As serinoproteases de peçonha de serpentes (SVSPs) agem sobre pontos específicos do sistema circulatório, sendo consideradas promissoras para o tratamento de uma diversidade de desordens hemostáticas. No presente estudo, é descrita a expressão de uma serinoprotease de Crotalus durissus collilineatus (Collineina-1) em Pichia pastoris, bem como a purificação dessa toxina a partir da peçonha de C. d. collilineatus e a caracterização estrutural e enzimática da Collineina-1 nas formas nativa e recombinante. O cDNA que codifica a serinoprotease foi amplificado a partir da biblioteca de cDNA da glândula de peçonha de C. d. collilineatus e ligado ao vetor pPICZ A. A linhagem KM71H de P. pastoris foi transformada com o plasmídeo recombinante e as colônias foram selecionadas por resistência à zeocina. A expressão heteróloga foi realizada em meio mínimo suplementado com metanol, resultando em um rendimento de 56 mg de proteína por litro de cultura. A proteína recombinante foi purificada por um protocolo baseado em técnicas cromatográficas de troca iônica e fase reversa. A purificação da serinoprotease a partir da peçonha de C. d. collilineatus foi realizada pela combinação de técnicas de cromatografia de exclusão molecular, troca iônica e fase reversa, e resultou no isolamento de duas isoformas, denominadas Collineina-1 e 2. Quando analisada por espectrometria de massas, a Collineina-1 recombinante apresentou massa molar de 28.868 Da, enquanto as enzimas Collineina-1 e 2 apresentaram massas de 29.475 Da e 29.388 Da, respectivamente. A partir do alinhamento das sequências parciais das serinoproteases, foi possível determinar 100% de identidade dos aminoácidos para a Collineina-1 nativa e recombinante. O alinhamento múltiplo da sequência deduzida de aminoácidos da Collineina-1 indica uma semelhança estrutural dessa proteína com outras serinoproteases de peçonha de serpente. As enzimas nativa e recombinante mostraram efeitos similares sobre fibrinogênio bovino por clivarem preferencialmente a cadeia A do fibrinogênio, liberando o fibrinopeptídeo A. Ambas as enzimas induziram a coagulação do plasma bovino de forma dose-dependente, sendo que a Collineina-1 recombinante apresentou maior potencial coagulante, com uma dose mínima coagulante (DMC) de 0,08 mg/uL contra 0,225 mgu/L para a proteína nativa. As serinoproteases foram capazes de hidrolisar os substratos cromogênicos S-2222, S-2238 e S2302, embora ambas as enzimas tenham demonstrado maior atividade sobre o substrato S-2302. A atividade esterásica sobre o TAME foi avaliada em diferentes condições de temperatura e na presença de íons divalentes. As duas enzimas demonstraram alta termoestabilidade e tiveram a atividade inibida na presença dos íons Zn2+ e Cu2+. A cinética enzimática de ambas as serinoproteases seguiram o modelo de Michaelis-Menten. A Collineina-1 nativa apresentou um valor de Km de 1,43 mM, contra 1,682 mM para a proteína recombinante, indicando que a proteína nativa apresenta maior afinidade pelo substrato TAME. No entanto, as enzimas apresentaram valores similares de Kcat/Km (250,69 mM.min-1 para a Collineina-1 e 248,03 mM.min-1 para a rCollineina-1), sugerindo que as serinoproteases não diferem significativamente na eficiência em hidrolisar o substrato. Estes resultados demonstraram a adequação do sistema de escolha na produção heteróloga da Collineina-1, já que a proteína recombinante foi expressa com integridade funcional sobre os parâmetros avaliados. / Snake venom serine proteases (SVSPs) act on specific points of the circulatory system and are promising for the treatment of a variety of hemostatic disorders. In the present study, we describe the expression of a serine protease from Crotalus durissus collilineatus (Collineina- 1) in Pichia pastoris, the purification of the native toxin from C. d. collilineatus venom and the structural and enzymatic characterization of Collineina-1 in native and recombinant forms. The cDNA encoding the serine protease was amplified from cDNA library of C. d. collilineatus venom gland and cloned into pPICZ A vector. KM71H P. pastoris strain was transformed with the recombinant plasmid and colonies were selected by zeocin resistance. Heterologous expression was carried out in minimal medium supplemented with methanol, resulting in a yield of 56 mg of protein per liter of culture. The recombinant protein was purified by ion exchange and reverse phase chromatography. Purification of the native serine protease was accomplished by combining techniques of molecular exclusion, ion exchange and reversed phase, and resulted in the isolation of two isoforms, named Collineina-1 and 2. When analyzed by mass spectrometry, the recombinant Collineina-1 showed a molar mass of 28,868 Da, while Collineina-1 and 2 presented masses of 29,475 and 29,388 Da, respectively. The alignment of partial sequences of the enzymes resulted in 100% of amino acid identity between native and recombinant Collineina-1. The multiple alignment of deduced amino acid sequence of Collineina-1 indicates structural similarity with other snake venom serine proteases. The native and recombinant forms of the enzyme showed similar effects on bovine fibrinogen by cleaving preferentially A chain, releasing fibrinopeptide A. Both enzymes induced coagulation of bovine plasma in a dose-dependent way, though recombinant Collineina-1 presented a higher coagulant potential, with a minimum coagulant dose (MCD) of 0.08 mg/uL against 0.225 mg/uL for the native form. The serine proteases hydrolyzed S- 2222, S-2238 and S2302 chromogenic substrates, although both enzymes demonstrated increased activity upon S-2302. The esterase activity on TAME was evaluated at different temperatures and in the presence of divalent ions. Both enzymes showed high thermostability and their activity were inhibited in the presence of Zn2+ and Cu2+. The enzyme kinetics of both serine proteases followed Michaelis-Menten model. The native Collineina-1 showed a Km value of 1.43 mM, against 1.682 mM for the recombinant form, indicating that the native protein has a higher affinity for TAME substrate. However, enzymes had similar values for Kcat/Km (250.69 mM.min-1 for Collineina-1 and 248.03 mM.min-1 for rCollineina-1), suggesting that the serine proteases did not differ significantly in the efficiency to hydrolyze the substrate. These results demonstrated the adequacy of the system of choice in producing the snake venom serine protease, since the recombinant protein was expressed with functional integrity on the evaluated parameters.

Ellipticine is an alkaloid isolated from Apocynaceae plants exhibiting significant antitumor and anti-HIV activities. Cytochromes P450 (CYP) and peroxidases are the enzymes participating in metabolism of ellipticine. This process provides activation and detoxication metabolites of ellipticine. The CYP enzymes, which participate in oxidation of ellipticine in different tissues (liver, lung and kidney) of rat, a model organism simulating the fate of ellipticine in humans have already been identified. In this work, the effects of ellipticine on contents and catalytic activities of CYPs and other components of the mixed-function oxidase (MFO) system in this animal system were studied. For detection of contents of CYPs and other components of the MFO system, spectroscopic and electrochemical methods were used. To determine catalytic activities of CYPs and NADPH:cytochrome P450 reductase, reactions with specific substrates of these enzymes were utilized. The results found in this study demonstrate that expression and catalytic activity of CYP1A is induced by ellipticine in all of the tested organs (liver, kidney and lung) of rats treated with the drug. Moreover in liver, the cytochrome b5 expression is also induced. In addition, in this organ, expression and catalytic activity of CYP3A was increased by...

Um dos eventos mais importantes durante o amadurecimento da banana é a degradação do amido, concomitante com o acúmulo de açúcares solúveis. Várias enzimas, que supostamente atuam na degradação do amido, já tiveram sua atividade/proteína específica detectadas nesta fase na banana. Entre elas a α-amilase, a β-amilase, as amido-fosforilases, as α-glicosidases e as isoamilases. A síntese do amido e, normalmente, sua degradação, ocorrem dentro do amiloplasto, que possui duas membranas a serem transpostas antes do acesso ao grânulo de amido ou aos produtos da ação de outras enzimas. Uma das isoformas da β-amilase em banana possui um peptídeo de transporte predito em sua seqüência, necessário para transpor estas membranas e entrar no amiloplasto. Uma maneira de contornar a dificuldade em estabelecer a real importância de cada enzima na degradação do amido é isolar os grânulos e as enzimas e submetê-lo à atividade seqüencial das enzimas supostamente responsáveis pela degradação. O ideal é utilizar a enzima endógena, mas o processo de purificação de enzimas em frutos é demorado e nem sempre bom em termos de pureza, quantidade e atividade. Estudos baseados na expressão heteróloga de genes da β-amilase permitiriam melhor compreender os mecanismos de atuação dessa enzima presente na polpa da banana. Assim, foram feitos ensaios de expressão heteróloga em Pichia pastoris na tentativa de produzir essa enzima em quantidade suficiente para purificação, aplicação nos grânulos de amido e produção de anticorpos policlonais. Foram testadas várias condições de indução da proteína, tais como aeração, temperatura, pH, concentração de metanol e tempo de indução, bem como a montagem de uma nova construção gênica com tag de histidina no vetor de expressão pPICZαA com confirmação do fenótipo dos transformantes positivos. Porém, a obtenção de β-amilase recombinante com atividade não foi bem sucedida, necessitando talvez de alterações nesses padrões de indução. / One of the most important events that occurs during ripening of banana is the starch degradation concomitantly with the accumulation of soluble sugars. Several enzymes, known by acting on starch degradation, had their activity and/or specific protein detected at this stage of banana. These include the α-amylase, the β-amylase, the starch-phosphorylases, the α-glucosidase and the isoamilases. The synthesis and starch degradation occur inside of the amyloplast that contain two membranes which has to be reach before accessing the starch granule or the products of the other enzymes. One of the isoforms of β-amylase in bananas has a transit peptide predicted in the sequence, required to access the amyloplast. To establish the real importance of each enzyme in the starch degradation it is necessary to isolate the granules and enzymes and submit them to the sequential activity to confirm the supposed degradation. The idea is to use the endogenous enzyme, but the process of purification of the enzyme in fruits demands a lot of time and the results of purity, quantity and activity are not guaranteed. Studies based on heterologous expression of the β-amylase genes allow us to understand the mechanisms of action of this enzyme present in the pulp of banana. Thus, tests were carried out with heterologous expression in Pichia pastoris in order to produce this enzyme in sufficient quantity to purification, and then, applied on starch granules and produce a polyclonal antibody. We tested different conditions of protein induction such as aeration, temperature, pH, methanol concentration and induction time, as well as the new genic construction with the histidine tag with an expression vector pPICZαA confirming the phenotype of positive transformants. However, recombinant β-amylase with activity was not obtained successfully, necessitating changes in these patterns of induction.

The wide diversity of plant secondary products results from different modifications undergone during biosynthesis, including glucosylation. These modification reactions result in production of the compounds actually found in plants and to unique chemical and biochemical properties, including some bitter compounds in grapefruit. While the presence of a PSPG box motif allows for identification of a clone as a putative glucosyltransferase (PGT), diversity of GT primary structures makes it difficult to accurately assign specific function. Our approach is to identify and isolate putative GT clones, express them heterologously, and biochemically characterize the proteins. Eleven putative GT clones have been isolated from Citrus paradise and some have been biochemically characterized. The current hypothesis being tested is that PGT3 and PGT9 clones are plant secondary product GTs. Due to issues with inclusion bodies when using E. coli, proteins were expressed in Pichia pastoris using the pPICZA vector. Recombinant protein expression was confirmed by Western blot and proteins were enriched by IMAC. Over 30 flavonoid and simple phenolic substrates, representing many compounds found in grapefruit, were screened for activity with PGT3 and PGT9 proteins. No significant activity was found and the biochemical function of the proteins encoded by these clones will be further investigated.

A produção de biocombustíveis e a obtenção de alguns compostos químicos a partir de materiais renováveis, como a biomassa lignocelulósica, ainda não são processos triviais, principalmente devido a recalcitrância destes materiais. Estudos recentes reconheceram as enzimas acessórias, como xilanases e enzimas com Atividade Auxiliar, como potencializadores da atividade de celulases no processo de despolimerização da lignocelulose. A prospecção de enzimas com características termoestáveis é vantajosa para este tipo de aplicação e além disso, estudos sobre o secretoma de diversos fungos cultivados em biomassa como fonte de carbono, tem encontrado enzimas com mecanismo oxidativo, dentre eles, o fungo termofílico Myceliophthora thermophila M77. Porém, estas enzimas tem sido pouco estudadas quanto a sua aplicação na sacarificação da biomassa. Sendo assim, este trabalho visou a expressão heteróloga, a caracterização bioquímica e a ação da enzima oxidativa celobiose desidrogenase do fungo M. thermophila (M77CDH) em conjunto com outras celulases no processo de sacarificação da biomassa. Pela análise filogenética a M77CDH prospectada foi classificada como pertencente a Classe IIB das CDHs. O gene que codifica esta enzima foi clonado no vetor pEXPYR e heterólogamente expresso em A. nidulans. A proteína recombinante M77CDH foi purificada e teve sua identidade confirmada por espectrometria de massas. Nas análises bioquímicas, apresentou atividade ótima a 65 °C e reteve mais de 80% da sua atividade a 50°C por 2 horas e pela análise de dicroísmo circular apresentou um desenovelamento da sua estrutura na temperatura de transição de 62,8 °C. Apresentou mais de 80% de atividade em uma faixa ampla de pH (4,5 - 9), em que o domínio citocromo mostrou maior afinidade em pHs alcalinos, característica incomum entre as CDHs descritas na literatura. A atividade da M77CDH foi ligeiramente aumentada pela adição de MgCl2 e Na2MoO4 e altamente afetada por CuSO4 e FeCl3. A eficiência catalítica (kcat/km=266 mM-1s-1) utilizando celobiose foi bastante similar aos valores indicados por CDHs da Classe IIA. O envelope da M77CDH gerado por SAXS foi satisfatório e conveniente com a literatura. Na sacarificação de bagaço de cana pré-tratado hidrotermicamente, utilizando coquetel de A. niveus suplementado com M77CDH, foi possível observar que a adição de M77CDH modificou o perfil de produtos liberados na desconstrução da biomassa. Por fim, na sacarificação do PASC observou-se a sacarificação e produção de ácido celobiônico. / The production of biofuels and chemicals from renewable materials such as lignocellulosic biomass are non-trivial processes mainly due to the recalcitrance of the material. Recent studies have recognized accessory enzymes such as xylanases and Auxiliary Activity enzymes as potentiators in cellulase activity during the depolymerization of lignocellulose. The prospection of thermostable enzymes can be an advantage the improve the depolymerization of these materials. In addition, several enzymes showing oxidative mode of action were found in the secretoma of the thermophilic fungus Myceliophthora thermophila strain M77. However, these enzymes are poor studied regarding their application in biomass saccharification. Therefore, this project aimed the heterologous expression and biochemical characterization of the oxidative enzyme cellobiose dehydrogenase of the fungus M. thermophila (M77CDH). By phylogenetic analysis the M77CDH was classified as belonging to Class IIB of CDHs. The gene encoding this enzyme was cloned and heterologously expressed in A. nidulans, the M77CDH was purified and had its identity confirmed by mass spectrometry. In the biochemical analyzes the M77CDH showed an optimum activity at 65 °C and retained more than 80% of its activity at 50 °C for 2 hours. The circular dichroism analysis showed a denaturation of its structure at the transition temperature of 62.8 ° C. M77CDH also kept more than 80% of its activity in a wide pH range (4.5 - 9), in which the cytochrome domain showed higher affinity at alkaline pH, an unusual behavior compared with other CDHs described in the literature. The activity of M77CDH was increased slightly in the presence of MgCl2 and Na2MoO4 and was highly affected by CuSO4 and FeCl3. The catalytic efficiency (kcat/km = 266 mM-1s-1) in cellobiose was quite similar to the values indicated by CDHs from Class IIA. The envelope of M77CDH generated by SAXS was satisfactory and convenient with the literature. In saccharification of sugarcane bagasse hydrothermally pretreated using A. niveus cocktail supplemented with M77CDH was possible to observe the addition of M77CDH modified the profile of released products in the deconstruction of the biomass. Finally, in the action on PASC was observed the saccharification and production of cellobionic acid.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase. In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants. In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins. We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.

No description available.

Microbial chlorate respiration plays an important role in the turnover of oxochlorates in nature and industrial waste management. This thesis deals with the characterization of the molecular components of chlorate respiration in Ideonella dechloratans. Chlorate respiration utilizes two soluble periplasmic enzymes, chlorate reductase and chlorite dismutase, to convert chlorate to chloride and oxygen. The genes encoding the enzymes participating in the chlorate degradation have been sequenced, and are found in close proximity, forming a gene cluster for chlorate metabolism. This work also includes the successful recombinant expression of three genes from Ideonella dechloratans. Two of the gene products, chlorite dismutase and the C subunit of chlorate reductase, participate in the chlorate respiration. The third gene, which is found close to the gene cluster for chlorate metabolism, encodes a soluble c-type cytochrome. The localization of the gene suggests the corresponding protein as a candidate for a role as electron donor to chlorate reductase. Also, the role of soluble periplasmic c cytochromes of Ideonella dechloratans in chlorate respiration was studied. At least one of the soluble c cytochromes was found capable of serving as electron donor for chlorate reduction. This c cytochrome, and several others, can also donate electrons to a terminal oxidase for subsequent reduction of oxygen, as required for the branched electron flow during chlorate respiration.

Orientador: João Pessoa Araujo Junior / Resumo: A suinocultura vem alcançando grande desenvolvimento de técnicas eficientes associadas ao melhoramento genético, nutrição, manejo e sanidade. Entretanto, devido aos métodos intensivos de criação, os suínos se tornaram mais susceptíveis a um grande número de doenças infecciosas. Entre os mais importantes patógenos que afetam a indústria suinícola mundial está o circovírus suíno 2 (PCV2), um pequeno vírus icosaédrico, não-envelopado, de DNA circular, de fita simples (ssDNA), ambisenso, composta por 1767-1768 nucleotídeos. Este vírus é altamente resistente a variações ambientais e agentes desinfetantes, é endêmico no mundo todo e está associado a várias manifestações clínicas distintas, que acarretam importantes perdas econômicas aos produtores. Um dos fatores possivelmente implicados na patogenicidade do PCV2 é a proteína Cap, a unidade fundamental constituinte do capsídeo deste vírus. Estudos realizados pela equipe do Prof. Dr. João Pessoa Araújo Jr., do Instituto de Biotecnologia da Unesp em Botucatu/SP, comprovaram que vírus com mutações em suas proteínas Cap isolados a partir de cultivo celular aumentavam a morte celular em culturas celulares infectadas. Estes resultados evidenciaram a importância do capsídeo nos mecanismos de infecção e patogenicidade do PCV2, tornando interessante a realização de estudos estruturais com as proteínas Cap mutantes. A execução de estudos estruturais in silico mostrou a baixa frequência das mutações identificadas na proteína Cap dos vírus pro... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Swine breeding has achieved a high development based on genetic improvement, nutrition, management and sanity. However, due to the intensive breeding methods, swine have become more susceptible to a higher number of infectious diseases. Among the most important pathogens that affect the swine world industry is the porcine circovirus 2 (PCV2), a small, icosahedral, non-enveloped virus, ambisense single-stranded circular DNA, composed by 1,767-1,768 nucleotides. This virus is highly resistant to environmental variations and disinfecting agents, endemic worldwide and has been associated to several distinct clinical manifestations that entail important economic losses to the producers. One of the factors possibly implicated on the PCV2 pathogenicity is the Cap protein, the fundamental unity that constitutes this virus capsid. Studies performed by the group of Dr. João Pessoa Araújo Jr. rom the Instituto de Biotecnologia da Unesp em Botucatu/SP, confirmed that viruses with mutated Cap proteins from cell culture increased cell death in infected cultures. Such results highlight the importance of capsid in the infection mechanisms and pathogenicity of PCV2 and the importance of structural and comparative studies with Cap protein structures. In silico structural studies showed the low frequency of the mutations identified in the mutant Cap proteins and also indicated a clear difference between the physico-chemical properties of the new amino acid residues in comparison to those found ... (Complete abstract click electronic access below) / Mestre

Submitted by Pâmela Merlo Marson (pam.marson@aluno.ibb.unesp.br) on 2018-06-05T17:11:43Z No. of bitstreams: 1 Dissertação_Mestrado_Pamela-Merlo-Marson_2018_vf.pdf: 1757229 bytes, checksum: ed7b8faed4d956e7a1ba89178199507d (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-06-07T14:10:28Z (GMT) No. of bitstreams: 1 marson_pm_me_bot.pdf: 1757229 bytes, checksum: ed7b8faed4d956e7a1ba89178199507d (MD5) / Made available in DSpace on 2018-06-07T14:10:28Z (GMT). No. of bitstreams: 1 marson_pm_me_bot.pdf: 1757229 bytes, checksum: ed7b8faed4d956e7a1ba89178199507d (MD5) Previous issue date: 2018-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A suinocultura vem alcançando grande desenvolvimento de técnicas eficientes associadas ao melhoramento genético, nutrição, manejo e sanidade. Entretanto, devido aos métodos intensivos de criação, os suínos se tornaram mais susceptíveis a um grande número de doenças infecciosas. Entre os mais importantes patógenos que afetam a indústria suinícola mundial está o circovírus suíno 2 (PCV2), um pequeno vírus icosaédrico, não-envelopado, de DNA circular, de fita simples (ssDNA), ambisenso, composta por 1767-1768 nucleotídeos. Este vírus é altamente resistente a variações ambientais e agentes desinfetantes, é endêmico no mundo todo e está associado a várias manifestações clínicas distintas, que acarretam importantes perdas econômicas aos produtores. Um dos fatores possivelmente implicados na patogenicidade do PCV2 é a proteína Cap, a unidade fundamental constituinte do capsídeo deste vírus. Estudos realizados pela equipe do Prof. Dr. João Pessoa Araújo Jr., do Instituto de Biotecnologia da Unesp em Botucatu/SP, comprovaram que vírus com mutações em suas proteínas Cap isolados a partir de cultivo celular aumentavam a morte celular em culturas celulares infectadas. Estes resultados evidenciaram a importância do capsídeo nos mecanismos de infecção e patogenicidade do PCV2, tornando interessante a realização de estudos estruturais com as proteínas Cap mutantes. A execução de estudos estruturais in silico mostrou a baixa frequência das mutações identificadas na proteína Cap dos vírus provenientes do cultivo in vitro e também indicou uma clara diferença entre as propriedades físico-químicas dos novos resíduos de aminoácidos em relação àqueles substituídos. Estas alterações, associadas à localização dos resíduos na superfície viral e a menor flexibilidade das proteínas Cap dos vírus mutantes, indicaram a possibilidade de alterações estruturais/funcionais relevantes, incluindo a alteração da afinidade por receptores celulares e diminuição da efetividade de anticorpos produzidos contra vírus vacinais. Foram também realizados trabalhos experimentais para a produção heteróloga da proteína Cap de um vírus selvagem, os quais envolveram ensaios de subclonagem da sequência de interesse em um vetor de expressão, testes de transcrição e experimentos de expressão protéica. Os resultados destes procedimentos foram compatíveis com a produção da proteína Cap, porém novos estudos são necessários para confirmar a produção da molécula alvo e melhorar o rendimento dos ensaios de expressão. / Swine breeding has achieved a high development based on genetic improvement, nutrition, management and sanity. However, due to the intensive breeding methods, swine have become more susceptible to a higher number of infectious diseases. Among the most important pathogens that affect the swine world industry is the porcine circovirus 2 (PCV2), a small, icosahedral, non-enveloped virus, ambisense single-stranded circular DNA, composed by 1,767-1,768 nucleotides. This virus is highly resistant to environmental variations and disinfecting agents, endemic worldwide and has been associated to several distinct clinical manifestations that entail important economic losses to the producers. One of the factors possibly implicated on the PCV2 pathogenicity is the Cap protein, the fundamental unity that constitutes this virus capsid. Studies performed by the group of Dr. João Pessoa Araújo Jr. rom the Instituto de Biotecnologia da Unesp em Botucatu/SP, confirmed that viruses with mutated Cap proteins from cell culture increased cell death in infected cultures. Such results highlight the importance of capsid in the infection mechanisms and pathogenicity of PCV2 and the importance of structural and comparative studies with Cap protein structures. In silico structural studies showed the low frequency of the mutations identified in the mutant Cap proteins and also indicated a clear difference between the physico-chemical properties of the new amino acid residues in comparison to those found in the wild-type virus. These mutations, associated with the location of the mutated residues on the viral surface and the lower mutated Cap protein flexibility, could lead to relevant structural/functional changes, including alteration of affinity for cellular receptors and decreased effectiveness of antibodies produced against vaccine viruses. Experimental works aiming the heterologous production of a wild-type Cap protein were also carried out, which involved expression vector subcloning, transcription tests and protein expression experiments. The results of these procedures were compatible with the production of the Cap protein, but further studies are needed to confirm the production of the target molecule and improve the yield of the expression assays.