Made available in DSpace on 2016-06-02T20:21:37Z (GMT). No. of bitstreams: 1 6599.pdf: 2881840 bytes, checksum: f0c91115276cbdeae047c039f2676980 (MD5) Previous issue date: 2015-02-27 / Universidade Federal de Sao Carlos / The sugar cane represents one of the most Important agricultural segments in Brazil, which is the largest producer and exporter of sugar in the world as well the second largest ethanol producer. The Sphenophorus levis (Curculionidae) is an important sugarcane pest which lacks effective methods of control. The larvae of this insect feed the sugar cane plant decreasing productivity and causing the plant death. In view of identify the insect digestive arsenal enzymes, a transcriptome study was previously performed from S. levis larvae. Thereby, an invertase (P-fructofuranosidases class) coding sequence was identified and characterized. Considering the scarcity of functional studies on insect P-fructofuranosidases and their apparent non-occurrence among coleopterans, the aim of the present study was to investigate the occurrence and characterize the P-fructofuranosidase transcript identified. To validate that the P-fructofuranosidase sequence (herein denominated Sl-fi-fruct) is indeed encoded by the S. levis genome, PCRs were performed using genomic DNA extracted from the larval fat body as well as DNA from the midgut with microbial content. Amplification of Sl-fi-fruct gene using larval fat body DNA indicated its presence in the insect's genomic DNA. Quantitative PCR (qRT-PCR) analyses indicated that the production of mRNA only occurs in the midgut and reaches the greatest expression level in 30-day-old larvae, which is the expected pattern for digestive enzymes. Chromatography of glycosidases from S. levis midguts showed two enzymes acting as P-fructofuranosidase, indicating the presence of a Sl-fi-fruct isoform or a P-fructofuranosidase from insect intestinal microbiota. Moreover, it was found that a-glucosidases do not act on sucrose hydrolysis. Phylogenetic analyses indicated this enzyme to be similar to enzymes found in other coleopteran and lepidopteran P-fructofuranosidases, but also closely similar to bacterial enzymes, suggesting potential horizontal gene transfer. Despite this, the enzyme seems to be restricted to different groups of bacteria, which suggests distinct origin events. The Sl-fi-fruct gene was cloned in Pichia pastoris to produce the recombinant enzyme (rSl-P-fruct). Molecular weight of the recombinant protein was about 64 kDa, indicating possible glycosylation, since the theoretical weight was 54.8 kDa. The substrate specificity test revealed that rSl-P-fruct hydrolyzes sucrose and raffinose, but not melibiose or maltose, thereby confirming invertase activity. The pH curve revealed greatest activity at pH 5.0, demonstrating rSl-P-fruct to be an acidic P-fructofuranosidase. The enzymatic characterization was done and the optimum temperature was 50 °C, thermal resistance at 36 °C and pH maximum resistance at 6.0. The Michaelis-Menten curve showed Km=20.02 μM, Kcat=520.9 s-1 and Vmax=105.7 μM.s-1. 5 mM of SDS and MgCl2 cause inhibition of rSl-β-fruct activity. The present study expands the concept of the occurrence of P-fructofuranosidase in insects. Despite the few descriptions of this gene in the animal kingdom, it is possible to state that P-fructofuranosidase is crucial to the establishment of some insects throughout their evolutionary history, especially members of the Lepidoptera and Coleoptera clades. Considering the rSl-P-fruct potential to industrial application, they are promising if the thermal properties are improved. / O coleóptero Sphenophorus levis é uma Importante praga da cana-de-açúcar, para o qual ainda não existe um método de controle adequado. Considerando a Importância da cultura canavieira no Brasil, maior produtor e exportador de açúcar e segundo maior produtor de etanol no mundo, um estudo transcriptômico das larvas do inseto foi realizado anteriormente a este trabalho para a identificação do seu arsenal digestivo. Dentre as prováveis enzimas digestivas, foram identificadas sequências que codificam uma enzima da classe das P-frutofuranosidases. O sequenciamento do clone de cDNA foi totalizado e verificou-se a presença de sinal de poliadenilação e cauda poli-A característica de eucariotos, bem como uma região codificadora para um provável peptídeo sinal para secreção da enzima. A comparação da sequência proteica deduzida com o banco de dados do NCBI aponta maior similaridade com invertases bacterianas. A amplificação do gene da P-frutofuranosidase de S. levis (Sl-fi-fruci) por PCR a partir de DNA genômico, livre de contaminação bacteriana, sugere que S. levis realmente codifica uma P-frutofuranosidase. Comparando-se sequências de aminoácidos de P-frutofuranosidases de coleópteros, observam-se regiões conservadas entre membros da família Curculionidae. O ensaio bioquímico das glicosidases intestinais de S. levis mostrou que existem provavelmente duas P-frutofuranosidases responsáveis pela digestão de sacarose. Portanto, o inseto pode apresentar uma isoforma da Sl-fí-fruct ou se beneficiar de uma invertase produzida pela própria microbiota. As análises de expressão via PCR quantitativo em tempo real revelaram que o gene é expresso em intestino médio, nas fases de alimentação do inseto, característica de uma enzima digestiva. As análises filogenéticas indicaram que Sl-P-fruct é similar a outras P-frutofuranosidases de lepidópteros e coleópteros, mas se apresenta mais próxima das enzimas bacterianas. Essa proximidade se dá a grupos distintos de bactérias, quando comparada com enzimas de lepidópteros, levando-nos a propor um evento de transferência horizontal independente do que ocorreu em lepidópteros. A fase aberta de leitura (ORF) codificadora da enzima, excluindo o peptídeo sinal, foi clonada no plasmídeo pPICZa-A para sua produção heteróloga em Pichia pastoris. A enzima produzida (rSl-P-fruc) apresentou-se glicosilada, com massa molecular aproximada de 65 kDa. Os ensaios de especificidade ao substrato confirmaram que a enzima é uma P-frutofuranosidase. A rSl-P-fruc apresentou pH de maior atividade próximo a 5,0 e temperatura de atividade máxima próxima a 50 °C. Os ensaios de termoestabilidade e resistência térmica sugerem uma baixa capacidade térmica para rSl-P-fruc, que se desnatura com facilidade. Os sais Dodecil Sulfato de Sódio (SDS) e MgCl2 provocam inibição da rSl-P-fruc na concentração de 1 mM. As constantes cinéticas estimadas para a rSl-P-fruct foram Km = 20,02 mM, Vmax = 105,7 |iM.s-1 e kcat = 520,9 s-1. O presente estudo expande o conceito da ocorrência de P-frutofuranosidases em coleópteros. Apesar dos poucos relatos desse gene no reino animal, é possível afirmar que a P-frutofuranosidase é importante e vem sendo mantida entre algumas espécies de lepidópteros e coleópteros. Tratando-se das potencialidades que a rSl-P-fruct apresenta para uma aplicação industrial, observa-se um potencial positivo caso haja melhorias em sua estabilidade térmica.

O papilomavirus humano (HPV) é o agente etiológico da infecção sexualmente transmissível mais comum na população mundial. A prevalência da infecção por HPV é estimada em 660 milhões de pessoas e mais de 50% das mulheres serão acometidas pela infecção ao menos uma vez na vida. O HPV é responsável por virtualmente todos os casos de câncer de colo de útero (99%), além de causar muitos outros tipos de câncer de mucosa. O câncer cervical afeta aproximadamente 1.4 milhões de mulheres e 500 mil novos casos surgem anualmente, resultando em 250 mil mortes por ano. Os maiores índices de incidência são observados na América Latina e na África. Duas vacinas contra o HPV são comercializadas desde 2006 por empresas estrangeiras. Entretanto, o custo mínimo da vacinação é superior a 600 reais. Este preço torna sua incorporação pelo sistema de saúde um processo economicamente inviável. Dessa forma, devem ser buscadas alternativas para a produção de uma vacina eficaz, de qualidade e de baixo custo no Brasil. A proteína L1, principal proteína do capsídeo do HPV, tem a propriedade de se auto agregar em partículas semelhantes às virais (VLPs), que são o principal componente das vacinas atuais e possuem muita semelhança com os vírions nativos, introduzindo inclusive, uma imunização predominantemente tipo-específica. Este projeto se propôs a construir e avaliar vetores de expressão visando à produção em larga escala da proteína L1 do HPV 16, o tipo de HPV de maior risco e incidência na população mundial. Foram construídos cinco plasmídeos para Pichia pastoris, uma das principais plataformas industriais de expressão. Eles se diferenciam pelos marcadores de seleção e pelas sequências de manutenção do vetor, mas têm exatamente o mesmo objetivo: gerar sistemas que possuam múltiplas cópias do gene da L1 e que não necessitem da utilização de antibióticos. Essa estratégia foi escolhida porque a produtividade de muitas das proteínas heterólogas tem grande dependência da dosagem gênica e porque o uso de antibióticos encarece muito o custo da produção. Na primeira etapa do projeto foi avaliado um sistema de expressão epissomal auxotrófico, mas ele não se manteve estável ao longo de 60 gerações. Na segunda parte, dois vetores de integração ao sítio do DNA ribossomal foram testados. O sistema mais estável e produtivo foi caracterizado quanto ao número de cópias integradas ao genoma da P. pastoris e ao nível de expressão. O sistema é capaz de produzir clones com mais de 30 cópias do gene da L1 e dois clones expressaram cerca de 20 a 30 miligramas/litro de L1. Adicionalmente outros dois vetores integrativos que utilizam sequências teloméricas para integração foram construídos. Ainda são necessários estudos conjuntos de fermentação em larga escala e purificação da proteína para verificar a viabilidade do sistema / The human papillomavirus (HPV) is the etiologic agent of the sexually transmitted infection most common worldwide. The prevalence of HPV infection is estimated at 660 million people and over 50% of women will be affected by the infection at least once in their lifetime. HPV is responsible for virtually all cervical cancers cases (99%), and causes many other types of mucosal cancer. Cervical cancer affects approximately 1.4 million women, and 500.000 new cases occur annually, resulting in 250.000 deaths per year. The highest incidence rates are seen in Latin America and Africa. Two HPV vaccines are marketed since 2006 by foreign companies. However, the minimum cost of vaccination is higher than 360 dollar. This price makes its incorporation by the Brazilian Health System economically unfeasible. Thus, alternatives must be found to produce an effective vaccine, with low cost and high-quality in Brazil. The L1 protein, the major capsid protein of HPV, has the ability to self aggregate into virus-like particles (VLPs) which are the main component of current vaccines. The VLPs are very similar to the native virions and can introduce an immunization predominantly type-specific. This project aimed to construct and evaluate expression vectors to produce, at large scale, the L1 protein of HPV 16, which is the HPV type at greater risk and incidence in the world. Five plasmids were constructed for Pichia pastoris, one of the major industrial expression platforms. They differ in the selection markers and the vector maintenance sequences, but they have exactly the same goal: to create systems with multiple copies of the L1 gene and that don\'t require the use of antibiotics. This strategy was chosen because the productivity of many heterologous proteins have heavy dependence on gene dosage and because the use of antibiotics is extremely expensive for its production. In the first stage of the project, it was rated an episomal auxotrophic expression system, but it was unstable after a period of 60 generations. In the second part, two vectors for integration in the ribosomal DNA locus were tested. The most stable and productive system was featured on the number of copies integrated into the P. pastoris genome and on the expression level. It was proved that the system is able to produce clones with more than 30 copies of the L1 gene and two clones expressed approximately 20-30 milligrams/liter of L1. Additionally, two other integrative vectors for integration in telomeric sequences were constructed for future testing. More studies on large-scale fermentation and protein purification will be necessary to determine the feasibility of the system.

Interleukin-12 (IL-12), an important immuno-modulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anti-cancer therapeutic. However, its clinical application is limited by lack of an effective bioproduction system and by toxicity associated with systemic administration of IL-12. The goals of this research were to determine whether plants can serve as an effective production system for bioactive IL-12, a complex 70kDa glycoprotein cytokine, and whether the plant lectin RTB can facilitate mucosal delivery of IL-12 to immune responsive sites. Transgenic tobacco plants expressing murine IL-12 were generated and characterized. To ensure stochiometric expression of the two separately encoded, disulfide-linked subunits of IL-12 (p35 and p40), a single-chain form of mouse IL-12 (mIL-12) was utilized. Hairy root cultures, as a fast-growing bioproduction system were developed from high expressers of mIL-12. A purification scheme was developed to purify plant-derived mIL-12 from hairy roots and purified mIL-12 was used to assess IL-12 bioactivity in vitro in mouse splenocytes and in vivo in mouse intranasal vaccination trials. Plant-derived mIL-12 triggered induction of interferon-gamma secretion from mouse splenocytes as well as stimulation of cell proliferation with comparable activities to those observed for the animal-cell-derived mIL-12. Mouse vaccination trials using GFP as the antigen and CT as the adjuvant suggested that plant-derived mIL-12 enhanced Th1 immunity and exhibited similar activity to animal-cell-derived mIL-12 in vivo. Plant-derived IL-12 itself was non-immunogenic suggesting conformational equivalency to endogenous mouse IL-12. Ricin B (RTB), the non-toxic carbohydrate-binding subunit of ricin, directs uptake of ricin into mammalian cells and the intracellular trafficking of ricin A, the catalytic subunit of ricin. RTB's function suggests that it may work as a molecular carrier for effective mucosal delivery of IL-12. To prove this hypothesis, transgenic plants producing RTB:IL-12 fusions were generated and characterized. Our results demonstrated that RTB fused to the carboxyl-terminus of IL-12 maintained full lectin activity and IL-12 bioactivity. RTB fused to the amino-terminus of IL-12 did not show lectin activity due to steric hinderance. Purified IL-12:RTB from transgenic plant tissue was tested in an in vitro mucosal-associated lymphoid tissue (MALT) assay. The results indicate that RTB facilitates the binding of IL-12 to the epithelial cells and presentation of IL-12 to immune responsive cells. In conclusion, my research has shown that transgenic plants are capable of producing valuable bioactive proteins, such as IL-12. Plant-derived mIL-12 exhibited similar activity to animal-cell-derived mIL-12 both in vitro and in vivo. Fusion of IL-12 with the RTB lectin facilitates the delivery of IL-12 to mucosal immune responsive cells and thus may serve as a molecular carrier to enhance IL-12 efficacy and reduce the side-effects associated with systemic administration of IL-12. / Ph. D.

abstract: ATP synthase is a large multimeric protein complex responsible for generating the energy molecule adenosine triphosphate (ATP) in most organisms. The catalysis involves the rotation of a ring of c-subunits, which is driven by the transmembrane electrochemical gradient. This dissertation reports how the eukaryotic c-subunit from spinach chloroplast ATP synthase has successfully been expressed in Escherichia coli and purified in mg quantities by incorporating a unique combination of methods. Expression was accomplished using a codon optimized gene for the c-subunit, and it was expressed as an attachment to the larger, more soluble, native maltose binding protein (MBP-c1). The fusion protein MBP-c1 was purified on an affinity column, and the c1 subunit was subsequently severed by protease cleavage in the presence of detergent. Final purification of the monomeric c1 subunit was accomplished using reversed phase column chromatography with ethanol as an eluent. Circular dichroism spectroscopy data showed clear evidence that the purified c-subunit is folded with the native alpha-helical secondary structure. Recent experiments appear to indicate that this monomeric recombinant c-subunit forms an oligomeric ring that is similar to its native tetradecameric form when reconstituted in liposomes. The F-type ATP synthase c-subunit stoichiometry is currently known to vary from 8 to 15 subunits among different organisms. This has a direct influence on the metabolic requirements of the corresponding organism because each c-subunit binds and transports one H+ across the membrane as the ring makes a complete rotation. The c-ring rotation drives rotation of the gamma-subunit, which in turn drives the synthesis of 3 ATP for every complete rotation. The availability of a recombinantly produced c-ring will lead to new experiments which can be designed to investigate the possible factors that determine the variable c-ring stoichiometry and structure. / Dissertation/Thesis / Ph.D. Biochemistry 2011

Orientadora: Profa. Dra. Maria Aparecida Sperança / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2016. / O arbovírus Mayaro (MAYV), encontrado nas regiões próximas a florestas e áreas rurais da América do Sul, é membro da família Togaviridae, gênero Alphavirus. Este gênero é distribuído amplamente, tendo dois grupos principais: Alphavirus do Velho Mundo (Chikungunya, Sindbis, O¿nyong-nyong, Ross River, Semliki Forest) e do Novo Mundo (Encefalite Equina Venezuelana, Encefalite Equina Ocidental), de acordo com a região na qual foram isolados originalmente. O mosquito Haemagogus spp. é o principal vetor do MAYV que também já foi isolado de mosquitos do gênero Aedes spp. Considerando a semelhança do MAYV com o vírus Chikungunya, recentemente inserido no Brasil, e transmitido por Ae. aegypti, há risco de que o MAYV possa ser transmitido em áreas urbanas. Cabe ressaltar que no Brasil já foram registrados casos de co-circulação de MAYV durante surtos epidêmicos de dengue. A infecção por MAYV causa sintomas semelhantes a outras doenças febris, como a febre da dengue, a febre do Chikungunya, e a malária, dificultando o diagnóstico preciso dos casos. O genoma de MAYV, com cerca de 11,7 kb, é organizado em duas principais regiões: domínio não estrutural (extremidade 5¿ do RNA), contendo os genes que codificam as proteínas não estruturais (nsP1-4); e domínio estrutural, contendo os genes que codificam as proteínas estruturais. As proteínas não estruturais dos Alphavirus são necessárias para o processamento da poliproteína e para síntese do RNA viral. Dentre as proteínas não estruturais, as proteases virais podem ser imunogênicas além de se constituírem em excelentes alvos terapêuticos. O estudo comparativo do genoma dos Alphavirus indica que a proteína nsP2 de MAYV possui diversas atividades enzimáticas, incluindo a atividade de cisteíno protease em sua região C-terminal. Portanto, com o intuito de obter um método de diagnóstico sorológico específico para MAYV, utilizando sistema de expressão recombinante em células de insetos, foi realizada a construção de três baculovírus recombinantes para a nsP2 de MAYV, para obtenção da proteína completa (nsP2FL), do domínio de cisteíno protease da porção C-terminal (Pro38), e do domínio N-terminal (NT51) como controle negativo da atividade proteolítica. A expressão de cada uma das formas recombinantes da nsP2 foi realizada em célula de inseto High Five¿. Os resultados foram analisados por meio de eletroforese em gel de poliacrilamida contendo SDS (SDS-PAGE) e Western blotting. Apenas a construção NT51 da nsP2 de MAYV foi detectada na fração celular da cultura de High Five¿ por Western blotting. A análise de RNA das células High Five¿ e Sf-9 infectadas com os baculovírus recombinantes para nsP2FL e Pro38, revelou a presença de transcritos das proteínas recombinantes, indicando que a ausência dos produtos proteicos poderia ser devido a perda da cauda de histidina presente nas construções por atividade de proteólise na extremidade Cterminal. Esta hipótese foi confirmada após expressão das proteínas nsP2FL e Pro38, em célula High Five¿ infectada com baculovírus recombinantes para os genes que codificam as respectivas proteínas, com cauda de histidina na porção N-terminal. / The arbovirus Mayaro (MAYV), found in regions close to forests and rural areas of South America, is a member of the family Togaviridae, genus Alphavirus. This genus is distributed widely, in two main groups, according to the region in which they were originally isolated: the Old World (Chikungunya, Sindbis, O'nyong-Nyong, Ross River, Semliki Forest) and the New World Alphavirus (Venezuelan Equine Encephalitis, Western Equine Encephalitis). The mosquito Haemagogus spp. is the main vector of MAYV which has also been isolated from mosquitoes of the genus Aedes spp. Considering the similarity of MAYV with Chikungunya virus, recently introduced in Brazil, and transmitted by Ae. aegypti, there is risk of MAYV urban transmission. Indeed, in Brazil, co-circulation of MAYV during dengue outbreaks have been related. Symptoms of MAYV fever are similar to other febrile diseases, such as dengue fever, Chikungunya fever, and malaria, making an accurate diagnosis, difficult. MAYV 11.7 kb genome is divided in two regions: non-structural domain (5'- RNA) containing the genes encoding the nonstructural proteins (nsP1-4); and structural domain containing genes encoding structural proteins. The Alphavirus nonstructural proteins are required for processing of the polyprotein and for viral RNA synthesis. Among the non-structural proteins, viral proteases may be immunogenic besides being excellent therapeutic targets. Genome comparative studies of Alphavirus indicates that the MAYV nsP2 protein has diverse enzymatic activities including a cysteine protease activity in its C-terminal region. Thus, with the objective to obtain a specific diagnostic method for MAYV, using a recombinant expression system in insect cells, a construction of three MAYV nsP2 recombinant baculoviruses to obtain the complete protein (nsP2FL), the C-terminal cysteine protease domain (Pro38), and the N-terminal (NT51) domain as a proteolysis activity negative control. The expression of each nsP2 construction was performed on Sf-9 and High Five¿ insect cells and the results were analyzed by polyacrylamide gel electrophoresis with SDS (SDS-PAGE) and Western blotting. Only the nsP2 NT51 construction was detected in the cellular fraction of Sf-9 and High Five¿ cultures by Western blotting. RNA analysis of Sf-9 cells infected with nsP2FL and Pro38 recombinant baculoviruses revealed the presence of transcripts, suggesting that the absence of the corresponding protein products occurred due the proteolysis of the C-terminal portion of the protein, resulting in histidine tail elimination. This hypothesis was confirmed by expression of nsP2FL e Pro38, proteins in Sf-9 and High Five¿ cells infected with recombinant baculoviruses for the genes encoding the respective proteins with histidine tail at the N-terminal portion.

Made available in DSpace on 2016-06-02T20:21:28Z (GMT). No. of bitstreams: 1 4058.pdf: 1157673 bytes, checksum: 2346bf567a5ddfbb9217c250df00ce8e (MD5) Previous issue date: 2011-09-09 / Universidade Federal de Sao Carlos / Saccharomyces cerevisiae is the most important microorganism used in alcoholic fermentation process. A strain of Saccharomyces cerevisiae widely used to produce alcohol in Brazil is the PE-2, due to its high fermentation capacity. The goal of this study was to develop an expression system for recombinant proteins using the industrial strain of Saccharomyces cerevisiae, PE-2. The protein chosen to be used as a model for this system was the Cane-CPI-1, an inhibitor of cysteine protease. Initially the construction of plasmids containing CaneCPI-1 gene was performed and yeast cells were transformed with pYADE4-CaneCPI-1 construction. The transformed strain was submitted to expression analyses during batch fermentative process with cell recycle. The recombinant protein was expressed in yeast cells and it was possible to purify the recombinant protein by affinity chromatography on nickel column. This purified protein was immunodetected using a polyclonal anti-CaneCPI-1antibody and monoclonal anti His-Tag antibody, which were also able to detect the CaneCPI-1 in a crude extract from Saccharomyces cerevisiae cells. Assays of inhibitory activity performed with the purified CaneCPI-1 revealed its ability to inhibit the catalytic activity of a cysteine proteinase. This study showed that the use of transformed strain did not affect the fermentation process and that the PE-2 industrial strain of S. cerevisiae can be a viable expression system for recombinant protein production and open perspectives for production of any protein of biotechnological applications during fermentation alcoholic process. / Saccharomyces cerevisiae é o microrganismo mais importante usado no processo de fermentação alcoólica. Uma linhagem de S. cerevisiae amplamente utilizado para produzir álcool no Brasil é a PE-2, devido à sua alta capacidade de fermentação e persistência no sistema. Neste trabalho utilizou-se esta linhagem industrial de levedura como um sistema de expressão de proteínas de interesse biotecnológico. A proteína escolhida como modelo para este sistema foi a CaneCPI-1, uma cistatina de cana-de-açúcar que possui ação inseticida. Inicialmente, foi realizada a construção de um plasmídeo contendo o gene que codifica para a proteína CaneCPI-1 e este foi utilizado para transformar células da levedura S.cerevisiae linhagem PE-2. Posteriormente foram realizados sucessivos ciclos de fermentação (5 ciclos) com esta levedura transformada e as células fermentadas foram submetidas a análises de expressão da proteína CaneCPI-1. Foi possível purificar a proteína recombinante por cromatografia de afinidade em coluna de níquel. Anticorpo policlonal anti-CaneCPI-1 e anticorpo monoclonal anti-His-Tag foram capazes de imunodetectar a proteína purificada e no extrato proteico bruto do último ciclo fermentativo. Ensaios da atividade inibitória mostraram que a CaneCPI-1 purificada foi capaz de inibir a atividade hidrolítica da papaína. A utilização da linhagem transformada não afetou a eficiência do processo fermentativo durante os quatro primeiros reciclos. A produção da CaneCPI-1 em uma linhagem industrial de S. cerevisiae ao longo do processo fermentativo se mostrou viável, abrindo-se novas perspectivas para a produção de qualquer proteína de aplicações biotecnológicas durante o processo de fermentação alcoólica.

No description available.

Recombinant preparation of TEAD transcription factor (abstract) The TEAD family transcription factors play an important role during devolopment of organisms, where their main purpose is to regulate organ size by activating expression of proteins involved in cell growth and differentiation and apoptosis inhibition. TEAD proteins activity is regulated by signalling pathways and interactions with coactivators. Disregulation of these mechanisms can lead to development of tumors, which is the reason why TEAD proteins became an interesting target for development of new anticancer drugs based on inhibiting their activity. There are several possibilities how to inhibit activity of a transcription factor including blocking its bond to DNA. To design a new drug that blocks transcription factors binding to DNA the structural basis of interaction of these two molecules has to be known first. In this thesis the DNA binding domain of human protein TEAD1 was prepared using the technique of recombinant expression in bacteria E. coli. Suitable conditions of protein production were found and the DNA binding domain of TEAD1 protein was purified so it will be possible to use it for structural analysis of its intraction with DNA.

The development of a biologic protein involves different stages and becomes a highly complex process which can be costly and time consuming to scale up for industrial production. Therefore, optimization is a necessary part of the production process development to lower the production expenses.An on-going project is working on upscaling the production of a protein derived from mussel adhesive proteins (MAPs) which has great properties to be used as a pharmaceutical drug or in medical devices. The protein is expressed in a bacterial host cell and the necessary post translational modifications (PTMs) are done in-vitro using enzymes. The work presented in this report was done to optimize both the protein production in lab scale bioreactors and the enzymatic reaction using an immobilized prolyl-4-hydroxylase (P4H) which does a post translational modification on prolyl-residues. Additionally, an interaction study was conducted to better understand the hydroxylation using the prolyl-4-hydroxylase.For the bioreactor optimization four initial trials were performed testing different growth and induction temperatures and also comparing exponential to linear feeding. From these trials it appeared that having 30 ℃ growth overnight and induction at the same temperature in combination with an exponential feeding rate gave the best results. The modifications done by the prolyl-4-hydroxylase were analysed by LC-MS and suggest that longer incubation time and more immobilized protein gives more modifications in the tested ranges and the possibilities of reusing the immobilized proteins looks promising. No conclusive data was discovered for the optimal substrate concentration. The interaction study revealed the importance of reagents used for catalysis with the enzyme to be present for interaction to occur, however more work needs to be done to discover an accurate KD for the interaction.

L’infertilité affecte environ 15% des couples en âge de se reproduire. Dans près de la moitié des cas, des facteurs masculins sont à la base de l’infertilité, quoique les causes exactes demeurent souvent inconnues. Les spermatozoïdes de mammifères subissent une série d’étapes de maturation avant d’acquérir la capacité de féconder un ovocyte. Les premiers changements ont lieu à l’intérieur de l’épididyme, où les spermatozoïdes gagnent la capacité de se mouvoir ainsi que de reconnaître et d’interagir avec l’ovocyte. Suite à l’éjaculation, ils doivent subir une seconde série de modifications à l’intérieur du tractus génital femelle, nommée capacitation. Nous avons préalablement démontré que chez le bovin, la famille de protéines BSP (Binder of SPerm) est essentielle à la capacitation. Des homologues des BSP ont aussi été isolés du fluide séminal de porc, de bouc, de bélier, de bison et d’étalon. Malgré la détection d’antigènes apparentés aux BSP dans le fluide séminal de souris et d’humain, les homologues des BSP n’ont jamais été caractérisés chez ces espèces. Nous avons émis l’hypothèse que des homologues des BSP seraient exprimés chez la souris et l’humain et joueraient un rôle dans la maturation des spermatozoïdes. Nous avons démontré que des séquences homologues aux BSP sont présentes dans les génomes murin et humain. Le génome murin contient trois séquences; Bsph1, Bsph2a et Bsph2b, tandis qu’une seule séquence (BSPH1) a été identifée chez l’humain. Les séquences d’ADNc de Bsph1, Bsph2a et BSPH1 ont été clonées, tandis que Bsph2b serait probablement un pseudogène. Les trois gènes sont exprimés uniquement dans l’épididyme et font partie d’une sous-famille distincte à l’intérieur de la famille des BSP. Chez les ongulés, les BSP sont exprimées par les vésicules séminales, sont ajoutées aux spermatozoïdes lors de l’éjaculation et représentent une proportion significative des protéines du plasma séminal. Au contraire, les BSP épididymaires ne sont retrouvées qu’en faibles quantités dans le fluide séminal. L’étude de leur rôle dans les fonctions spermatiques était donc plus difficile que chez les ongulés, où l’isolement des protéines natives du plasma séminal à l’aide de techniques de chromatographie était possible. Afin d’étudier sa fonction, nous avons exprimé BSPH1 recombinante dans E. coli. Les ponts disulfure des domaines de type-II caractéristiques de ces protéines ont fait en sorte que l’expression de BSPH1 fusionnée à une étiquette hexahistidine ou glutathion-S-transférase a donné lieu à des protéines insolubles dans les corps d’inclusion. La production de BSPH1 soluble a été possible grâce à l’ajout d’une étiquette thiorédoxine et l’expression dans une souche au cytoplasme oxidatif. BSPH1 a été purifiée par affinité et sa liaison aux partenaires connus des BSP, la phosphatidylcholine, les lipoprotéines de faible densité et la membrane des spermatozoïdes, suggérait que la protéine recombinante possédait sa conformation native et pouvait être utilisée pour des essais fonctionnels. La forme native de BSPH1 a été détectée dans le plasma séminal humain suite au fractionnement par gel filtration. La liaison de BSPH1 native à une colonne d’affinité à l’héparine a indiqué qu’elle partage aussi cette propriété de liaison avec la famille des BSP, et pourrait lier les GAGs semblables à l’héparine du tractus génital féminin. Une colonne d’immunoaffinité anti-BSPH1 a été préparée à l’aide d’anticorps générés contre des protéines recombinantes, et a permis d’isoler BSPH1 native à partir d’extraits de spermatozoïdes humains. Nos résultats montrent que BSPH1 native serait localisée dans les microdomaines « rafts » de la membrane. Sa masse moléculaire apparente était de 32 kDa, ce qui est supérieur à la masse prédite selon sa séquence en acides aminés, indiquant la présence probable de modifications post-traductionnelles, ou d’une migration anormale. L’effet de BSPH1 recombinante et des anticorps anti-BSPH1 sur la motilité, la viabilité et la capacitation a aussi été étudié. Les deux dernières variables ont été mesurées par un essai de cytométrie en flux, optimisé dans cette étude. Aucun effet des protéines recombinantes ou des anticorps sur la motilité et la viabilité des spermatozoïdes n’a été noté. Quoiqu’une stimulation modeste, quoique significative, de la capacitation ait été observée à la plus faible concentration de BSPH1, les concentrations plus élevées n’ont pas montré d’effet. De la même manière, les anticorps anti-BSPH1 n’ont pas eu d’effet significatif sur la capacitation. Ces résultats suggèrent que BSPH1 produite dans E. coli n’affecte pas la capacitation de façon marquée. Cependant, puisque BSPH1 native possède probablement des modifications post-traductionnelles, une protéine recombinante produite dans des cellules de mammifères pourrait affecter les fonctions spermatiques. De manière alternative, les BSP épididymaires remplissent peut-être un rôle différent dans les fonctions spermatiques que celles sécrétées par les vésicules séminales des ongulés. Les résultats décrits dans cette thèse pourraient contribuer à améliorer le diagnostic de l’infertilité masculine, ainsi que les techniques de reproduction assistée et éventuellement, pourraient mener au développement de contraceptifs masculins. / Infertility affects approximately 15% of couples of reproductive age. In nearly half the cases, male factors are responsible, although causes underlying male infertility often remain unknown. Mammalian sperm undergo a series of maturational steps before acquiring the capacity to fertilize an oocyte. The first changes take place inside the epididymis, where sperm gain motility and the ability to recognize and interact with the oocyte. After ejaculation, sperm go through a second maturation event named capacitation, taking place inside the female reproductive tract. We previously showed that in the bovine species, proteins of the BSP (Binder of SPerm) family are essential for capacitation. Homologs of these proteins have also been isolated from boar, ram, goat, bison and stallion seminal fluid. Although BSP-related antigens have been detected in mouse and human seminal fluid, BSP homologs have never been characterized in these species. We hypothesized that BSPs would indeed be expressed in mice and humans and could be involved in sperm maturation. Our studies demonstrated that BSP-homologous sequences are present in the mouse and human genomes. The mouse genome contains three BSP-like sequences, Bsph1, Bsph2a and Bsph2b, whereas only one sequence (BSPH1) was identified in the human genome. The complete cDNA sequences of Bsph1, Bsph2a and BSPH1 were cloned, whereas Bsph2b is probably a pseudogene. The two murine and sole human genes are expressed uniquely in the epididymis, and are part of a distinct sub-family within the BSP superfamily. The BSPs of ungulates are expressed in the seminal vesicles, are added to sperm upon ejaculation and represent a significant proportion of seminal plasma proteins. In contrast, BSP proteins expressed in the mouse and human epididymides are found in very small quantities in seminal fluid. The study of their role in sperm functions was therefore less straightforward than for ungulate species, where direct isolation of the native proteins from seminal plasma was feasible using various chromatography techniques. In order to investigate the role of the human BSP protein, BSPH1, we expressed the recombinant protein in E. coli. Probably due to the multiple disulfide bonds within the fibronectin type-II domains characteristic of these proteins, expression of BSPH1 with a hexahistidine or glutathione-S-tranferase tag gave rise to insoluble protein trapped inside bacterial inclusion bodies. Successful expression of soluble BSPH1 was achieved when the protein was fused to a thioredoxin tag and expressed in a bacterial strain that possesses an oxidizing cytoplasm. This protein was purified using affinity chromatography techniques and tested for binding to known ligands of BSP proteins: phosphatidylcholine, low-density lipoproteins and the human sperm membrane. Since recombinant BSPH1 displayed all three binding properties, we concluded that it had assumed its native conformation and could be used in subsequent functional assays to determine its role in sperm functions. The native form of BSPH1 was detected in human seminal plasma after fractionation on a gel filtration column. Native BSPH1 also bound to a heparin-affinity column, indicating that it shares this binding property with the BSP family and may also bind heparin-like GAGs of the female reproductive tract. An anti-BSPH1 immunoaffinity column was prepared using antibodies generated with bacterially expressed recombinant proteins and was used to isolate native BSPH1 from human sperm extracts. In addition, our results show that BSPH1 probably localizes to detergent-resistant microdomains of the human sperm membrane. Its apparent molecular weight was 32 kDa, which is superior to that predicted by its amino acid sequence. Therefore, BSPH1 probably undergoes post-translational modifications or migrates abnormally during electrophoresis. The effect of recombinant BSPH1 protein and anti-BSPH1 antibodies on human sperm motility, viability and capacitation were also investigated. The latter two sperm functions were assayed using a flow cytometry technique optimized in this study. No effect of recombinant BSPH1 or antibodies on sperm motility or viability was noted. Although a modest yet significant stimulation of capacitation was observed at lower BSPH1 protein concentrations, higher concentrations showed no effect. In the same fashion, anti-BSPH1 antibodies showed no significant effect on capacitation. These results suggest that recombinant BSPH1 produced in E. coli does not appreciably affect capacitation. However, since native BSPH1 may be subject to post-translational modifications, it is possible that BSPH1 expressed in a mammalian system would affect sperm capacitation. Alternatively, epididymally expressed BSPs may play a somewhat different role in sperm functions than those secreted by the seminal vesicles of ungulates. The results described in this thesis could aid in better diagnosing male infertility, improving assisted reproduction and eventually, developing male contraceptives.