The methylotrophic yeast, Pichia pastoris, has been used as a protein expression system to express over 500 heterologous proteins. P. pastoris provides many advantages over other organisms that have been utilized for this purpose. In this project, we developed a new host/selectable marker and auxotrophic strains of P. pastoris based on methionine biosynthesis to increase P. pastoris's versatility as a host for homologous protein expression. This was accomplished by selecting for a yeast that is deficient in methionine biosynthesis, P. pastoris (yJC239), and gene complementation through transformation with a genomic DNA library. Bioinformatics show that the P. pastor is MET gene has 54% amino acid identity with 68% similarity to the S. cerevisiae MET2 gene, which codes for homoserine O-transacetylase. We have constructed expression vectors for intracellular and extracellular expression of proteins with the MET2 marker and have also constructed strains with various auxotrophs including me/2.

Pichia pastoris is a methylotrophic yeast gaining acclamation for its capabili ties in heterologous protein expression. In contrast to other host organisms such as bacteria or mammalian cells, P. pastoris offers many advantages over its counterparts. For example, P. pastoris is cost-effective in that it can grow to high cell densities on simple media. The optional use of a constitutive (GAP) or inducible (A OXI) promoter and the ab ility to perfo1m post-translational protein modifications are additional qualities that make for a powerful heterologous expression system. This study focuses on harnessing the benefits described to develop a high-throughput reporter system for the screening of potential super-secreting mutant strains of P. pastoris. Plasmid constructs were engineered with the lacZ reporter gene, which encodes for the β-galactosidase protein, and fused to the S. cerevisiae MATa signal sequence. Expression plasmids were successfully transformed in P. pastoris strain yGS 115 followed by induction. Western blot analyses confirm the expression of β-galactosidase and colorimetric activity assays further validate enzymatic function. A mutant library containing cis- and/or trans-acting mutations was created by treating P. pas loris clones harboring the β-galactosidase expression plasmid with ultraviolet (UV) radiation. A colorimetric plate assay was combined with a replica-plating system that enabled the screening of thousands of potential super-secreting mutant colonies in a high-throughput format. This study sheds light onto our current understanding of secretion in yeast and further contributes to developing P. pastoris into a valuable heterologous protein expression system.

The methylotropic yeast Pichia pastoris has been used for many applications, particularly for its ability to produce and readily secrete heterologous proteins. Nonetheless, there are obstacles in making this useful yeast into a more efficient secretion system that readily secretes problem proteins. In the Lin-Cereghino lab, mutant strains were developed by the method of restriction enzyme mediated integration. These mutants have the ability to secrete β-galactosidase at higher levels in comparison to the wild type. This study focused on characterizing the specific mutant ah2 for its ability to secrete HRP, SLPI, and CALB lipase proteins, as well as using transmission electron microscopy to observe the effect of the pREMI-Z mutation on the morphology. Analysis of the Ah2 protein resulted in a comparative β-galactosidase secretion study, as well as a growth rate study, between the original pREMI-Z ah2 mutant and ah2 mutant cells that were transformed with pKanB-AH2 rescue construct. Lastly, a cell localization experiment was done to examine where Ah2p localizes. By these analyses, we gain a bit more understanding of the P. pastoris secretion pathway, while also outlining a procedure by which to characterize the other pREMI-Z mutants.

No description available.

<p> Tissue plasminogen activator (tPA), is commonly used as thrombolytic agent for the treatment of various cardiovascular diseases. This thesis constitutes the first report on cloning and expression of tPA in the methylotrphic yeast Pichia pastoris. </p> <p> The tPA gene was first cloned into an E. coli/Pichia shuttle plasmid and then integrated into the Pichia genome. The recombinant Pichia was able to express tPA intracellularly, under methanol induction. The tPA produced by the Pichia had a similar molecular weight as native tPA and it had serine protease activity. At the shake flask scale, the recombinant Pichia strain was able to produce twice as much tPA as reported for E. coli. </p> <p> Elastin-like polypeptides (ELP) are proteins that have a peculiar characteristic: they are able to undergo a reversible inverse phase transition temperature within a very narrow temperature range. On a second aspect of heterologous protein, a construct composed of thioredoxin-intein-ELP was used to provide direct evidence, for the first time, that protein folding and activity, in this case the intein, was maintained when the tripartite fusion was present in the aggregated state. These results are important, since they provide the necessary degree of confidence to stimulate future work directed towards expression and maintenance of proper folding of aggregation-prone proteins when expressed in-vivo E. Coli as ELP directed inclusion bodies. It is also shown that the intein-ELP system may be a very interesting system to be used as a drug delivery vehicle. </p> / Thesis / Master of Applied Science (MASc)

The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (kLa) and subsequently increased the cell mass and protein production compared to conventional fermentation. Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. Maximum cell mass productivity in the conventional 350 rpm was only 0.37 g / (L·h), while the maximum value in MBD 350 rpm was 2.0 g / (L·h), which was similar to 2.2 g / (L·h) in the conventional 750 rpm. Biomass yield on glycerol Ys (g cell/ g glycerol) was 0.334 g / g in the conventional 350 rpm, 0.431 g / g in MBD 350 rpm and 0.438 g / g in the conventional 750 rpm. Protein production in MBD 350 rpm was 7.3 times higher than that in the conventional 350 rpm, but similar to the conventional 750 rpm. Maximum protein productivity in the conventional 350 rpm was 0.37 mg / (L·h), 2.8 mg / (L·h) in MBD 350 rpm, and 3.3 mg / (L·h) in the conventional 750 rpm. Protein yield on methanol Yp (mg protein / g methanol) was 1.57 mg /g in the conventional 350 rpm, 5.02 in MBD 350 rpm, and 5.21 in the conventional 750 rpm. The volumetric oxygen transfer coefficient kLa was 1011.9 h-1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h-1) but was similar to the conventional 750 rpm (1098 h-1). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging. Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter. / Master of Science

ATPDases ou ATP-difosfohidrolases são enzimas que clivam o ATP e o ADP a AMP e Pi e estão envolvidos em inibição da agregação plaquetária. No parasita Schistosoma mansoni nosso grupo identificou e clonou o gene da ATPDase1, e a proteína foi localizada na superfície do tegumento. Recentemente, clonamos o gene da ATPDase2 usando a informação do banco de dados de ESTs de S. mansoni e imunolocalizamos a sua proteína também no tegumento. ATPDase2 foi encontrada em ambas as membranas do tegumento basal e apical juntamente com a ATPDase1, entretanto ATPDase2 somente foi encontrada no espaço sincicial do tegumento. A presença de ambas as enzimas sobre a superfície externa do tegumento sugere um maior papel sobre a regulação de abundância de nucleotídeos. Análise da expressão de ambos os genes foram realizadas por RT- PCR em tempo real usando RNA de ovos, miracídeo, cercária, esquistossômulo e verme adulto. Os resultados mostraram que o gene da ATPDase1 foi mais expresso em ovos (7 vezes), adulto (6 vezes), cercária (3,5 vezes) e esquistossômulo (1,5 vezes) quando comparado ao miracídio, que foi tomado como referência. O gene da ATPDase2 foi mais expresso em ovos (16 vezes), cercária (11 vezes), miracídio (7 vezes) e verme adulto (2 vezes) quando comparado a esquistossômulo, mostrando que ambos os genes são modulados ao longo de seus estágios de ciclo de vida . Para maior caracterização destas enzimas, elas foram expressas heterologamente na levedura Pichia pastoris como proteínas de fusão com cauda de 6 histidinas e as proteínas recombinantes foram purificadas por cromatografia de afinidade com resina de Ni-NTA. As ATPDases recombinantes foram obtidas de forma ativa e medições de atividade enzimática foram realizadas. ATPDase1 - mostrou atividades ATPásica e ADPásica em torno de 650 e 160 nmoles Pi.min -1. mg-1 , respectivamente. ATPDase2 teve atividades ATPásica e ADPásica na faixa de 1050 e 250 nmoles Pi.min-1.mg-1 , respectivamente. Adicionalmente, atividades UTPásica e UDPásica também foram encontradas nestas enzimas. Estudos de dicroísmo circular com estas duas enzimas elucidaram suas estruturas secundárias. Com isto, ATPDase1 (S66 to Q507 ) teve alfa-hélice (7 %), folha-beta (45 %) e estrutura randômica (48 %), e a ATPDase2 (N83 a K564 ) mostrou conter alfa-hélice (14 %), folha-beta (33 %) e estrutura randômica (53 %). Nós mostramos que a ATPDase2 é secretada pelo parasita no meio, de forma similar como descrito para as ATP-difosfohidrolases humanas CD39L2 e CD39L4. Adicionalmente, em ensaios de inibição de penetração (cercária em camundongo) usando anticorpo anti- ATPDase1 foi mostrada uma redução em 20 % da capacidade de penetração através da pele das cercárias previamente incubadas com o anti-soro. Devido a que a expressão do gene da ATPDase2 estava mais alta em miracídio e cercária, estágios que infectam caramujos e humanos, respectivamente, postulamos que ATPDase2 poderia ajudar no processo de invasão do parasita. No estágio ovo ambos os genes estão altamente expressados sugerindo um possível envolvimento das ATPDases na resposta de proteção contra o sistema imune humano. Ensaios de proteção contra S. mansoni em camundongos, usando as ATPDases 1 e 2 como antígenos, resultaram em uma baixa proteção obtendo-se não mais que 20% na redução da carga parasitária. / ATPDases or ATP-diphosphohydrolases are enzymes that cleave ATP and ADP to AMP and Pi and are involved in inhibition of platelet aggregation. In the parasite Schistosoma mansoni our group had identified and cloned the ATPDase1 gene and localized the protein on the tegument surface. Recently, we cloned the ATPDase2 gene using S. mansoni EST databank information and we immunolocalized it also in the tegument. ATPDase 2 was found on both the apical and basal tegument membranes together with ATPDase1, but only ATPDse2 was found in the syncytium space of the tegument. The presence of both enzymes on the tegumental outer surface suggests a major role in regulation of nucleotides abundance. Expression analysis of both genes was performed by Real Time RT- PCR using RNA from eggs, miracidia, cercariae, schistosomula and adult worms. The results showed that ATPDase1 gene was more expressed in eggs (7-fold), adults (6-fold), cercariae (3.5-fold) and schistosomula (1.5-fold) when compared to miracidia, which was taken as the reference. ATPDase2 gene was more expressed in eggs (16-fold), cercariae (11-fold), miracidia (7-fold) and adult worms (2-fold) when compared to schistosomula, showing that both genes are modulated along the life cycle stages. For further characterization of these enzymes, they were expressed heterologously in the yeast Pichia pastoris as fusion proteins with hexa-histidine tags and the recombinant proteins were purified by Ni-NTA affinity chromatography. The recombinant ATPDases were obtained in active form and activity measurements were performed. ATPDase1 did show ATPase and ADPase activities about 650 and 160 nmoles Pi.min-1.mg-1 , respectively. ATPDase2 had ATPase and ADPase activities in the range of 1050 and 250 nmoles Pi.min-1.mg-1 , respectively. These results were obtained in the presence of calcium as cofactor. Additionally, UTPase and UDPase activities were found for both enzymes. Circular dichroism studies with these enzymes elucidated their secondary structures; ATPDase1 (S66 to Q507 ) has alpha helix (7%), beta sheet (45%) and random coil (48%), whereas ATPDase2 (N83 a K564 ) showed alpha helix (14%), beta sheet (33%) and random coil (53%). We found that ATPDase2 was secreted by the parasite to the medium, similar to what has been described for human CD39-L2 and CD39-L4 ATP-diphosphohydrolases. Additionally, a penetration assay (cercaria to mice) using antibody anti-ATPDase1 did show a decrease of 20% in the penetration capacity through mice skin of cercaria previously incubated with this antiserum. Because ATPDase2 gene expression was increased in miracidia and cercariae, the stages that infect snail and human, respectively, we postulate that ATPDase2 may help the parasite\'s invasion process. In the egg stage both genes were highly expressed suggesting a possible involvement of the ATPDases in the protection response of eggs against the human immune system. Assays of protection against S. mansoni in mice, using recombinant ATPDase1 and 2 as antigens, resulted in low protection obtaining no more than 20% of parasite burden reduction.

Neste trabalho, construiu-se linhagens recombinantes de Pichia pastoris e Saccharomyces cerevisiae que expressam hormônio de crescimento de carpa (GHc). Para expressão de GHc em P. pastoris, o cDNA de GH de H. molitrix foi inserido nos plasmídeos pHILD2 (expressão interna) e pPIC9K (expressão e secreção) dando origem aos plasmídeos pHILGH e pPICGH. A expressão em S. cerevisiae foi controlada pelo promotor e terminador PGK e o cassete de expressão ladeado por elementos &#948;, dirigindo a integração em múltiplas cópias no genoma. Ainda, construiu-se um plasmídeo onde a sequência de GHc foi fusionada a uma sequência sinal modificada (sequência sinal de proteína de K. lactis, fusionada a fragmento de interleucina humana), visando altos níveis de secreção. Os transformantes (expressão interna) expressaram proteína recombinante de 20 KDa. Um dos transformantes de S. cerevisiae secreta cerca de 1045,0 µg/ml de GHc. As proteínas recombinantes apresentaram hibridação específica contra anticorpo anti GHc comercial indicando que são biologicamente ativas. / In order to achieve the GHc expression in P. pastoris, the GH cDNA of the carp H. molitrix was introduced into the plasmids pHILD2 (intracellular expression) and pPIC9K (expression and secretion) originating the plasmids pHILGH and pPICGH. The expression of the GH cassette in S. cerevisiae was driven by the PGK promoter and terminator and the cassette was flanked by the &#948; elements, which provides multiple copies integration into the genome. We also constructed a plasmid in which the GHc cDNA sequence was fused to a modified signal sequence (K. lactis protein signal sequence fused to a human interleukin fragment), aiming to reach high levels of secretion. P. pastoris and S. cerevisiae recombinant clones (intracellular expression) were shown to express a recombinant protein of 20 KDa. One of the S. cerevisiae recombinant clones secreted around 1045.0 µg/ml of GHc. The recombinant proteins showed hybridization of the bands against antibody anti-commercial GHc. The results indicate that the recombinant proteins produced in this work are biologically active.

Papilomavírus humanos (HPVs) são vírus de DNA que infectam células epiteliais, podendo ser responsáveis pelo aparecimento de lesões benignas e malignas. Dentre os mais de 120 tipos identificados, o HPV -16 constitui o principal agente etiológico do câncer cervical, que é uma das maiores causas de morte por câncer em mulheres no mundo. Sendo assim, infecções associadas ao HPV devem ser prevenidas por vacinas indutoras de resposta imune vírus-específicas. A proteína L1 do capsídio viral é capaz de arranjar-se em partículas morfologicamente e antigenicamente semelhantes ao vírus, denominadas \"virus-like particles\" (VLPs), que induzem altos títulos de anticorpos neutralizantes. Neste trabalho, foram clonados os genes L1 selvagem e otimizado de HPV -16 em vetores de expressão de leveduras metilotróficas como Hansenula polymorpha e Pichia pastoris. Foi observada uma expressão consistente da proteína recombinante apenas em P. pastoris, com o gene L1 otimizado. Foram realizadas diversas tentativas de purificação da proteína heteróloga, empregando técnicas de cromatografia e ultracentrifugação em gradiente descontínuo de sacarose. A correta montagem das VLPs foi confirmada por microscopia eletrônica. Problemas de agregação, heterogeneidade e adsorção a superfícies apresentados pela proteína L1 foram resolvidos após utilização de surfactante não-iônico e de um procedimento de desmontagem e remontagem das partículas, gerando preparações mais homogêneas. Ensaios de hemaglutinação e inibição da hemaglutinação comprovaram a apresentação de epítopos conformacionais na superfície das VLPs. Este trabalho demonstrou pela primeira vez a expressão da proteína L1 de HPV -16 em P. pastoris, visando ao desenvolvimento de uma vacina profilática de baixo custo para o sistema público de saúde. / Human papillomaviruses (HPVs) are DNA viruses that infect epithelial cells and can cause both benign and malignant lesions. From over 120 types catalogued so far, HPV-16 is the main etiologic agent of cervical cancer, which is the one of the most common causes of cancer deaths among women worldwide. Thus, HPV -associated infections might be prevented by vaccine inducing virus-specific immune responses. The L1 major capsid protein can self assemble into virus-like particles (VLPs), which are morphologically and antigenically indistinguishable from native viruses and induce high titers of neutralizing antibodies. In this work, we have cloned wild-type and codon-optimized L1 genes from HPV-16 in expression vectors of the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris. Consistent L1 expression was only observed in P. pastoris transformed with the construction containing the codon-optimized gene. Many attempts to purify the heterologous protein were made, including chromatography and ultracentrifugation in sucrose density gradients. The correct assembly of VLPs was confirmed by electron microscopy. Some problems presented by recombinant L1 like aggregation, surface adsorption and heterogeneity were solved by using non-ionic surfactants and a procedure of disassembly and reassembly of the particles. Hemagglutination and hemagglutination inhibition assays corroborated the display of surface conformational epitopes by VLPs. This work showed for the first time the expression of the HPV-16 L1 protein in P. pastoris, aiming the development of a prophylactic vaccine free of charge for the public health system in Brazil.

A leucemia linfóide aguda (LLA) é considerada uma doença grave dos glóbulos brancos, sendo mais comum e mais agressiva em crianças e adolescentes. O tratamento para a LLA tem avançado devido aos estudos para a otimização de drogas já utilizadas em quimioterapias. Entre essas drogas estão as enzimas L- asparaginases (ASPases) que atuam reduzindo a concentração de L-asparagina (Asn) na corrente sanguínea, impedindo a proliferação das células cancerosas, visto que essas não conseguem sintetizar quantidades apropriadas desse aminoácido. No entanto, o medicamento por ser oriundo de um procarioto causa severas reações alérgicas aos usuário, afim de diminuir a imunogenicidade deste quimioterápico, é importante gerar um biofármaco oriundo de um eucarioto. Neste âmbito, obtivemos a Pichia pastoris recombinante responsável pela produção da enzima ASPase intermembranar, oriunda do gene ASP3 de Saccharomyces cerevisiae. Através do planejamento experimental, foi possível ter um aumento de 5 vezes na atividade obtida na condição inicial. O clone Mut+ alcançou sua melhor atividade de 8,6 U/g de célula nas seguintes condições: 20°C, pH inicial 6 e 1,5% de concentração de indutor. / Acute lymphoblastic leukemia (ALL) is considered a serious disease of white blood cells, is more common and more aggressive in children and adolescents. Treatment for ALL has advanced due to studies for drug optimization already used in chemotherapy. Among these drugs are the enzymes L-asparaginases (ASPases) which act by reducing the concentration of L-asparagine (Asn) in the bloodstream, preventing the proliferation of cancer cells, since these can not synthesize appropriate amounts of this amino acid. However, the drug to be derived from a prokaryote causes severe allergic reactions to the user, in order to decrease the immunogenicity of the chemotherapy, it is important to generate a biopharmaceutical derived from a eukaryote. In this context, we obtained the recombinant Pichia pastoris responsible for producing the enzyme ASPase intermembrane, coming from the ASP3 gene of Saccharomyces cerevisiae. Through the experimental design, it was possible to have a 5-fold increase in activity obtained at the initial condition. The Mut + clone achieved their best activity of 8.6 U/g cell under the following conditions: 20 °C, initial pH 6 and 1.5% of inducer concentration.