein expression. Protein purification. Shikimate dehydrogenase.

PAGES: 96
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

Plasmodium falciparum is known to cause severe malaria. Current treatment consists in artemisinin-based combination therapy, but resistance can lead to treatment failure. The knowledge of essential proteins of P. falciparum can be used on the search for new antimalarials, among these is shikimate dehydrogenase (SDH), part of a pathway responsible for the production of endogenous aromatic amino acids. SDH from P. falciparum (PfSDH) is unknown to the scientific community, therefore, this study aims to establish the first protocol for active PfSDH expression. Putative PfSDH nucleotide sequence was used to construct the optimized expression vector pET28a+PfSDH inserted in E. coli BL21(DE3). Optimal expression conditions were acquired by varying IPTG and time while temperature was fixed at 37 °C. Western Blot analysis was applied to verify appropriate PfSDH expression. Solubilization and purification started with lysis followed by double IMAC purification. SDS-PAGE was applied over various steps. Enzyme activity was measured spectrophotometrically by NADPH oxidation. Tanimoto coefficient and pharmacophore pattern similarity were applied to the in silico search for ligands employing the substrate DHS as template. Optimal PfSDH expression occur at 0.1 mM IPTG for 48 hours of growth at 37 °C and shaking at 200 rpm. Recombinant PfSDH obtained after purification was soluble, pure and its physiological catalysis was confirmed. Among all possible ligands obtained, 15 molecules had greater relevance when combining both methods applied. Thus, this study describes the first protocol for heterologous expression of PfSDH in soluble and active form, likewise instigating for a higher comprehension related to target-ligant interactions.

COMMITTEE MEMBERS:
Externo à Instituição - SPARTACO ASTOLFI FILHO - UFAM
Interno - ***.429.088-** - CAROLINA BIONI GARCIA TELES - FIOCRUZ
Presidente - ***.646.382-** - FERNANDO BERTON ZANCHI - UNIR
Externa à Instituição - JULIANE CORRÊA GLÓRIA - FIOCRUZ
Externo à Instituição - MARCO ALBERTO MEDEIROS