In this study, effects of different carbon sources and their feeding strategies on recombinant human growth hormone (rhGH) production by Pichia pastoris were investigated by means of cell growth, recombinant protein production and expression levels of hGH and alcohol oxidase (AOX) genes. In this content, firstly, the strain to be used for high level rhGH production was selected between the two phenotypes, i.e., P. pastoris hGH-Mut+ and P. pastoris hGH-MutS. In this selection both phenotypes were compared in two different media containing glycerol/methanol or sorbitol/methanol and P. pastoris-hGH-Mut+ strain grown on medium containing 30 g/L sorbitol with 1% (v/v) methanol was found to have the highest hGH expression level and rhGH production level, 9.84x109 copies/mg CDW and 120 mg/L, respectively. Thereafter, effects of sorbitol, mannitol, fructose, lactose, sucrose, citric acid, lactic acid and acetic acid were investigated by using P. pastoris hGH-Mut+ strain in laboratory scale bioreactors. Among them sorbitol and sucrose were selected to be compared for production in pilot scale bioreactors by adding them batch-wise at the beginning of induction phase with fed batch methanol feeding scheme at &amp / #956 / =0.03h-1. It was shown that sucrose does not support cell growth as sorbitol although it does not repress recombinant protein production. Then three different feeding strategies were applied to develop sorbitol/methanol mixed feeding i) single sorbitol addition at t=0, ii) besides at t=0, adding second batch-wise sorbitol at t=9 h, iii) giving pulse methanol at t=24 h to trigger AOX promoter. These three strategies were compared with a production without addition of co-substrate sorbitol. Substrate consumption, cell growth, recombinant protein production and expression levels of hGH and AOX were investigated for these different feeding strategies. The highest cell concentration was achieved in third strategy as 55 g/L where the highest extracellular rhGH production (301 mg/L) was achieved in the second strategy, with addition of two times of sorbitol. For this highest recombinant protein production case, overall cell and product yield on total substrate were found as 0.17 g/g and 1.71 mg/g, respectively. Moreover, the highest hGH and AOX expression levels were obtained in this strategy.

Recombinant Pichia pastoris is one of the important methylotropic yeast due to its robustness and ability to produce hormones like human chorionic gonadotropin (hCG), luteinizing hormone (LH) extracellularly. High growth on glycerol and strong protein expression on methanol by insertion of alcohol oxidase (AOX) promoter demand the fermentation to be a multistage operation. Methylotropic pathway demands more oxygen as methanol has to be converted to formaldehyde with half mole of oxygen. Moreover as fermentation progresses cell density in the reactor also increases. In case of Pichia pastoris fermentation cell density usually reaches very high (above 100 gm/lit) at the end of fermentation. Both these contribute in the increased oxygen demand in the fermentation and oxygen transfer turns out to be a limiting step. The present study focuses on the oxygen transfer process and its improvement in the fermentation. Oxygen transfer in bioreactor is a multistep process and involves different kinetic as well as mass transfer steps. In case of fermentation especially at high cell densities, oxygen transfer from bubbles to the broth becomes limiting step. The interface transport is governed by many physical as well as kinetic parameters. It is essential to screen these parameters from the whole set to identify the key parameters. Sensitivity analysis is carried out by using Metabolic Control Analysis (MCA) to quantify the effects of different parameters. It is found that bubble size and oxygen partial pressure are two such key parameters which can be manipulated. Use of pure oxygen to increase partial pressure and thereby solubility of oxygen in broth is a common approach. This work focuses on bubble size manipulation to increase the oxygen transfer rates.The idea behind this work is on to generate micron sized bubbles and utilize them effectively in the fermentation. There are many techniques reported to generate microbubble dispersions. In this work ’Spinning Disc microbubble Generator’ is fabricated to generate microbubbles. A flat disc surrounded by baffles with 5 mm gap in between, when subjected to 5000 rpm generates microbubbles. Some modifications are done to the set up to achieve desired properties of the bubbles. The bubbles generated fall in the range of 30-300 micron with mean size of about 60 micron. Use of Tween-20 surfactant stabilize the bubbles and hence offer a good resistance to coalescence and breakage. The liquid fraction in the bubbles can be as high as 40%. Contineous addition of this dispersion unnecessarily can dilute the fermentation broth. To overcome this volume constrain, a recirculation system is designed. Microbubble dispersion is added contineously to the reactor and equivalent fermentation broth is pumped back to the microbubble generator to achieve steady state to the liquid volume in both the vessels. Mass transfer studies with microbubbles show the potential of microbubble dispersion (MBD) to enhance mass transfer significantly. Decrease in volumetric mass transfer coefficient (KLa) due to surfactant is overcompensated by the increase in the interfacial area and net effect is, potential enhancement in KLa. The enhance- ment factor, that is, ratio of mass transfer coefficient with MBD to mass transfer coefficient with conventional sparging, is obtained to be about 4 to 5. Prior to utilization of bubbles in the recirculation system, cells are checked for the shear sensitiveness. Negligible lysis losses and almost no effect on growth patterns in shake flask culture confirm that the cells used are mechanically stable at operating conditions. Better growth patterns in shake flask are observed when microbubbles are pumped for predetermined duration in the broth. It shows possible use of MBD as oxygen carriers. Glycerol batch phase with MBD and conventional sparging is studied at different initial cell densities. Conventional sparging fails to grow the cells and Dissolved Oxygen (DO) levels close to zero suggest high oxygen demands which can not be sustained by conventional sparging. The same batch is run using MBD. Reasonably good growth patterns are observed. DO levels are well above 70% for most of the time during operation. High oxygen demand which can not be sustained by conventional sparging alone can be sustained by MBD. In this way in high den- sity cultures utilization of MBD can be a good alternative to fulfill required oxygen demand in fermentation.

The methylotrophic yeast Pichia pastoris is widely used for recombinant protein production due to its ability to grow to high cell densities as well as possession of an inducible methanol utilization pathway (MUT). The expression of genes encoding enzymes of the MUT pathway is very tightly regulated. These genes are turned on when methanol but not glucose is used as the sole carbon source. Thus, P. pastoris cells can be grown to high densities in glucose containing medium and expression of genes of MUT pathway can be turned on by changing the carbon source to methanol. This strategy is widely used for recombinant protein production wherein the gene of interest is cloned downstream of the methanol-inducible promoter of the gene encoding the first enzyme of the MUT pathway, alcohol oxidase I (AOXI). Despite production of a large number of recombinant proteins using the AOXI promoter, the mechanism of transcriptional activation of AOXI is not very well understood. It is only recently that a zinc finger protein known as Mxr1p (methanol expression regulator 1) was shown to play a key role in the regulation of AOXI as well as other genes of methanol utilization pathway (1) P. pastoris strains that do not express Mxr1p (mxr1) are unable to grow on peroxisomal substrates such as methanol and oleic acid. Methanol-inducible expression of genes involved in MUT pathway as well as those involved in peroxisome biogenesis (peroxins,) is severely impaired in mxr1 strains. While Mxr1p is constitutively expressed in cells cultured on glucose as well as methanol, it is cytosolic in glucose-grown cells, but nuclear in methanol-grown cells (1). The exact nucleotide sequence to which Mxr1p binds and regulates the expression of genes of MUT pathway is not known. The aim of this thesis is to map the Mxr1p binding sites in the promoters of methanol-inducible genes of P. pastoris. As a first step towards understanding the mechanism of transcriptional regulation of AOXI and other methanol inducible genes of P. pastoris by Mxr1p, the N-terminal region comprising of 150 amino acids, including the zinc finger DNA binding domain of Mxr1p was cloned into an E. coli expression vector and the recombinant protein was purified from E. coli cells. This recombinant protein (referred to as Mxr1p in this study) was used in an electrophoretic mobility shift assay (EMSA) to identify Mxr1p binding sites in the AOXI promoter. EMSA was carried out with sixteen different oligonucleotides spanning AOXI promoter region between -940 and -114 bp. Such studies led to the identification of six Mxr1p binding sites in AOXI promoter. Using a combination of DNase I footprinting as well as EMSA with chimeric double stranded oligonucleotides, the minimal Mxr1p binding site was identified as a 20 bp DNA sequence containing a core 5’CYCC 3’ sequence. Using methylation interference as well as extensive mutagenesis studies, nucleotides critical for Mxr1p binding were identified. Comparative analysis of Mxr1p binding sites identified in our study with the AOXI promoter deletion studies of Hartner et al (2) suggested that the Mxr1p binding sites identified in our study are likely to function as methanol-inducible enhancers in vivo, since deletion of AOXI promoter regions comprising Mxr1p binding sites results in a significant loss of methanol-inducible promoter activity. Thus, Mxr1p binding sites are likely to function as Mxr1p response elements (MXREs) in vivo. Mxr1p is considered to be the P. pastoris homologue of S. cerevisiae Adr1p (alcohol dehydrogenase II [ADH2] synthesis regulator). Adr1p is a key regulator of S. cerevisiae genes involved in the metabolism of glycerol, ethanol and oleic acid. The DNA binding domains of Adr1p and Mxr1p share 82% similarity and 70% identity. We therefore examined whether Mxr1p can bind to the Adr1p binding site of ADH2 promoter(ADH2-UAS1). Our studies indicate that Mxr1p does not bind to ADH2-UAS1. Interestingly, a single point mutation restores Mxr1p binding to ADH2-UAS1. Since Mxr1p is involved in the regulation of a number of genes including AOXI, we examined whether promoters of other Mxr1p-regulated genes also harbour MXREs similar to those identified in AOXI promoter. The promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8) were chosen for this purpose. A detailed analysis of Mxr1p binding to these promoter sequences led to the conclusion that DHAS and PEX8 promoters also harbour Mxr1p binding sites similar to those of AOXI promoter. Based on these studies, we have derived a consensus sequence for Mxr1p binding. This study is the first report on detailed characterization of Mxr1p binding sites in three methanol-inducible promoters of P. pastoris and thus provides the molecular framework by which this transcription factor functions as a master regulator of genes involved in methanol utilization pathway of P. pastoris. Our study provides the blue print for mapping Mxr1p binding sites in the promoters of other Mxr1p-regulated genes.

<p>The aim of this work was to develop a production process for the enzyme xyloglucan <i>endo</i>-transglycosylase from <i>Populus tremula x tremuloides</i> (<i>Ptt</i>XET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale.</p><p>This work also shows how the wildtype <i>Ptt</i>XET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction.</p><p>A heterologous production system for <i>Ptt</i>XET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of <i>Ptt</i>XET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB).</p><p>For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF).</p>

Grundlage der vorliegenden Arbeit ist die Überlegung, dass eine Möglichkeit, die Spezifität der bisher angewendeten Verfahren zur West-Nil-Virus-Diagnostik zu verbessern, in der Anwendung rekombinanter WNV-spezifischer Antigene besteht. Die unter anderem auf bioinformatischen Methoden basierende Identifikation von potenziellen B-Zell-Epitopen und Auswahl entsprechender Sequenzabschnitte richtete sich dabei gezielt auf immunogene Bereiche, die innerhalb der Gruppe der Flaviviren einen ausreichenden Sequenzunterschied zu allen weiteren sequenzverwandten Erregern, zusammengefasst im Japanische Enzephalitis-Serokomplex, boten. Drei ausgewählte Bereiche innerhalb der Strukturproteinsequenz, bezeichnet als prM, Cnat und Cme, sollten mit Hilfe des Expressionssystems Pichia pastoris bzw. Escherichia coli rekombinant exprimiert werden. Nach Erarbeitung optimaler Expressionsbedingungen folgte die affinitätschromatografische Reinigung der im weiteren Verlauf zur Immunisierung von Balb/c-Mäusen eingesetzten Polypeptide. Die gewonnenen Seren der nach verschiedenen Immunisierungsprotokollen geimpften Mäuse wurden im Anschluss immunologisch untersucht. Es zeigte sich, dass die rekombinanten Derivate des Capsid-Proteins eine deutliche Serokonversion hervorriefen. Analysen der mit Cnat und MBP-Cme immunisierten Mausseren wiesen vorhandene peptidspezifische sowie virusspezifische Antikörper nach. Der Einsatz dieser gewonnenen Peptidantigene im indirekten ELISA-Testsystem zur Detektion WNV-spezifischer Antikörper unter Verwendung humaner WNV-IgG-positiver Serumproben zeigte positive Resultate. Im Gegensatz hierzu führte die Immunisierung mit prM lediglich zu einer unspezifischen murinen Antikörperbildung. Die Unterscheidung zwischen WNV-positiven und WNV negativen Humanseren war unter Verwendung des rekombinanten Antigens prM nicht möglich. Im Ergebnis zeigten zwei der drei in dieser Arbeit rekombinant erstellten Strukturproteinabschnitte ihr immunologisches Potenzial in der Generierung muriner WNV spezifischer Antikörper. Zudem konnte mit der Expression der WNV-spezifischen C Protein Antigene ein Beitrag zur Etablierung eines indirekten ELISA-Testsystems zur Detektion WNV-bedingter Humaninfektionen geleistet werden.

Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by a defect in alpha-N-acetylglucosaminidase (NAGLU), a lysosomal enzyme involved in the degradation of heparan sulphate. Dysfunctional NAGLU gives rise to a clinical phenotype of severe and progressive mental retardation, often accompanied by hyperactivity and aggressive behaviour. At present, there is no effective treatment for MPS IIIB. However, cloning of the human NAGLU cDNA has made the potential production of human recombinant enzyme for use in enzyme replacement therapy (ERT) a viable option. The work outlined herein focuses on attempts to produce human recombinant NAGLU (rNAGLU) using both yeast and insect cell based expression systems; with the major focus on yeast based expression. Use of a humanized yeast strain, codon optimisation of a portion of the NAGLU gene, selection of Mut+, MutS and multiple integrant strains, and growth at decreased temperature were explored to optimise NAGLU expression in the methylotrophic yeast, Pichia pastoris. As none of these measures resulted in abundant NAGLU production, Sf9 and Tni insect cell lines were investigated as an alternate expression system. Additionally, a protein transduction domain (PTD) was fused to NAGLU (NTAT) to circumvent current problems faced in delivering therapeutic enzymes to the brain. NAGLU protein, with and without a fused PTD, were expressed using stable transfection and baculovirus infection techniques. Small scale experiments utilizing the baculovirus expression vector system (BEVS) have yielded promising results, generating functionally active NAGLU and NTAT protein of the expected approximately 80-85 kDa molecular mass. This preliminary success indicates the BEVS may be an attractive option for the large scale production of rNAGLU and rNTAT.

Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by a defect in alpha-N-acetylglucosaminidase (NAGLU), a lysosomal enzyme involved in the degradation of heparan sulphate. Dysfunctional NAGLU gives rise to a clinical phenotype of severe and progressive mental retardation, often accompanied by hyperactivity and aggressive behaviour. At present, there is no effective treatment for MPS IIIB. However, cloning of the human NAGLU cDNA has made the potential production of human recombinant enzyme for use in enzyme replacement therapy (ERT) a viable option. The work outlined herein focuses on attempts to produce human recombinant NAGLU (rNAGLU) using both yeast and insect cell based expression systems; with the major focus on yeast based expression. Use of a humanized yeast strain, codon optimisation of a portion of the NAGLU gene, selection of Mut+, MutS and multiple integrant strains, and growth at decreased temperature were explored to optimise NAGLU expression in the methylotrophic yeast, Pichia pastoris. As none of these measures resulted in abundant NAGLU production, Sf9 and Tni insect cell lines were investigated as an alternate expression system. Additionally, a protein transduction domain (PTD) was fused to NAGLU (NTAT) to circumvent current problems faced in delivering therapeutic enzymes to the brain. NAGLU protein, with and without a fused PTD, were expressed using stable transfection and baculovirus infection techniques. Small scale experiments utilizing the baculovirus expression vector system (BEVS) have yielded promising results, generating functionally active NAGLU and NTAT protein of the expected approximately 80-85 kDa molecular mass. This preliminary success indicates the BEVS may be an attractive option for the large scale production of rNAGLU and rNTAT.

Made available in DSpace on 2016-06-02T20:21:36Z (GMT). No. of bitstreams: 1 6407.pdf: 1899401 bytes, checksum: d7929c149990e2a77bfc5e204bc566f5 (MD5) Previous issue date: 2014-09-04 / Universidade Federal de Minas Gerais / Cysteine proteases (CPs) are proteolytic enzymes which have a cysteine residue at its active site. Plant legumains are CPs known as vacuolar processing enzymes and they play key roles in seed maturation, germination, senescence, stress response, programmed cell death during development and defense against pathogens. Although there are many studies about plant legumains, most of them is related to legumain functions in seeds of dicotyledonous. To date, only one legumain from sugarcane has been described. In this study, it was performed the characterization of a new sugarcane legumain, named CaneLEG2. The recombinant CaneLEG2 was produced in the heterologous expression system Pichia pastoris and its kinetic characterization showed that it exhibits self-activation and activity under acidic pH, which are common features of plant legumains. This study also demonstrated that the sugarcane cystatin CaneCPI-3 has a strong inhibition over the CaneLEG2 activity, suggesting that this cystatin may participate of the regulation of endogenous cysteine proteases. The results obtained in this work will support the understanding of the functions of CaneLEG2 and CaneCPI-3 in sugarcane. / As cisteino-peptidases (CPs) são enzimas proteoliticas que possuem um resíduo de cisteina em seu sitio ativo. As legumainas de plantas são CPs conhecidas como enzimas de processamento vacuolar (VPE) e participam nos processos de maturação de sementes, germinação, senescência, resposta a estresses, morte celular programada no desenvolvimento da planta ou em resposta ao ataque de patógenos. Embora existam diversos estudos com legumainas de plantas, a maioria deles esta relacionada as funções das legumainas em sementes de dicotiledoneas. Ate o momento, apenas uma legumaina de cana-de-açúcar havia sido descrita. Neste presente trabalho, foi realizada a caracterização de uma nova legumaina de cana-de-açúcar, denominada CaneLEG2. A CaneLEG2 recombinante foi produzida em sistema de expressão heterologa Pichia pastoris e sua caracterização cinetica mostrou que ela apresenta auto-ativação e atividade em pH ácido, caracteristicas comuns em legumainas de plantas. Esse estudo ainda demonstrou que a cistatina de cana-de-açúcar CaneCPI-3 exerce uma forte inibição sobre a atividade da CaneLEG2, sugerindo que essa cistatina pode atuar na regulação de cisteino-peptidases endógenas. Os resultados obtidos neste trabalho ajudam no entendimento das funções exercidas pela CaneLEG2 e CaneCPI-3 na cana-de-açúcar.

Made available in DSpace on 2014-08-20T13:32:46Z (GMT). No. of bitstreams: 1 tese_paula_telmo.pdf: 809734 bytes, checksum: c94f446bc6a605fddb45c97bbeedb7ec (MD5) Previous issue date: 2013-04-09 / The toxocariasis is a widespread zoonosis worldwide, thus becoming an important public health problem. The diversity of clinical conditions associated with the different sites (liver, lungs, brain, eyes, lymph nodes, etc.) of the Toxocara canis larvae in the human body, hamper the diagnosis of this disease. In this context, immunological methods for detecting anti-T. canis serum antibodies were developed. Currently, enzyme-linked immunosorbent assay (ELISA) associated with the excretorysecretory antigens of T. canis larvae (TES), and sera previously adsorved with somatic antigen of Ascaris spp. has been used as a standard method for testing of IgG anti-T. canis serum antibodies. The antigen TES is obtained from T. canis larvae cultivation and demand four months of expensive and laborious work. Given the above, recombinant antigens are being tested, aiming at improving the immunodiagnosis of human toxocariasis. The objective of this work was to produce recombinant excretion/secretion 30kDa T. canis larval antigen (TES30) in two heterologous protein expression systems, and evaluate them in the immunodiagnosis of human toxocariasis. The recombinants proteins rTES30E and rTES30P, respectively, produced in Escherichia coli and Pichia pastoris, were characterized by un indirect ELISA to detect anti-T. canis IgG in experimentally infected mice serum, which showed 100% effectiveness compared to native TES. For detecting anti-T. canis IgG antibodies in human sera, the rTES30E showed sensitivity and specificity (95% CI) of 95.8% and 82.6%, while rTES30P showed 95.4% and 92.8%, respectively, in comparison to native TES. Thus, we conclude that both recombinants proteins have antigenic potential, becoming an alternative to the use of native TES in immunodiagnosis of human toxocariasis. / A toxocaríase humana é uma zoonose difundida em todo o mundo, constituindo-se em um importante problema de saúde coletiva, porém é negligenciada. A diversidade de quadros clínicos associada aos diferentes sítios (fígado, pulmões, cérebro, olhos, gânglios linfáticos, etc.) em que as larvas de Toxocara canis podem se alojar no organismo humano, dificulta o diagnóstico desta parasitose. Neste contexto, métodos imunológicos para a detecção de anticorpos anti-T. canis foram desenvolvidos. Atualmente, o ensaio imunoenzimático (ELISA) associado ao antígeno de excreção e secreção de T. canis (TES), com adsorção prévia de soros com antígeno somático de Ascaris spp., tem sido utilizado como método padrão para pesquisa de anticorpos IgG séricos anti-T. canis. O antígeno TES é obtido a partir do cultivo de larvas de T. canis e demanda, aproximadamente, quatro meses de trabalho dispendioso e fastidioso. Diante do exposto, antígenos recombinantes estão sendo testados, visando o aperfeiçoamento do imunodiagnóstico da toxocaríase humana. Assim, o objetivo deste trabalho foi produzir o antígeno de excreção/secreção de 30kDa de T. canis (TES30) em dois sistemas heterólogos de expressão protéica, e avaliá-los no imunodiagnóstico da toxocaríase humana. As proteínas recombinantes rTES30E e rTES30P produzidas em Escherichia coli e Pichia pastoris, respectivamente, foram caracterizadas através de ELISA-IgG com soros de camundongos infectados experimentalmente, demonstrando 100% de eficácia em comparação ao TES nativo. Na análise dos soros humanos, com ELISA-IgG a rTES30E apresentou sensibilidade e especificidade (IC 95%) de 95,8% e 82,6%, enquanto a rTES30P apresentou 95,4% e 92.8%, respectivamente, em comparação ao TES nativo. Assim, conclui-se que ambas as proteínas recombinantes apresentam potencial antigênico, constituindo-se em uma alternativa ao uso do TES nativo no imunodiagnóstico da toxocaríase humana.

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 tese_lorena_leonardo_souza.pdf: 798251 bytes, checksum: b0213429356f32517eb7b25ac76b2eeb (MD5) Previous issue date: 2009-12-17 / The rabies virus has two major antigens: the nucleoprotein, a conserved internal protein antigenically and genetically and glycoprotein, a protein responsible for the external adsorption of virus two the host cell and induction of neutralizing antibodies. The development of recombinant DNA technology has opened a new perspective on the control of rabies, since recombinant vaccines have residual pathogenicity and are produced with the antigenic proteins of the virus, without their presence. Furthermore, recombinant proteins can be expressed in order to be used in diagnosis. The objective of this study was to review the literature about rabies, cloning and express the nucleoprotein and glycoprotein of rabies virus using the system Pichia pastoris and evaluate the antigenicity and the immunogenicity of these proteins by Dot blotting, SDS page, Western blotting and ELISA. Glycoprotein synthetic antigen proved to be recognized by anti-rabies from animals experimentally infected with rabies virus strain CVS. And recombinant nucleoprotein expression was confirmed by the techniques of Dot blotting and Western blotting to be recognized by monoclonal anti-histidine. Thus, we conclude that the cloning and expression of synthetic glycoprotein and cloinig and expression nucleoprotein rabies virus by the yeast P. pastoris has been effective, which makes these products an alternative for the production of immunobiological. / O vírus da raiva apresenta dois antígenos principais: a nucleoproteína, uma proteína interna conservada antigênica e geneticamente e a glicoproteína, uma proteína externa responsável pela adsorção do vírus à célula hospedeira e pela indução da produção de anticorpos neutralizantes. O desenvolvimento da tecnologia do DNA recombinante iniciou uma nova perspectiva no controle da Raiva, já que vacinas recombinantes não têm patogenicidade residual e são produzidas com as proteínas antigênicas do vírus, sem sua presença. Além disso, as proteínas recombinantes podem ser expressas com a finalidade de serem usadas em diagnóstico. O objetivo deste trabalho foi clonar e expressar a glicoproteína e a nucleoproteína do vírus da raiva utilizando o sistema Pichia pastoris e avaliar a antigenicidade e imunogenicidade destas proteínas através do Dot blotting, SDS page, Western blotting, inibição da imunofluorescência e ELISA. A glicoproteína demonstrou ser antigênica ao ser reconhecida por anticorpos anti-rábicos provenientes de animais experimentalmente infectados com o vírus rábico cepa CVS. A nucleoproteína recombinante teve sua expressão confirmada pelas técnicas de Dot blotting e Western blotting ao ser reconhecida por anticorpos monoclonais anti-histidina. Podemos concluir que a levedura P. pastoris é um sistema eficiente para clonagem e expressão da nucleoproteína e glicoproteína do vírus rábico.