L’hexokinase (HK) est la première enzyme du métabolisme des hexoses et catalyse la réaction qui permet aux hexoses d’entrer dans le pool des hexoses phosphates et donc par le fait même la glycolyse. Bien que le glucose soit son principal substrat, cette enzyme peut aussi phosphoryler le mannose et le fructose. Malgré son importance dans le métabolisme primaire, l’HK n’a jamais été purifiée à homogénéité sous forme native. Le but de ce travail était donc de purifier une isoforme d’HK à partir de tubercule de Solanum tuberosum et par la suite de caractériser ses propriétés cinétiques. Bien avant que je commence mon travail, un groupe de recherche avait déjà séparé et partiellement purifié trois isoformes d’HK de S. tuberosum. Un protocole d’extraction était donc disponible, mais l’HK ainsi extraite était peu stable d’où le besoin d’y apporter certaines modifications. En y ajoutant certains inhibiteurs de protéases ainsi qu’en modifiant les concentrations de certains éléments, le tampon d’extraction ainsi modifié a permis d’obtenir un extrait dont l’activité HK était stable pendant au moins 72h après l’extraction, en empêchant la dégradation. À l’aide du tampon d’extraction optimisé et d’une chromatographie sur colonne de butyl sépharose, il a été possible de séparer 4 isoformes d’HKs. Par la suite, une isoforme d’HK (HK1) a été purifiée à l’homogénéité à l’aide de 5 étapes de chromatographie supplémentaires. En plus de caractériser les propriétés cinétiques de cette enzyme, l’analyse de séquençage par MS/MS a permis de l’associer au produit du gène StHK1 de Solanum tuberosum. Avec une activité spécifique de 10.2 U/mg de protéine, il s’agit de l’HK purifiée avec l’activité spécifique la plus élevée jamais rapportée d’un tissu végétal.L’ensemble des informations recueillies lors de la purification de HK1 a ensuite été utilisée pour commencer la purification d’une deuxième isoforme (HK3). Ce travail a permis de donner des lignes directrices pour la purification de cette isoforme et certains résultats préliminaires sur sa caractérisation enzymatique. / Hexokinase (HK) catalyzes the first step of hexose metabolism by phosphorylating hexose to generate the corresponding hexose phosphate thereby allowing hexose entrance in glycolysis. Even though glucose is the main substrate, HK can also phosphorylate a broad spectrum of hexoses. Despite its importance this enzyme has never been purified to homogeneity in a native form. The aim of this work was therefore to purify this enzyme from Solanum tuberosum tubers and subsequently characterize its kinetic properties. Before I started this work, another group had already separated and partially purified 3 HK isoform from S. tuberosum. An extraction protocol was available but improvement was necessary since the extracted HK had little stability. By adding some protease inhibitors and by modifying the concentration of certain components in the extraction buffer we were able to obtain an extract with a HK activity stable for at least 72 h after extraction by preventing degradation. With this buffer and chromatography on butyl sepharose it was possible to separate 4 HK isoforms from S. tuberosum. After 5 more chromatographic steps, one HK isoform was purified to homogeneity (HK1). This enzyme was characterized and sequenced by MS/MS. We were able to associate this protein sequence with the gene product of StHK1 from S. tuberosum. With a specific activity of 10.2 U/mg of protein, this is the HK with the highest specific activity ever reported from a plant tissue. All the information gathered while purifying HK1 was used to undertake the purification of a second isoform (HK3). We were able to obtain preliminary results on its kinetic properties.

Résumé La ribonucléase P (RNase P) est une ribonucléoprotéine omniprésente dans tous les règnes du vivant, elle est responsable de la maturation en 5’ des précurseurs des ARNs de transfert (ARNts) et quelques autres petits ARNs. L’enzyme est composée d'une sous unité catalytique d'ARN (ARN-P) et d'une ou de plusieurs protéines selon les espèces. Chez les eucaryotes, l’activité de la RNase P cytoplasmique est distincte de celles des organelles (mitochondrie et chloroplaste). Chez la plupart des espèces, les ARN-P sont constituées de plusieurs éléments structuraux secondaires critiques conservés au cours de l’évolution. En revanche, au niveau de la structure, une réduction forte été observé dans la plupart des mtARN-Ps. Le nombre de protéines composant la RNase P est extrêmement variable : une chez les bactéries, environ quatre chez les archéobactéries, et dix chez la forme cytoplasmique des eucaryotes. Cet aspect est peu connu pour les formes mitochondriales. Dans la plupart des cas, l’identification de la mtRNase P est le résultat de longues procédures de purification comprenant plusieurs étapes dans le but de réduire au minimum le nombre de protéines requises pour l’activité (exemple de la levure et A. nidulans). Cela mène régulièrement à la perte de l’activité et de l’intégrité des complexes ribonucléo-protéiques natifs. Dans ce travail, par l’utilisation de la technique de BN-PAGE, nous avons développé une procédure d’enrichissement de l’activité RNase P mitochondriale native, donnant un rendement raisonnable. Les fractions enrichies capables de cette activité enzymatique ont été analysées par LC/MS/MS et les résultats montrent que l’holoenzyme de la RNase P de chacune des fractions contient un nombre de protéines beaucoup plus grand que ce qui était connue. Nous suggérons une liste de protéines (principalement hypothétiques) qui accompagnent l’activité de la RNase P. IV De plus, la question de la localisation de la mtRNase P de A. nidulans a été étudiée, selon nos résultats, la majorité de la mtRNase P est attachée á la membrane interne de la mitochondrie. Sa solubilisation se fait par l’utilisation de différents types de détergent. Ces derniers permettent l’obtention d’un spectre de complexes de la RNase P de différentes tailles. / Abstract RNase P is a ribonucleo-protein complex (an RNA enzyme or ribozyme) that cleaves 5’ leader sequences of precursor tRNAs and a few other small RNAs. It occurs in all three domains of life, Bacteria, Archaea and Eukarya, with the latter containing distinct nuclear and organellar (mitochondrial or plastid) activities. In most instances, the complex contains a single, well-conserved RNA subunit that carries the active center of the enzyme. Yet, compare to bacterial and nuclear P RNA, most mtP RNAs are structurally highly reduced. The number of P proteins is highly variable: one in Bacteria, about four in Archaea, and ten in the cytoplasmic form of Eukarya. Much less is known in the case of mitochondria. MtRNase P is usually purified by using numerous separation steps that include unphysiological conditions, leading to complexes having a minimum number of subunits (e.g., in yeast and Aspergillus nidulans), that often loose their activity. Here, using BN PAGE, we have developed an enrichment procedure for A. nidulans mtRNase P that avoids some of the most disruptive conditions. The protein composition of active fractions was identified with LC/MS/MS, indicating that the RNase P holoenzyme is much larger than previously thought. Finally, the question of mtRNase P localization within mitochondria was investigated, by tracing its RNA subunit by RT PCR. We found that mtRNase P of A. nidulans is a predominantly membrane-attached enzyme; it is in part solubilized by detergents such as digitonin and Triton.

- 5 - Abstract Hydrogen/deuterium exchange coupled to mass spectrometry (HX-MS) utilizes the spontaneous exchange of protein backbone amide hydrogens for deuterium atoms from solution to gain information about changes in protein structure. To localize these changes to specific areas of the protein, enzymatic digestion by aspartate proteases is used. The proteases' ability to produce small overlapping peptides and to provide full sequence coverage of the studied protein is essential for pinpointing the protein regions of interest. In this study recombinant proteases nepenthesin I (Nepenthes gracilis) and rhizopuspepsin (Rhizopus chinensis) were prepared and compared to commercially available proteases porcine pepsin A and aspergillopepsin (Aspergillus saitoi). The comparison was performed using various activity assays, where the effects of pH, temperature and denaturing and reducing agents on the activity of the proteases were studied. All four proteases were also immobilized on a polymeric resin POROS and their activity in an online HX-MS digestion setup was tested using myoglobin as a model substrate.

A proteína A de superfície de pneumococo (PspA) é encontrada na superfície de todas as cepas de Streptococcus pneumoniae e candidata promissora para novas vacinas pneumocócicas. Foi desenvolvido um processo de purificação da PspA4Pro cujas etapas iniciais foram: ruptura da biomassa celular, precipitação do homogenato obtido com o detergente brometo de cetiltrimetilamônio (CTAB) e remoção do precipitado por centrifugação. Foram avaliadas cromatografias de troca iônica (aniônica, catiônica), afinidade por metais, interação hidrofóbica e mista de troca catiônica e hidrofóbica. Utilizando precipitação com CTAB, cromatografia de troca aniônica, crioprecipitação em pH4,0 e cromatografia de troca catiônica atingiu-se a pureza requerida de PspA4Pro (>95%) com recuperação entre 14% e 33%. O processo alcançou níveis aceitáveis de endotoxina no produto final e a PspA4Pro purificada foi reconhecida por anticorpos anti-PspA4, manteve sua atividade e sua estrutura secundária. / Pneumococcal surface protein A (PspA) is found in all Streptococcus pneumoniae strains and is a promising candidate to be used in new pneumococcal vaccines. A purification process for PspA4Pro which inicial steps were: cell disruption, precipitation of the homogenate with the cationic detergent cetyltrimethylammonium bromide (CTAB) and pellet removal by centrifugation. The chromatographic techniques tested were ion exchange (anionic and cationic), immobilized metal affinity, hydrophobic interaction and mix mode with hydrophobic and cationic ligands. Using CTAB precipitation, anion exchange chromatography, crioprecipitation in pH4.0 and cation exchange chromatography the PspA reached the required purity (>95%) with recovery between 14% and 33% . The process reached acceptable levels of endotoxin in the final product and the purified PspA4Pro was recognized by anti-PspA4 antibodies and manteined its activity and secondary structure.

Sterine sind essentielle Komponenten der Zellmembran, deren Konzentration und Lokalisierung genau kontrolliert wird. Die Hefe Saccharomyces cerevisiae ist ein fakultativ anaerober Organismus, der in Abwesenheit von Sauerstoff auxotroph für Sterine wird. Die Proteine Aus1p und Pdr11p gehören zur Familie der ABC Proteine und spielen eine wichtige Rolle in diesem Prozess, da die gleichzeitige Deletion beider Protein die Aufnahme von Sterinen unter anaeroben Wachstumsbedingungen blockiert.In dieser Arbeit wurde das Gen AUS1 in voller Länge kloniert. Methoden für die Extraktion und Reinigung dieses Transporters wurden entwickelt, damit dieser detailliert charakterisiert werden kann. Mit Hilfe von Detergenzien wurde das Protein löslich gemacht und zeigte ATP-Bindung und -Hydrolyse. Die ATP-Hydrolyse konnte durch die Mutation eines konservierten Lysins zu Methionin im Walker A Motif verhindert. Genauso konnte die ATP-Hydrolyse auch durch klassische Inhibitoren von ABC Transportern inhibiert werden. Nach der Rekonstitution von Aus1p in Proteoliposomen wurde die ATPase Aktivität spezifisch durch Phosphatidylserin in einer stereoselektiven Weise stimuliert.Zusätzlich konnte gezeigt werden, dass Änderungen im zellulären PS Spiegel die Aus1p-abhängige Aufnahme von Sterin an die Membran beeinflussen. Diese Ergebnisse schlagen eine für die Aktivität des Transporters wichtige, direkte Interaktion zwischen Aus1p und PS vor.Da es sich bei der Aufnahme von Sterin um einen komplexen Prozess handelt, könnten Komponenten exisitieren, die mit Aus1p interagieren. Der Hefestamm, der die Immunpräzipitation von Aus1p mit seinem Interaktionspartner ermöglicht, wurde erzeugt und der Einfluß von Mannoproteinen auf Sterinaufnahme wurde getestet. Außerdem wurde eine Methode entwickelt, mit der Aus1p in Giant Unilamellar Vesicles rekonstituiert werden kann. Mit diesen Liposomen kann das Verhalten und die Aktivität von Aus1p in Membranen mit einer komplexen Lipidzusammensetzung untersucht werden. / Sterols are essential components of cellular membranes and their concentration and localization are tightly controlled. Saccharomyces cerevisiae is a facultative anaerobic organism which becomes auxotrophic for sterols in the absence of oxygen. However, the precise mechanism of sterol uptake remains to be revealed. Two proteins belonging to ABC protein family, Aus1p and Pdr11p were proposed to play a critical role in this process as simultaneous deletion of both of them blocks sterol uptake under anaerobiosis. In the present work, the full length AUS1 gene was cloned. An extraction and purification procedures were then developed to allow for detailed characterization of the transporter. The detergent solubilized protein was shown to bind and hydrolyse ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1p was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. Furthermore, it was demonstrated that Aus1p-dependent sterol uptake, but not Aus1p expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1p and PS which is critical for the activity of the transporter. Because of the complexity of sterol incorporation process efforts were made to identify additional components of the sterol uptake machinery that interact with Aus1p protein. The yeast strain allowing for immunopercipitation of Aus1p with its interaction partners was generated and previously proposed influence of mannoproteins on the sterol uptake was tested. Additionally, method was developed to reconstitute Aus1p protein into Giant Unilamellar Vesicles. These liposomes can be used further for testing of the behaviour and activity of Aus1p in the membranes with complex lipid composition.

O exossomo é um complexo multiproteico conservado evolutivamente de archaea a eucariotos superiores que desempenha funções celulares essenciais tais como: atividade exoribonucleolítica 3\'→5\', regulação dos níveis de mRNA, maturação de RNAs estruturais e controle de qualidade de RNAs durante os vários estágios do mecanismo de expressão gênica. Em Archaea, o exossomo é composto por até quatro subunidades diferentes, duas com domínios de RNase PH, aRrp41 e aRrp42, e duas com domínios de ligação a RNAs, aCsl4 e aRrp4. Três cópias das proteínas aRrp4 e/ou aCsl4 se associam com o núcleo hexamérico catalítico do anel de RNase PH e completam a formação do complexo. A proteína PaNip7 é um cofator de regulação do exossomo da archaea Pyrococcus abyssi e atua na inibição do complexo enzimático ligando-se simultaneamente ao exossomo e a RNAs. Neste projeto, a reconstituição in vitro do exossomo da archaea Pyrococcus abyssi formado pela proteína de topo PaCsl4 foi obtida. Para tanto foram realizadas análises de interação proteica usando as técnicas de cromatografia de afinidade, gel filtração e SDS-PAGE. Em adição à formação da isoforma PaCsl4-exossomo, um fragmento peptídico correspondente à região C-terminal da PaNip7 foi sintetizado pelo método da fase sólida, purificado por RP-HPLC e o purificado foi caracterizado por LC/ESI-MS almejando realizar futuros experimentos de interação com o exossomo. / The exosome is a multiprotein complex evolutionarily conserved from archaea to higher eukaryotes that performs essential cellular functions such as: 3\'→5\' exoribonucleolytic activity, regulation of mRNA levels, maturation of structural RNAs and quality control of RNAs during the various stages of the gene expression mechanism. In Archaea, the exosome is composed of up to four different subunits, two with RNase PH domains, aRrp41 and aRrp42, and two with RNAs binding domains, aCsl4 and aRrp4. Three copies of the aRrp4 and/or aCsl4 proteins associate with the hexameric catalytic core of the RNase PH ring and complete the formation of the complex. The PaNip7 protein is a regulating cofactor of the Pyrococcus abyssi archaeal exosome and acts in the inhibition of the enzyme complex by binding simultaneously to the exosome and RNAs. In this project, the reconstitution in vitro of the Pyrococcus abyssi archaeal exosome formed by the PaCsl4 top protein was achieved. To this end protein interaction analyses were performed using affinity chromatography, gel filtration and SDS-PAGE techniques. In addition to the formation of the PaCsl4-exosome isoform, a peptide fragment corresponding to the C-terminal region of PaNip7 was synthesized by solid-phase method, purified by RP-HPLC and the purified peptide was characterized by LC/ESI-MS aiming to perform future binding experiments with the exosome.

A proteína A de superfície de pneumococo (PspA) é indispensável para a virulência da bactéria e foi escolhida para a elaboração de uma nova vacina conjugada contra S. pneumoniae. Para tanto foi desenvolvido um processo industrial de produção e purificação do fragmento recombinante da PspA clado 3 em E. coli. Cultivos descontínuos alimentados foram estabelecidos com glicose ou glicerol em reator de 5L, obtendo-se 62g/L de células secas e 3g/L de PspA3. As células foram lisadas por homogeneizador contínuo de alta pressão com eficiência de 96,7%. A centrifugação foi definida como etapa de clarificação. A sequência cromatográfica troca aniônica seguida de afinidade por Ni+2 rendeu os melhores resultados de pureza (81%) e recuperação (70%). A cromatografia de troca catiônica foi selecionada como terceira etapa do processo, definindo assim um processo de produção e purificação escalonável que possibilitou a obtenção de PspA3 com alto grau de pureza (90%). / The pneumococcal surface protein A (PspA) is indispensable for virulence of S. pneumoniae and it was the first choice as carrier for a new conjugated vaccine against S.pneumoniae. Hence, the purpose of this work was to develop an industrial production and purification process of a recombinant fragment PspA clade 3 (rfPspA3) in E. coli. Fed-batch cultivations in 5 L bioreactors with defined medium were carried out using glucose or glycerol as carbon sources. It was obtained 62 g/L of dry cell weight and 3 g/L of rfPspA3. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. Centrifugation was defined for the clarification step. The sequence with Q- followed by IMAC-Sepharose yielded the best purity and recovery of rfPspA3 (81 and 70%, respectively). Cation exchange was chosen for the last chromatography. In conclusion, an industrial production and purification process was developed and rfPspA3 was obtained with high purity (90%).

A produção de proteínas recombinantes, principalmente para uso farmacêutico, tem sido intensamente estudada, juntamente com suas propriedades físico-químicas, o que possibilita uma melhor escolha da técnica de purificação, e assim, evitar perdas no rendimento e custos elevados. A glicocerebrosidase (GCR) é uma enzima lisossomal, e sua deficiência ocasiona um distúrbio autossômico recessivo denominado doença de Gaucher. Atualmente o tratamento para essa patologia é por meio da Terapia de Reposição Enzimática (TRE), a qual tem sido realizada com grande êxito. Tendo em vista fornecer dados que possam auxiliar na redução do número de etapas cromatográficas no processo de downstream, este trabalho teve como objetivo, a purificação da GCR expressa por células de Ovário de Hamster Chinês (CHO), em meio livre de soro fetal bovino (SFB). Para se alcançar tais resultados, realizou-se o cultivo das células CHO-GCR em meio quimicamente definido livre de SFB (CHO-S-SFM II). Foram realizadas três técnicas cromatográficas (troca iônica, interação hidrofóbica e afinidade), com base em suas propriedades físico-químicas. E para o ensaio da atividade enzimática, foi utilizado o substrato fluorogênico 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G). As células CHO-GCR cultivadas em meio CHO-S-SFM II, apresentando viabilidade maior que 95%, e produção da GCR ativa, durante todo o período de cultivo. Os protocolos aplicados para a purificação da GCR, não apresentaram resultados significativos. Com o volume não retido após cromatografia por interação hidrofóbica, se estimou os valores de KM 2,13 e VMAX 0,0295 UFR/h para as constantes cinéticas da GCR. A diálise no processo de purificação mostrou ser uma etapa necessária para a atividade enzimática da GCR. No cultivo das células CHO-GCR para a formação do banco de trabalho, nos meios RPMI 1640 e α-MEM, ambos com a adição de 10% SFB, não houve diferença significativa no crescimento entre eles, e apresentaram 100% de viabilidade durante todo o período de cultivo. Porém, ao analisar de forma isolada a fase exponencial de cada curva, notou-se que às células cultivadas no meio RPMI 1640, apresentaram taxa de crescimento superior, às cultivadas em meio α-MEM. Concluiu-se que a expressão da GCR em meio livre de SFB, proporciona amostras menos complexas, em relação aos meios de cultura que necessitam de suplementação com SFB, o que pode reduzir o número de etapas cromatográficas, melhorando o rendimento e a redução da perda da atividade da GCR. O meio basal RPMI 1640 com a adição de SFB foi uma alternativa satisfatória para o cultivo das células CHO-GCR. Este estudo forneceu dados que podem contribuir para a melhoria do processo de purificação da GCR. Novas pesquisas podem ser desenvolvidas a fim de melhorar o processo de purificação da GCR. / The production of recombinant proteins, mainly for pharmaceutical use, has been intensively studied along with its physico-chemical properties, which allows a better choice of the purification technique, and thus avoid losses in yield and high costs. Glucocerebrosidase (GCR) is a lysosomal enzyme, and its deficiency causes an autosomal recessive disorder called Gaucher\'s disease. Currently the treatment for this pathology is through Enzymatic Replacement Therapy (ERT), which has been successfully performed. In order to provide data that may help reducing the number of chromatographic steps in the downstream process, this work aimed to purify GCR expressed by Chinese Hamster Ovary (CHO) cells in fetal bovine serum free (FBS). To achieve such results, the CHO-GCR cells were cultured in chemically defined Serum-free medium (CHO-S-SFM II). Three chromatographic techniques (ion exchange, hydrophobic interaction and affinity) were performed, based on their physicochemical properties. And for the enzymatic activity assay, the fluorogenic substrate 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G) was used. CHO-GCR cells cultured in CHO-S-SFM II medium, presenting viability greater than 95% and GCR production active, throughout the culture period. The protocols applied for GCR purification did not present significant results. With the volume not retained after chromatography by hydrophobic interaction, KM values of 2.13 and VMAX 0.0295 UFR/h were estimated for GCR kinetic constants. Dialysis in the purification process was shown to be a step necessary for the enzymatic activity of GCR. In the culture of the CHO-GCR cells for the formation of the working bank, in the media RPMI 1640 and α-MEM, both with the addition of 10% FBS, there was no significant growth difference between them, and they showed 100% viability during all the growing period. However, when analyzing in isolation, the exponential phase of each curve, it was observed that the cells grown in RPMI 1640 medium showed higher growth rates, tham grown in α-MEM medium. It was concluded that the expression of GCR in serum-free medium provides less complex samples, relative to the culture media requiring FBS supplementation, which may reduce the number of chromatographic steps, improving yield and loss reduction of GCR activity. The basal medium RPMI 1640 with the addition of FBS was a satisfactory alternative for culturing the CHO-GCR cells. This study provided data that may contribute to the improvement of the GCR purification process. New research can be developed to improve the GCR purification process.

Os primeiros vasos sanguíneos do embrião de vertebrados, formados de novo a partir de células originadas da mesoderme, originam os vasos linfáticos em um processo denominado vasculogênese. Já no adulto, novos vasos são formados principalmente através da angiogênese (ou linfoangiogênese) a partir da vasculatura pré-existente. Em indivíduos saudáveis, a arquitetura vascular é relativamente estática, sendo que o excesso ou a insuficiência de vasos são comumente relacionados à angiogênese patológica, vinculada a diversas doenças, como câncer, degeneração macular relacionada à idade, isquemia de membros e muitas outras. Dessa forma, o controle local da densidade de vasos sanguíneos torna-se interessante para o tratamento de condições patológicas visando melhoria de prognóstico e cura. Dentre os diversos fatores de crescimento conhecidos, o fator de crescimento endotelial vascular, VEGF, destaca-se como o principal regulador do processo de angiogênese através de isoformas pró-angiogênicas (VEGFxxx) e antiangiogênicas (VEGFxxxb) do gene VEGF-A. Consequentemente, as proteínas codificadas por esse gene constituem um alvo com alto potencial terapêutico. No presente estudo, propusemos a produção das isoformas proteicas recombinantes rhVEGF165, rhVEGF165b e rhVEGF121, oriundas do gene VEGF-A humano, visando à geração de biofármacos que podem ser utilizados para terapia molecular e engenharia tecidual. As sequências codificadoras das isoformas rhVEGF165 e rhVEGF121 foram amplificadas a partir do cDNA total sintetizado de amostras de RNA total de pulmão humano, enquanto que a da isoforma rhVEGF165b foi gerada através da mutação sítio-dirigida da sequência de rhVEGF165. As sequências foram clonadas no vetor de clonagem pGEM®-T Easy, sendo em seguida subclonadas no vetor pLV-eGFP, um vetor plasmidial de transferência lentiviral, que permite a expressão de transgenes em células de mamíferos e, também, da proteína repórter eGFP. Células humanas HEK293 em cultura aderente foram independentemente cotransfectadas com cada uma das construções geradas (pLV-rhVEGF165, pLV-rhVEGF165b e pLV-rhVEGFl21) juntamente com o vetor pTK-Hyg na proporção de 40:1 (m/m), viabilizando a seleção dos transfectantes com o antibiótico higromicina B, além da detecção de eGFP. Os clones celulares superprodutores das proteínas de interesse foram avaliados quanto à cinética de expressão em meio carente de soro e adaptados para a cultura em suspensão estática na presença de meio na ausência de componentes derivados de animais, demostrando a capacidade de expressão das isoformas rhVEGFs nestas condições de cultivo. Para o nosso conhecimento, este é o primeiro trabalho a descrever a expressão de isoformas de VEGF-A em células HEK293 mantidas em suspensão. As isoformas rhVEGF165 e rhVEGF165b foram purificadas por cromatografia de afinidade a heparina, a partir do meio condicionado pelos clones superprodutores gerados. Ensaios in vitro, utilizando o AngioPhaseTM Kit, e in vivo, através do ensaio da membrana corioalantoide em embriões de galinha (CAM Assay), ambos próprios para avaliação da atividade pró- e antiangiogênica de diferentes compostos, demonstraram que a isoforma rhVEGF165 possui atividade biológica, enquanto a isoforma rhVEGF165b não apresentou a atividade esperada (inibição da angiogênese). Estas isoformas foram testadas em modelo murino de engenharia tecidual do intestino curto, com indícios de que poderiam contribuir para o uso terapêutico neste contexto. A purificação da isoforma rhVEGF121, bem como as análises estruturais das proteínas produzidas, estão em processo de otimização. / The first blood vessels of the vertebrate embryo are formed de novo from mesoderm-derived cells and give rise to lymph vessels in a process termed vasculogenesis. In the adult, new blood vessels are formed mainly through angiogenesis (or lymphangiogenesis) from the pre-existing vasculature. In healthy individuals, the vascular architecture is fairly static, and both the excess and the insufficiency of vessels comprise a pathological angiogenic state, to which is credited the onset and/or progression of several diseases such as cancer, age-related macular degeneration, limb ischemia, and many others. Therefore, locally controlling the blood vessel density becomes interesting for the treatment of pathological conditions aiming at prognosis improvement and cure. Among the various known growth factors, the vascular endothelial growth factor, VEGF, stands out as the major regulator of the angiogenic process. This process is mediated through the action of pro- (VEGFxxx) and antiangiogenic (VEGFxxxb) isoforms, which are derived from the VEGF-A gene. Consequently, the proteins encoded by this gene are potential therapeutic targets. In this work, we set out to produce the recombinant protein isoforms rhVEGF165, rhVEGF165b, and rhVEGF121, which originate from the human VEGF-A gene, with the aim of generating biopharmaceuticals to be used for molecular therapy and tissue engineering. The rhVEGF165 and rhVEGF121 coding sequences were amplified from total cDNA sythesized from human lung total RNA. Conversely, the rhVEGF165b coding sequence was generated by site-directed mutagenesis of the rhVEGF165 sequence. The sequences were cloned into the pGEM®-T Easy cloning vector. These cDNAs were then subcloned into pLV-eGFP, a plasmid lentiviral transfer vector that allows for expression of transgenes and the eGFP reporter protein in mammalian cells. Human HEK293 cells cultivated under adherent conditions were independently co-transfected with each of the obtained constructs (pLV-rhVEGF165, pLV-rhVEGF165b, and pLV-rhVEGF121) and the pTK-Hyg vector at a proportion of 40:1 (m/m), enabling for the selection of transfectants with hygromycin B, apart from the detection of eGFP. The cell clones overexpressing the proteins of interest were evaluated for expression kinetics in serum-deprived conditioned media and adapted to static suspension culture in medium free of animal-derived components, demonstrating that expression of the protein isoforms was possible in these culture conditions. To the best of our knowledge, this is the first work that describes the expression of VEGF-A isoforms in HEK293 cells in suspension culture. The rhVEGF165 and rhVEGF165b isoforms were purified by affinity chromatography from media previously conditioned by the overexpressing cell clones. The biological activity of rhVEGF165 was demonstrated in vitro, by the AngioPhaseTM Kit assay, and in vivo with the CAM (chorioallantoic membrane) assay, both of which are suitable for evaluating the pro- and antiangiogenic activity of different compounds. The rhVEGF165b did not show the expected antiangiogenic activity. These isoforms were tested in a model of murine tissue-engineered small intestine, indicating a possible contribution to therapeutic use in this context. The purification of rhVEGF121, as well as the structural analysis of all three proteins, are in the process of being optimized.

A produção de proteínas recombinantes, principalmente para uso farmacêutico, tem sido intensamente estudada, juntamente com suas propriedades físico-químicas, o que possibilita uma melhor escolha da técnica de purificação, e assim, evitar perdas no rendimento e custos elevados. A glicocerebrosidase (GCR) é uma enzima lisossomal, e sua deficiência ocasiona um distúrbio autossômico recessivo denominado doença de Gaucher. Atualmente o tratamento para essa patologia é por meio da Terapia de Reposição Enzimática (TRE), a qual tem sido realizada com grande êxito. Tendo em vista fornecer dados que possam auxiliar na redução do número de etapas cromatográficas no processo de downstream, este trabalho teve como objetivo, a purificação da GCR expressa por células de Ovário de Hamster Chinês (CHO), em meio livre de soro fetal bovino (SFB). Para se alcançar tais resultados, realizou-se o cultivo das células CHO-GCR em meio quimicamente definido livre de SFB (CHO-S-SFM II). Foram realizadas três técnicas cromatográficas (troca iônica, interação hidrofóbica e afinidade), com base em suas propriedades físico-químicas. E para o ensaio da atividade enzimática, foi utilizado o substrato fluorogênico 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G). As células CHO-GCR cultivadas em meio CHO-S-SFM II, apresentando viabilidade maior que 95%, e produção da GCR ativa, durante todo o período de cultivo. Os protocolos aplicados para a purificação da GCR, não apresentaram resultados significativos. Com o volume não retido após cromatografia por interação hidrofóbica, se estimou os valores de KM 2,13 e VMAX 0,0295 UFR/h para as constantes cinéticas da GCR. A diálise no processo de purificação mostrou ser uma etapa necessária para a atividade enzimática da GCR. No cultivo das células CHO-GCR para a formação do banco de trabalho, nos meios RPMI 1640 e α-MEM, ambos com a adição de 10% SFB, não houve diferença significativa no crescimento entre eles, e apresentaram 100% de viabilidade durante todo o período de cultivo. Porém, ao analisar de forma isolada a fase exponencial de cada curva, notou-se que às células cultivadas no meio RPMI 1640, apresentaram taxa de crescimento superior, às cultivadas em meio α-MEM. Concluiu-se que a expressão da GCR em meio livre de SFB, proporciona amostras menos complexas, em relação aos meios de cultura que necessitam de suplementação com SFB, o que pode reduzir o número de etapas cromatográficas, melhorando o rendimento e a redução da perda da atividade da GCR. O meio basal RPMI 1640 com a adição de SFB foi uma alternativa satisfatória para o cultivo das células CHO-GCR. Este estudo forneceu dados que podem contribuir para a melhoria do processo de purificação da GCR. Novas pesquisas podem ser desenvolvidas a fim de melhorar o processo de purificação da GCR. / The production of recombinant proteins, mainly for pharmaceutical use, has been intensively studied along with its physico-chemical properties, which allows a better choice of the purification technique, and thus avoid losses in yield and high costs. Glucocerebrosidase (GCR) is a lysosomal enzyme, and its deficiency causes an autosomal recessive disorder called Gaucher\'s disease. Currently the treatment for this pathology is through Enzymatic Replacement Therapy (ERT), which has been successfully performed. In order to provide data that may help reducing the number of chromatographic steps in the downstream process, this work aimed to purify GCR expressed by Chinese Hamster Ovary (CHO) cells in fetal bovine serum free (FBS). To achieve such results, the CHO-GCR cells were cultured in chemically defined Serum-free medium (CHO-S-SFM II). Three chromatographic techniques (ion exchange, hydrophobic interaction and affinity) were performed, based on their physicochemical properties. And for the enzymatic activity assay, the fluorogenic substrate 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G) was used. CHO-GCR cells cultured in CHO-S-SFM II medium, presenting viability greater than 95% and GCR production active, throughout the culture period. The protocols applied for GCR purification did not present significant results. With the volume not retained after chromatography by hydrophobic interaction, KM values of 2.13 and VMAX 0.0295 UFR/h were estimated for GCR kinetic constants. Dialysis in the purification process was shown to be a step necessary for the enzymatic activity of GCR. In the culture of the CHO-GCR cells for the formation of the working bank, in the media RPMI 1640 and α-MEM, both with the addition of 10% FBS, there was no significant growth difference between them, and they showed 100% viability during all the growing period. However, when analyzing in isolation, the exponential phase of each curve, it was observed that the cells grown in RPMI 1640 medium showed higher growth rates, tham grown in α-MEM medium. It was concluded that the expression of GCR in serum-free medium provides less complex samples, relative to the culture media requiring FBS supplementation, which may reduce the number of chromatographic steps, improving yield and loss reduction of GCR activity. The basal medium RPMI 1640 with the addition of FBS was a satisfactory alternative for culturing the CHO-GCR cells. This study provided data that may contribute to the improvement of the GCR purification process. New research can be developed to improve the GCR purification process.