Banca de QUALIFICAÇÃO: MARCOS ANTÔNIO OLIVEIRA FILHO

Uma banca de QUALIFICAÇÃO de MESTRADO foi cadastrada pelo programa.
DISCENTE : MARCOS ANTÔNIO OLIVEIRA FILHO
DATA : 21/12/2017
HORA: 09:00
LOCAL: AUDITÓRIO DO CATRE
TÍTULO:

"Recuperation and partial purification of antigen 503 of Leishmania i. chagasi from recombinant E. coli M15 and simultaneous LPS removal using Aqueous Two-Phase Systems"

PALAVRAS-CHAVES:

Leishmaniasis, ATPS, antigen 503, endotoxin, LPS removal.

PÁGINAS: 67
GRANDE ÁREA: Engenharias
ÁREA: Engenharia Química
RESUMO:

The antigen 503, a protein found in the amastigote phase of Leishmania i. chagasi, can integrate vaccines and specific diagnosis assays for visceral leishmaniasis (VL). However, a problem on the protein purification is the presence of endotoxins (LPS), natural compound of bacterial expression systems, which triggers immune response in human body and interacts with the proteins impairing possible chromatography steps. To solve this problem, Aqueous Two-phase Systems (ATPS) can be employed with the advantages of eliminate clarification steps on the whole process, indeed it also concentrates the molecule of interest and reduces the concentration of LPS, preparing the product for a more efficient adsorption in the following steps. Thus, the present work aimed evaluate ATPS based on PEG1500/Salt, Ethanol/Salt (K₂HPO₄ and (NH₄)₂SO₄) and Acetonitrile/Dextrose on the partial purification of antigen 503 expressed in recombinant E. coli M15 and simultaneous removal of LPS. For that, firstly, binodal curves of these systems were constructed by cloud point titration method at room temperature (25 ± 2 ºC). The parameters of the equation purposed by Merchuk were calculated by these experimental data and the Tie-Line Lengthes (TLL) were obtained. Generally, the TLL rose with an increase of the concentration of organic solvent. When the cell homogenate from E. coli was applied, the system which exhibited the best results was 30% PEG1500 and 10% K₂HPO₄ (m/m) achieving 116,96% of recuperation and 1,55 of purification factor (assay P3). The removal of endotoxins was promising for all the systems evaluated, as it was kept above 90,0%. The assay P3 also stood out for the LPS removal, being able to remove 99,90%. Thus, ATPS can be a precedent step to the chromatography for the removal of high LPS concentration and grow the purity of recombinant proteins from E. coli.

 

MEMBROS DA BANCA:
Externo ao Programa - 2378605 - CRISTIANE FERNANDES DE ASSIS
Presidente - 1346198 - EVERALDO SILVINO DOS SANTOS
Externo ao Programa - 3652554 - FRANCISCO CANINDE DE SOUSA JUNIOR