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Master's Dissertation
DOI
10.11606/D.87.2016.tde-11032016-112447
Document
Author
Full name
Flávia Garcia Borges
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2015
Supervisor
Committee
Alcalde, Felipe Santiago Chambergo (President)
Ferreira Júnior, José Ribamar dos Santos
Marana, Sandro Roberto
Title in Portuguese
Análise do promotor quimérico regulado por zinco para a expressão de proteínas recombinantes em Saccharomyces cerevisiae.
Keywords in Portuguese
Fungos
Leveduras
Promotor
Sistema de expressão
Zinco
Abstract in Portuguese
O desenvolvimento de sistemas de expressão através da modificação de fortes promotores conhecidos vem sendo amplamente utilizado para a produção de proteínas com potencial utilização biotecnológica em diversos hospedeiros como S. cerevisiae. O objetivo do trabalho foi construir um promotor quimérico através de modificações do promotor cbh1 de T. reesei e inserção de elementos de resposta a metais provenientes do promotor ZRT1 de S. cerevisiae. O promotor desenhado foi utilizado na construção de um sistema de expressão, que foi inserido na levedura e teve sua atividade analisada pelo teste de ONPG. Foi possível observar que, conforme a concentração de zinco aumenta, a atividade do promotor diminui. O promotor teve maior atividade no meio sem zinco e menor no meio com 1000 μM de zinco. Esses resultados confirmam que o sistema funciona de forma eficiente em S. cerevisiae. Também foi possível observar que os mutantes crescidos em meio com limitação de zinco e, consequentemente com maior atividade do promotor, tiveram sua taxa de crescimento alterada. O que é esperado devido ao fenômeno conhecido como estresse metabólico, comum durante a produção de proteínas recombinantes em leveduras, na qual a cultura apresenta uma diminuição significativa do crescimento.
Title in English
Analysis of chimeric promoter regulated by zinc for the expression of recombinant proteins in Saccharomyces cerevisiae.
Keywords in English
Expression system
Fungi
Promoter
Yeasts
Zinc
Abstract in English
The development the expression systems by modifying strong promoters has been widely used for the production of proteins with potential biotechnological use in various hosts such as S. cerevisiae. This study aimed to construct an expression system constituted of the chimeric promoter built with cbh1 promoter modified by inserting metal responsive elements from the promoter of ZRT1 gene of S. cerevisiae. The S. cerevisiae was transformed and the system was induced with different concentrations of zinc and was tested using ONPG as substrate. It was observed that under high zinc concentrations promoter activity is low. At low zinc concentrations the opposite effect is observed, and the promoter reaches its highest activities. These results confirm that the system functions efficiently in S. cerevisiae. It was also observed that the mutant grown in environment with zinc limitation, hence with higher activity of the promoter, showed reduced growth rate. Indeed, this is expected due to the phenomenon known as metabolic burden, characterized by a joint stress during the production of recombinant proteins in yeast, under conditions which the culture has a significant growth reduction.
 
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Publishing Date
2016-03-11
 
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