Banca de DEFESA: Letícia Santos Abrunhosa

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : Letícia Santos Abrunhosa
DATE: 31/08/2023
TIME: 09:30
LOCAL: A definir
TITLE:

Cloning and expression of L-asparaginase from Fusarim proliferatum in Eschericha coli

KEY WORDS:

L-asparaginase, Eschericha coli, recombinant protein, Fusarium proliferatum, heterologous expression

PAGES: 100
BIG AREA: Ciências da Saúde
AREA: Farmácia
SUMMARY:

L-asparaginase is an enzyme used for the treatment of Acute Lymphoblastic Leukemia, which mostly affects children and teenagers. Currently, Brazil imports L-asparaginase without National Health Surveillance Agency registration, making it important to have a national production of this enzyme and the need to search for alternative production methods. One alternative would be cloning and expressing L-asparaginase from Fusarium proliferatum in Escherichia coli, with the aim of obtaining higher enzymatic productivity and reducing production costs. Another important factor would be the attenuation of adverse effects caused by treatment with enzyme obtained from prokaryotic sources, since Fusarium proliferatum is an eukaryotic microorganism. Therefore, the objective of this study was to clone L-asparaginase obtained from Fusarium proliferatum and express it in Escherichia coli BL21(DE3). The L-asparaginase gene from Fusarium proliferatum was identified and synthesized in pET-28a(+) vector with codon optimization for E. coli and NdeI/XhoI restriction sites. The vector was then transformed for replication in E. coli DH10B, with subsequent transformation for expression in E. coli BL21(DE3). After confirming the insertion of the vector in the expression system, clones were selected for evaluation of L-asparaginase production. SDS-PAGE and Western Blot were used to select the clone with the highest enzyme expression, followed by evaluation of the best cell lysis method. Once the clone and the most efficient cell lysis condition were defined, screening was performed to optimize cultivation conditions, varying the IPTG inducer concentration, post-induction time and induction temperature.The soluble and insoluble fractions were used for enzymatic assay, where asparaginase activity was measured by Nessler and ß-hydroxamate aspartic acid methods.

BANKING MEMBERS:
Presidente - 2329402 - ANGELICA AMORIM AMATO
Interna - 1562111 - PEROLA DE OLIVEIRA MAGALHAES DIAS BATISTA
Externa ao Programa - 1529483 - MARIA DE FATIMA BORIN
Externo à Instituição - FELIX GONÇALVES DE SIQUEIRA