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Master's Dissertation
DOI
10.11606/D.9.2002.tde-11072003-110847
Document
Author
Full name
Eb Chiarini
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2002
Supervisor
Committee
Penna, Thereza Christina Vessoni (President)
Hirata, Rosario Dominguez Crespo
Pessoa Júnior, Adalberto
Title in Portuguese
Comparação entre os métodos físico e químico de permeação e de extração da proteína verde fluorescente (GFPuv) de culturas de Escherichia coli dh5-alpha
Keywords in Portuguese
Escherichia coli
GFPuv
Proteínas
Abstract in Portuguese
A proteína verde fluorescente (GFPuv), pelo fato de ter como característica a emissão de luz verde fluorescente brilhante quando exposta à luz ultravioleta, tem sido utilizada como marcador em diversos campos de pesquisa. Células transformadas de Escherichia coli DH5-a expressando a GFPuv foram submetidas aos tratamentos: (i) permeação seletiva (congelamento/ descongelamento/ sonicação) associada à extração por partição em três fases (TPP) e posterior purificação em coluna cromatográfica de interação hidrofóbica (HIC), e (ii) extração direta das células por TPP e posterior purificação em coluna HIC. Este trabalho teve por objetivo a extração da GFPuv com a aplicação das metodologias citadas para a extração da proteína, avaliando a influência da permeação seletiva das células de Escherichia coli no processo de extração da GFPuv. Na análise dos resultados observou-se que apesar da permeação seletiva ser uma metodologia mais laboriosa, mostrou-se mais eficiente pela obtenção de aproximadamente 9 vezes mais GFPuv em relação à extração direta das células por TPP.
Title in English
Comparison between physical and chemical methods for permeation and extraction of green fluorescent protein (GFPuv) from 'Escherichia coli' DH5-alpha
Keywords in English
Escherichia coli
GFPuv
Protein.
Abstract in English
The green fluorescent protein (GFPuv), for the fact of having as characteristic the emission of brilliant green fluorescent light when exposed to the ultraviolet light, it has been used as marker in several research fields. Transformed cells of Escherichia coli DH5-a expressing GFPuv was subjected to: (i) selective permeation (freezing / thawing/ sonication) associated to the extraction for partition in three phases (TPP) and subsequent purification in hydrophobic interaction chromatography column (HIC), and (ii) direct extraction of the cells for TPP and subsequent purification in HIC column. This work had for objective the extraction of GFPuv with the application of the methodologies mentioned for the extraction of the protein, evaluating the influence of the selective permeation in the cells of Escherichia coli in the process of GFPuv extraction. In the analysis of the results was observed that in spite of the selective permeation to be a more laborious methodology, it was shown more efficient for the obtaining of approximately 9 more times GFPuv in relation to the direct extraction of the cells for TPP.
 
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Publishing Date
2007-02-01
 
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