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Master's Dissertation
DOI
10.11606/D.87.2010.tde-13082010-163827
Document
Author
Full name
Fernanda Resende Gomes
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2010
Supervisor
Committee
Ho, Paulo Lee (President)
Soares, Irene da Silva
Winter, Lucile Maria Floeter
Title in Portuguese
Expressão do fator estimulador de colônia de granulócito humano recombinante (rhG-CSF) em Escherichia coli.
Keywords in Portuguese
Escherichia coli
Corpos de Inclusão
Expressão heteróloga
Fatores biológicos
Genética bacteriana
Granulócitos (Humano)
Granulócitos citoplasmáticos
rhG-CSF
Abstract in Portuguese
O Fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) produzido em Escherichia coli é uma proteína não glicosilada com 175 aminoácidos, de grande importância clínica para o tratamento de neutropenias. O presente trabalho propõe a construção de dois sistemas de expressão em E. coli, um sistema para obtenção do rhG-CSF no citoplasma e outro para secreção da proteína recombinante no meio de cultura utilizando a sequência sinal da L-asparaginase II. Os dois sistemas de expressão foram testados e comparados. A partir desses dados, passou-se para as etapas de obtenção do rhG-CSF com o sistema de expressão sem a sequência sinal. As etapas de renaturação e purificação foram eficientes obtendo-se uma proteína com adequado grau de pureza, integridade estrutural e atividade biológica. Essa proteína também foi utilizada com sucesso para a produção de anticorpos policlonais em camundongos. Com os resultados obtidos, a proteína rhG-CSF mostrou-se viável para estudos posteriores em bioreatores e produção em escala-piloto.
Title in English
Expression of recombinant human colony stimulating factor (rhG-CSF) in Escherichia coli.
Keywords in English
Escherichia coli
Bacterial genetics
Biological factors
Granulocytes (Human)
Granulocytes cytoplasmic
Heterolog expression
Inclusion body
rhG-CSF
Abstract in English
The recombinant human granulocyte colony stimulating factor (rhG-CSF) is a non-glycosylated protein with 175 amino acids. This factor plays an important role in hematopoietic cell proliferation and has been widely used for treating neutropenia. The purpose of this work is to construct two expression systems in E. coli; a system for obtaining rhG-CSF in the cytoplasm and the other for secretion of recombinant protein in the culture medium using the signal sequence of L-asparaginase II. The two expression systems were tested and compared. From these data, the next steps for obtaining the rhG-CSF were done with the expression system without the signal sequence. The refolding and purification steps were efficient, resulting in a protein with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of polyclonal antibodies in mice. With these results, the protein rhG-CSF was feasible for further studies in bioreactors and pilot scale production.
 
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Publishing Date
2010-09-16
 
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