The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N -terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris , a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N -terminal partner to several C -terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C -terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C -terminal of the MBP domain, including the spacer and cargo regions, but the MEP domain could still act to enhance the secretion of certain cargo proteins. The attempt to identify the unknown protease was unsuccessful. However, in contrast to other fusion partners, MBP was secreted with the cargo when it was fused as a C -terminal peptide to an N -terminal cargo protein. These studies provide insights into the role of proteases and fusion partners in the secretory mechanism of P. pastoris , suggesting new strategies to optimize this expression system.

A produção de proteínas e peptídeos heterólogos representa um dos mais importantes segmentos do setor biotecnológico. Apesar das diferentes tecnologias hoje disponíveis para a (bio) síntese de proteínas e peptídeos (como a síntese química ou o emprego de microrganismos, animais e plantas transgênicos), nenhuma destas é capaz de produzir uma quantidade suficiente de proteínas e peptídeos para suprir as necessidades do mercado por apresentarem altos custos de produção e comercialização. Diante disso, este projeto visou utilizar os sistemas genéticos alternativos ou não convencionais existentes nas células da levedura Saccharomyces cerevisiae para a produção de peptídeos e proteínas heterólogas em larga escala, com um alto grau de pureza necessário às aplicações clínicas e com um custo de produção relativamente baixo para uso comercial. Estes sistemas não convencionais, representados especialmente por partículas virais intracelulares de S. cerevisiae, são conhecidos há várias décadas por conferirem o fenótipo killer em diferentes linhagens de leveduras, tanto para os isolados ambientais quanto para as linhagens utilizadas industrialmente. As características moleculares destes sistemas virais são bem conhecidas, sendo que os genomas dos diferentes tipos de partículas virais de S. cerevisiae já foram completamente seqüenciados. Assim, este trabalho avaliou a capacidade de leveduras industriais e laboratoriais em produzir proteínas e peptídeos heterólogos funcionais utilizando para tanto, um sistema viral naturalmente presente na levedura, que compreende um mutante natural do vírus helper L-A denominado de sistema viral X de S. cerevisiae. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-06-02T19:51:46Z No. of bitstreams: 1 Dissertacao Queli Defaveri Varela.pdf: 4371110 bytes, checksum: 9992e73e45f4352a46630f52b980c259 (MD5) / Made available in DSpace on 2014-06-02T19:51:46Z (GMT). No. of bitstreams: 1 Dissertacao Queli Defaveri Varela.pdf: 4371110 bytes, checksum: 9992e73e45f4352a46630f52b980c259 (MD5) / The production of heterologous proteins and peptides represents one of the most important segments of the biotechnology industry. Despite the different technologies available today for (bio) synthesis of proteins and peptides (such as chemical synthesis or the use of microorganisms, transgenic animals and plants), none of these are capable of producing sufficient quantities of proteins and peptides to the needs of the market because they present high costs of production and marketing. Thus, this project aims to use the alternative genetic systems or unconventional existing in the cells of the yeast Saccharomyces cerevisiae for the production of heterologous proteins and peptides on a large scale, with a high degree of purity needed for clinical applications and at a cost of production compared low for commercial use. These non-conventional systems, particularly represented by intracellular viral particles of S. cerevisiae, are known for several decades to give the killer phenotype in different strains of yeast, both for environmental isolates and to the strains used industrially. The molecular characteristics of these viral systems are well known, and the genomes of different types of viral particles from S. cerevisiae have been completely sequenced. Thus, this work assessed the capacity of industrial and laboratory yeasts to produce heterologous proteins and peptides using a viral system naturally present in yeast that is a natural mutant of the helper virus L-A, called X system of S. cerevisiae.

A produção de proteínas e peptídeos heterólogos representa um dos mais importantes segmentos do setor biotecnológico. Apesar das diferentes tecnologias hoje disponíveis para a (bio) síntese de proteínas e peptídeos (como a síntese química ou o emprego de microrganismos, animais e plantas transgênicos), nenhuma destas é capaz de produzir uma quantidade suficiente de proteínas e peptídeos para suprir as necessidades do mercado por apresentarem altos custos de produção e comercialização. Diante disso, este projeto visou utilizar os sistemas genéticos alternativos ou não convencionais existentes nas células da levedura Saccharomyces cerevisiae para a produção de peptídeos e proteínas heterólogas em larga escala, com um alto grau de pureza necessário às aplicações clínicas e com um custo de produção relativamente baixo para uso comercial. Estes sistemas não convencionais, representados especialmente por partículas virais intracelulares de S. cerevisiae, são conhecidos há várias décadas por conferirem o fenótipo killer em diferentes linhagens de leveduras, tanto para os isolados ambientais quanto para as linhagens utilizadas industrialmente. As características moleculares destes sistemas virais são bem conhecidas, sendo que os genomas dos diferentes tipos de partículas virais de S. cerevisiae já foram completamente seqüenciados. Assim, este trabalho avaliou a capacidade de leveduras industriais e laboratoriais em produzir proteínas e peptídeos heterólogos funcionais utilizando para tanto, um sistema viral naturalmente presente na levedura, que compreende um mutante natural do vírus helper L-A denominado de sistema viral X de S. cerevisiae. / The production of heterologous proteins and peptides represents one of the most important segments of the biotechnology industry. Despite the different technologies available today for (bio) synthesis of proteins and peptides (such as chemical synthesis or the use of microorganisms, transgenic animals and plants), none of these are capable of producing sufficient quantities of proteins and peptides to the needs of the market because they present high costs of production and marketing. Thus, this project aims to use the alternative genetic systems or unconventional existing in the cells of the yeast Saccharomyces cerevisiae for the production of heterologous proteins and peptides on a large scale, with a high degree of purity needed for clinical applications and at a cost of production compared low for commercial use. These non-conventional systems, particularly represented by intracellular viral particles of S. cerevisiae, are known for several decades to give the killer phenotype in different strains of yeast, both for environmental isolates and to the strains used industrially. The molecular characteristics of these viral systems are well known, and the genomes of different types of viral particles from S. cerevisiae have been completely sequenced. Thus, this work assessed the capacity of industrial and laboratory yeasts to produce heterologous proteins and peptides using a viral system naturally present in yeast that is a natural mutant of the helper virus L-A, called X system of S. cerevisiae.

La production de protéines hétérologues permet de développer une nouvelle génération de vaccins. La bactérie Escherichia coli est l’un des organismes hôtes les plus utilisés pour la production de protéines hétérologues, appelées également protéines recombinantes. Le déclenchement de la production de protéine altère la croissance bactérienne en réponse à la réallocation des ressources métaboliques vers la synthèse de la protéine ; ce qui peut conduire à l’arrêt complet de la croissance. Le maintien de la croissance bactérienne durant la production de la protéine recombinante est pourtant essentiel pour améliorer significativement la quantité et la fonctionnalité des protéines produites. Dans une démarche rationnelle visant à développer un système biologique robuste et performant pour la production d’une grande diversité de protéines recombinantes chez E. coli, les contraintes métaboliques liées à leur production ont été quantifiées. A partir de ces résultats, le système d’expression T7 a été intégré à la régulation métabolique et traductionnelle de la bactérie E. coli BL21 (DE3) afin d’adapter la vitesse de production avec les capacités métaboliques de la souche. Ce nouveau système biologique de production a ainsi permis d’augmenter considérablement les quantités de protéines produites et offre la possibilité de développer de nouveaux procédés performants de production semi-continus et continus en milieu chimiquement défini. / The production of heterologous proteins offers the ability to develop a new generation of vaccines. The most used organism for the production of heterologous proteins, also called recombinant proteins, is the bacterium Escherichia coli. However, the induction of the production often alleviates the bacterial growth by the new allocation of metabolic resources toward the production of the recombinant protein. Even, this may also lead to growth arrest. The production of high quantities of functional recombinant proteins requires a good balance between of bacterial growth and production of the recombinant protein.In order to rationally develop a robust and an efficient biological system for the production of a large variety of recombinant proteins in E. coli, the metabolic constraints associated to their production have been quantified. From this observation, the T7 expression system has been integrated into the metabolic and translational regulation of the E. coli BL21 (DE3) in order to adjust as perfect as possible the protein production rate to the metabolic capacities of the strain. This new biological production system has made it possible to significantly increase the quantities of proteins produced and opens up the possibility of developing performant semi-continuous and continuous production processes in a chemically defined medium.