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Master's Dissertation
DOI
https://doi.org/10.11606/D.87.2008.tde-08012009-163245
Document
Author
Full name
Jessica Paola Fuentes Rivera Navarro
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2008
Supervisor
Committee
Vicente, Elisabete Jose (President)
Cruz, Sandra Helena da
Santos, Manoel Armando Azevedo dos
Title in Portuguese
Construção de sistema que permite a ancoragem de proteína recombinante à superfície celular de levedura.
Keywords in Portuguese
Saccharomyces cerevisiae
Âncora FLO1p
Biotecnologia
Glicoamilase
Sistema de ancoragem de proteínas
Superfície celular
Abstract in Portuguese
Sistemas do tipo cell surface display vêm sendo desenvolvidos para expressão de proteínas heterólogas ancoradas à superfície celular de microrganismos. Várias aplicações foram reportadas destes sistemas, incluindo o emprego como biocatalizador celular, desenvolvimento de vacinas e biosorventes celulares. Neste trabalho foi desenvolvido um sistema que permite ancoragem da proteína glicoamilase de Aspergillus awamori à superfície da parede celular da levedura Saccharomyces cerevisiae. O gene codificador da glicoamilase com sua seqüência sinal foi fusionado ao fragmento do gene codificador da região C-terminal da proteína Flo1p (Flo428), que foi utilizada como âncora (fragmento CG*FC). As células de levedura foram transformadas com o fragmento híbrido CG*FC e os transformantes foram capazes de degradar amido e liberar glicose. A atividade da glicoamilase não foi detectada no meio de cultura, porém está presente no sedimento celular. Estes resultados demonstram que a glicoamilase foi ancorada à parede celular da nova linhagem recombinante de levedura.
Title in English
Construction of a system that allows anchoring of recombinant protein to the cell surface of yeast.
Keywords in English
Saccharomyces cerevisiae
Anchor Flo1p
Anchoring protein system
Biotechnology
Cell surface
Glucoamylase
Abstract in English
Cell surface display systems have being developed for expression of heterologous proteins anchored to the cell surface of microorganisms. Several applications of these systems have been reported, including employment as whole-cell biocatalysts, development of vaccines and cellular biosorvents. In this work it was developed a system that allows the anchoring of the Aspergillus awamori glucoamylase protein to the cell wall surface of the yeast Saccharomyces cerevisiae. The gene encoding glucoamylase with its secretion signal was fused to the gene fragment encoding the C-terminal region of Flo1 protein, used as an anchor (CG*FC fragment). Yeast cells were transformed with hybrid CG*FC fragment and transformants were able to degrade starch and release glucose. Glucoamylase activity was not detected in the culture medium, but only in sedimented cells. These results demonstrate that glucoamylase was anchored to the cell wall of the new yeast recombinant strain.
 
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Publishing Date
2009-03-19
 
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