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Master's Dissertation
DOI
10.11606/D.9.2016.tde-15122015-082825
Document
Author
Full name
Rafaela Coelho Correia
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2015
Supervisor
Committee
Pessoa Junior, Adalberto (President)
Ebinuma, Valéria de Carvalho Santos
Oliveira, Ricardo Pinheiro de Souza
Title in Portuguese
Produção biotecnológica de L-asparaginase(ASP3) de Saccharomyces cerevisiae em sistema de expressão heterólogo Pichia pastoris
Keywords in Portuguese
Biotecnologia
L-Asparaginase
Leucemia linfóide aguda
Pichia pastoris
Proteína recombinante
Abstract in Portuguese
A leucemia linfóide aguda (LLA) é considerada uma doença grave dos glóbulos brancos, sendo mais comum e mais agressiva em crianças e adolescentes. O tratamento para a LLA tem avançado devido aos estudos para a otimização de drogas já utilizadas em quimioterapias. Entre essas drogas estão as enzimas L- asparaginases (ASPases) que atuam reduzindo a concentração de L-asparagina (Asn) na corrente sanguínea, impedindo a proliferação das células cancerosas, visto que essas não conseguem sintetizar quantidades apropriadas desse aminoácido. No entanto, o medicamento por ser oriundo de um procarioto causa severas reações alérgicas aos usuário, afim de diminuir a imunogenicidade deste quimioterápico, é importante gerar um biofármaco oriundo de um eucarioto. Neste âmbito, obtivemos a Pichia pastoris recombinante responsável pela produção da enzima ASPase intermembranar, oriunda do gene ASP3 de Saccharomyces cerevisiae. Através do planejamento experimental, foi possível ter um aumento de 5 vezes na atividade obtida na condição inicial. O clone Mut+ alcançou sua melhor atividade de 8,6 U/g de célula nas seguintes condições: 20°C, pH inicial 6 e 1,5% de concentração de indutor.
Title in English
Biotechnological production of L- asparaginase ( ASP3 ) of Saccharomyces cerevisiae in a heterologous expression system Pichia pastoris
Keywords in English
Acute lymphoblastic leukemia
Biotechnology
L-asparaginase
Pichia pastoris
Recombinant protein
Abstract in English
Acute lymphoblastic leukemia (ALL) is considered a serious disease of white blood cells, is more common and more aggressive in children and adolescents. Treatment for ALL has advanced due to studies for drug optimization already used in chemotherapy. Among these drugs are the enzymes L-asparaginases (ASPases) which act by reducing the concentration of L-asparagine (Asn) in the bloodstream, preventing the proliferation of cancer cells, since these can not synthesize appropriate amounts of this amino acid. However, the drug to be derived from a prokaryote causes severe allergic reactions to the user, in order to decrease the immunogenicity of the chemotherapy, it is important to generate a biopharmaceutical derived from a eukaryote. In this context, we obtained the recombinant Pichia pastoris responsible for producing the enzyme ASPase intermembrane, coming from the ASP3 gene of Saccharomyces cerevisiae. Through the experimental design, it was possible to have a 5-fold increase in activity obtained at the initial condition. The Mut + clone achieved their best activity of 8.6 U/g cell under the following conditions: 20 °C, initial pH 6 and 1.5% of inducer concentration.
 
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Publishing Date
2016-02-15
 
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