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1
Superina, Mariella. "Natural history of the pichi (Zaedyus pichiy) in Mendoza Province, Argentina." ScholarWorks@UNO, 2007. http://scholarworks.uno.edu/td/604.
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The pichi Zaedyus pichiy (Xenarthra, Dasypodidae) is a poorly known, diurnal armadillo inhabiting arid and semi-arid regions of Argentina and Chile that has endured substantial population declines. My objective was to elucidate different aspects of the natural history of Z. pichiy as a first step towards establishing a conservation plan. Wild and captive pichis were studied. Body temperature of wild pichis averaged 35.2±1.2 °C and was highly variable (range 32.2 – 38.3 °C). Temperature measurements of semi-captive males showed that pichis can survive energetically challenging periods by entering hibernation or daily torpor. Stomach contents of poached animals revealed that pichis feed predominantly on insects but also ingest plant material, vertebrates and arachnids. This opportunistic, omnivorous feeding strategy allows them to thrive where food type and availability vary seasonally. The reproductive cycle of pichis was studied by means of histological and fecal hormone analyses. Pichis are seasonal breeders that produce one yearly litter of 1 to 2 offspring, and the initiator of breeding season seems to be an increase in daylength. The absence of regular estrous cycles and corpora lutea in non-pregnant females, and immediate mating attempts after pairing, all suggest that pichis are induced ovulators. Clinical examinations and hematological, serological and coproparasitological analyses of free-ranging pichis, and necropsies and histological examinations of confiscated pichis and roadkills, indicate that the populations are currently in good health. While parasites were often found, no severe pathologies were observed. Infections with potentially zoonotic diseases were rare: only a few pichis were seropositive for Trypanosoma cruzi, none had antibodies against Toxoplasma gondii, and none of the histologically examined individuals presented lesions attributable to these pathogens. Elevated ambient humidity levels often caused moist dermatitis with epidermal detachment in captive pichis. Poaching is currently considered to have a much higher negative impact on the wild populations than disease epidemics. Mortality due to heavy poaching activity may be difficult, if not impossible, to compensate by the current birth rates. This preliminary database on the natural history and reproduction of pichis will assist efforts to conserve this little-known species of armadillo.
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Pichon, Samuel. "Système de sécrétion de type IV et protéines à domaines ankyrines dans les interactions Wolbachia-arthropodes." Poitiers, 2009. http://theses.edel.univ-poitiers.fr/theses/2009/Pichon-Samuel/2009-Pichon-Samuel-These.pdf.
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Wolbachia est une bactérie Gram(-) intracellulaire modifiant la reproduction de nombreux arthropodes. Chez l'isopode Armadillidium vulgare, la souche wVulC entraîne la féminisation des mâles. Nous avons caractérisé deux opérons vir s’exprimant dans tous les tissus hôtes et codant un système de sécrétion de type IV (T4SS) pouvant permettre d'exporter des effecteurs bactériens vers le cytoplasme de l'hôte. La comparaison des séquences et de l'organisation des gènes de 37 souches de Wolbachia a révélé la forte conservation des deux opérons vir suggérant l'importance du T4SS dans la biologie de la bactérie. Nous avons également identifié, dans le génome en cours de séquençage de wVulC, 66 gènes codant des protéines à domaines ankyrines. Ces motifs forment des sites d'interactions protéine-protéine chez les eucaryotes et sont supposés être impliqués chez Wolbachia dans l'interaction avec des protéines de l’hôte. Nous avons montré qu'une des trois copies du gène pk2 de wVulC, n'est exprimée que chez des souches féminisantes mais chez aucune des 3 souches induisant l’incompatibilité cytoplasmique chez les isopodes terrestres. Ce produit du gène pk2 pourrait être impliqué dans la féminisation de l’hôte. Toutefois, nous avons réalisé des tests d'interaction par double-hybride en levures et par la méthode CRAfT (Crerecombinase Reporter Assay for Translocation) entre les protéines du T4SS et cinq protéines à domaines ankyrines dont Pk2 afin de savoir si ces dernières étaient sécrétées par ce système. Les résultats montrent qu'aucun des cinq produits de gènes ank testés n'est sécrété par la bactérie mais se révèlent encourageants pour identifier les effecteurs de Wolbachia<br>Wolbachia are intracellular Gram(-) bacteria that are reproductive manipulators of many arthropods. In the isopod Armadillidium vulgare, the Wolbachia wVulC strain induces male feminization. Here, we characterized two vir operons which are expressed in all host tissues and which encode a type IV secretion system (T4SS) used to translocate bacterial effectors into host cytoplasm. Gene organization and sequence comparison in 37 Wolbachia strains highlighted the high conservation of both vir operons and their importance for the biology of the bacteria. We also identified in the on-going assembly of the wVulC genome, 66 ankyrin domain-encoding genes. Ankyrin motifs are known to mediate protein-protein interactions in eukaryotic organisms and thus are suggested to mediate in Wolbachia the interaction with host molecules. We showed that one of the three copies of the wVulC pk2 gene is only expressed in feminizing strains but not in the three strains inducing cytoplasmic incompatibility in terrestrial isopods. The associated Pk2 protein could be involved in male feminization. We thus tested the interaction between three T4SS proteins and five ankyrins (including Pk2) via the yeast twohybrid and CRAfT (Cre-recombinase Reporter Assay for Translocation) methods. None of the five ankyrin proteins were revealed to be secreted by the wVulC strain. Nevertheless, this promising approach may enable us to identify Wolbachia effectors
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Chaharmahali, Pegah M. "Calcium homeostasis in Pichia pastoris." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/179.
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Pichia pastoris is a methylotrophic yeast that has been used widely in biological and industrial researches. P. pastoris is able to produce from milligram to gram quantities of protein. In this study, we examined the effects of calcium and magnesium on growth, heterologous protein expression, and calcium homeostasis in P. pastoris . The divalent cations calcium and magnesium are responsible for diverse roles in in the function of the cells in eukaryotes. Although it is known that calcium is responsible for many biological functions in eukaryotes, there is a limited understanding of the calcium homeostasis in P. pastoris . In this study, we found that addition of calcium and magnesium to yeast extract peptone dextrose (YPD) does not increase the cell growth of wild type P. pastoris . However, the original concentrations of calcium and magnesium in YPD were critical to cells, as removing calcium and magnesium using EGTA and EDTA, respectively, decreased the cell growth of wild type P. pastoris . Changes to cytoplasmic calcium concentration in P. pastoris was studied using the fluorescent calcium sensitive dye, indo-1 (10 μM) and fluorescent spectrophotometer. The intracellular calcium concentration increased with addition of calcium chloride, magnesium chloride, and phosphate buffered saline (PBS). PBS (standard 1x) induced a significantly higher increase in intracellular calcium concentration compared to inductions with calcium chloride (1.2 mM). Our results showed that the addition of calcium and magnesium into the growth medium did not increase the expression of alcohol oxidase-1 driven β-galactosidase expression. However, calcium and magnesium may still play crucial roles in protein expression as EDTA and EGTA caused an increase in β-galactosidase expression as indicated by higher β-galactosidase activity. Our findings suggest that EDTA and EGTA may also increase the expression of other heterologous proteins in P. pastoris .
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Rampasio, Raquel Rodrigues 1986. "Estudos sobre a clonagem e expressão do gene SEH1 (epóxido hidrolase) de Pichia stipitis EM Pichia pastoris." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/248442.
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Previous issue date: 2012<br>Resumo: Epóxidos enantiopuros e dióis vicinais têm sido utilizados na síntese de inúeras moléculas bioativas. Dessa forma, as epóxido-hidrolases microbianas capazes de hidrolisar enantioseletivamente epóxidos racêmicos emergiram como uma alternativa promissora na obtenção destes compostos. Recentemente, a linhagem P. stipitis CCT 2617 foi selecionada por apresentar atividade hidrolítica frente a epóxidos terminais e teve seu genoma completo publicado. Assim, esta levedura foi selecionada para o trabalho de clonagem e expressão de sua epóxido hidrolase. Neste trabalho, a clonagem do gene SEH1, que codifica para a epóxido hidrolase de P. stipitis, foi efetuada com sucesso em P. pastoris, tanto no vetor pPICZa A, quanto no vetor pPICZ B. A clonagem da proteína com a cauda de histidina deve auxiliar na detecção da expressão. A detecção de uma banda, referente a uma proteína de 46 kDa, no gel de eletroforese foi um indício de que a expressão da enzima SEH (contendo o fator a) ocorreu, porém, não conseguimos reproduzir este resultado posteriormente. Além disso, buscamos melhores alternativas para a detecção da atividade enzimática, como o teste de adrenalina e o ensaio baseado em substrato fluorogênico, que devem ser aperfeiçoados para a utilização com células íntegras. A modelagem computacional da estrutura tridimensional da PSEH resultou em um modelo contendo 40% de hélices a e 12% de folhas b. Determinamos que os resíduos que devem fazer parte do sítio ativo são Tyr319, Asp209, Asp352 e His383 e, tendo em vista que a PSEH deve se apresentar na forma de um homodímero com sítio ativo similar ao das EHs de P. aeruginosa, A. radiobacter e A. niger, nossa hipótese é que esta enzima deve hidrolisar epóxidos pouco volumosos e aromáticos com algum nível de enantiosseletividade<br>Abstract: Enantiopure epoxides and vicinal diols have been used to prepare a number of bioactive molecules. Thus, the microbial epoxide hydrolases able to enantioselectivity hydrolyze racemic epoxides emerged as a promising alternative in the synthesis of these compounds. Recently, the P. stipitis CCT2617 strain was selected due to the presence of hydrolytic activity against terminal epoxides and had its genome completely described. Therefore, this yeast was selected for cloning and expression of the gene SEH1, which was annoted as epoxide hydrolase. In this work, the cloning of SEH1 gene, codifying for the epoxide hydrolase of P. stipitis, was done with success in P. pastoris, both in pPICZa A and pPICZ B vectors. The cloning of the protein with a histidine tag should help in the detection of expression. The detection of a protein with 46 kDa evidenced that the expression of SEH enzyme (containing the a factor) is occurring, however, this result was not reproducible due to the sample degradation. Furthermore, better alternatives for the detection of enzyme activity were performed, as adrenaline test and fluorogenic assay, which must be optimized for application with whole cells. The 3D structure computational modeling of PSEH resulted in a model that contains 40% of a helices and 12% of b sheets. Our hypothesis is that the residues that make part of the active site are Tyr319, Asp209, Asp352 and His 383. And, considering that the PSEH should be in the homodimeric form with an active site similar to that of the EHs of P. aeruginosa, A. radiobacter and A. niger, this enzyme should hydrolyze small and aromatic epoxides probably with some enantioselectivity<br>Mestrado<br>Quimica Organica<br>Mestre em Química
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Bravo, Huaynates Cecilia Milagro. "Asibilación en el Asháninka del Pichis." Bachelor's thesis, Universidad Nacional Mayor de San Marcos, 2013. https://hdl.handle.net/20.500.12672/14413.
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Publicación a texto completo no autorizada por el autor<br>El documento digital no refiere asesor<br>Evidencia un proceso fonológico registrado en algunas variedades de asháninka (una lengua amazónica peruana perteneciente a la familia Arawak). Este proceso se denomina asibilación y consiste en la realización de la consonante coronal sorda /t/ como el segmento africado [ts] cada vez que esta se presenta frente a la vocal alta /i/. La bibliografía en torno a la gramática del asháninka muestra que la asibilación se presenta en el asháninka del Gran Pajonal (Vílchez, 1996), el Perené (Mihas, 2010), el Ene (Falcón, 1994) y el Pichis; mientras que, por otro lado, este proceso está ausente en las variedades habladas en las regiones del Apurucayali (Payne, Payne y Sánchez; 1982) y el Tambo (Kindberg, 1980). Los datos que forman parte del análisis de esta tesis provienen de la variedad de asháninka del Pichis y permitirán evidenciar que la asibilación es un proceso fonológico que se produce en esta variedad tanto en interior de palabra como en límite morfológico; de manera que el objetivo principal de esta investigación consistirá en explicar por qué se produce la asibilación en el asháninka del Pichis.<br>Tesis
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Böttner, Mewes. "Die Expression humaner Proteine in der Hefe Pichia pastoris: Hochdurchsatzverfahren und bioinformatische Identifizierung von Expression-beeinflussenden Sequenzmerkmalen." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972858717.
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Aizemberg, Raquel [UNESP]. "Produção de glicerol quinase em Pichia pastoris." Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/88349.
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aizemberg_r_me_arafcf.pdf: 685654 bytes, checksum: d058342c2d66649275266dc34dcb968d (MD5)<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>Universidade Estadual Paulista (UNESP)<br>A levedura Pichia pastoris vem sendo largamente utilizada como um eficiente sistema de expressão para a produção de proteínas heterólogas, pois é um sistema seguro, fácil e mais barato que sistemas de expressão de outros eucariotos. Neste trabalho, a enzima de interesse é a glicerol quinase (GK), que cataliza a transferência do fosfato terminal do ATP para o glicerol originando glicerol-3-fosfato e ADP. Esta reação pode ser utilizada na determinação da concentração de glicerol, subproduto da fermentação alcoólica. A leitura do consumo de glicerol é realizada pela determinação espectrofotométrica do NADH gerado na reação de oxido-redução catalizada pela enzima glicerol-3-fosfato desidrogenase. Este estudo de indução foi realizado em diferentes condições de crescimento da levedura Pichia pastoris. Os resultados mostraram a seleção do melhor clone da levedura Pichia pastoris para a expressão extracelular da enzima glicerol quinase, e a determinação das melhores condições do meio de cultura para a produção da enzima de interesse foram: concentração do meio de cultura BMMY (20 vezes), densidade inicial de célula (0,1 mg/mL), concentração de metanol na fase de indução (1%), natureza do tampão (fosfato de potássio), pH (6,0), suplementação de glicerol no meio BMMY (1%), peptona (marca Difco), sem adição de sulfato de amônio, caseína e glicina, uso do meio BMMY e liofilização do mesmo. Estudos de parâmetros cinéticos foram realizados e a atividade máxima da GK foi obtida em pH 9,8, a 50ºC e 2,5 μM de substrato, por metodologia clássica, além da presença de sulfato de magnésio e diluição da enzima de 30 vezes. A enzima apresentou alta estabilidade térmica ― a atividade foi completamente...<br>The yeast Pichia pastoris has been widely used as an efficient expression system for production of heterologous proteins because it is a safe, easy and cheaper than expression systems in other eukaryotes.In this studie, the enzyme of interest is glycerol kinase (GK), which catalizes the transfer of terminal phosphate from ATP to glycerol resulting glycerol-3-phosphate and ADP. This reaction can be used in determining the concentration of glycerol, a byproduct of fermentation. The reading of the consumption of glycerol is carried out by spectrophotometric determination of NADH generated in the redox reaction catalyzed by the enzyme glycerol-3-phosphate dehydrogenase. This study of induction was performed in different conditions of growth of the yeast Pichia pastoris. The results show that selecting the best clone of the yeast Pichia pastoris for the expression of extracellular enzyme glycerol kinase, and determining the best conditions of the culture medium for producing the enzyme of interest were: concentration of the culture medium BMMY (20 times), initial cell density (0.1 mg/mL), methanol concentration in the induction phase (1%), nature of buffer (potassium phosphate), pH (6.0), glycerol supplementation in BMMY medium (1%), peptone (Difco), without addition of ammonium sulfate, casein and glycine in BMMY and lyophilized medium. Studies of kinetic parameters were conducted and the GK maximum activity was obtained at pH 9.8 at 50°C and 2.5 μM substrate by conventional method, besides the presence of magnesium sulfate and diluting the enzyme 30 times. The enzyme showed high thermal stability - the activity was fully maintained up to 50°C for one hour - and at pH 7.0 for 7 days and kept under refrigeration, freeze-dried extract showed a decrease in enzymatic activity. Calculated by... (Complete abstract click electronic access below)
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Boudreau, Vanessa. "Production inductible d'anhydrase carbonique par pichia pastoris." Thesis, Université Laval, 2011. http://www.theses.ulaval.ca/2011/28711/28711.pdf.
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Felix, Wagner Pereira. "ProduÃÃo heterÃloga de frutalina em Pichia pastoris." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=2314.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico<br>A frutalina, lectina α-D-galactose ligante, foi expressa e processada num sistema heterÃlogo para expressÃo de proteÃnas utilizando a levedura metilotrÃfica Pichia pastoris. Para atingir os objetivos esperados foram desenvolvidas trÃs estratÃgias: identificar o gene que codifica para a frutalina a partir do DNA genÃmico obtido de folhas de A. incisa; sintetizar, por uma empresa especializada, o gene que codifica para as cadeias polipeptÃdicas da frutalina, otimizando-o para a expressÃo em P. pastoris e isolar o gene que codifica para a frutalina, a partir do mRNA presente em sementes maduras e frescas, para obtenÃÃo do cDNA utilizando a tÃcnica da RT-PCR. Enquanto na primeira estratÃgia, foi obtido apenas o gen da cadeia beta, nas duas outras estratÃgias o gen completo foi obtido. No entretanto, a estratÃgia que mostrou melhor expressÃo da frutalina recombinante em P. pastoris foi a da obtenÃÃo do gene que codifica para a frutalina, a partir do mRNA. A expresÃo foi tempertura dependente e a frutalina recombinante apresentou uma massa molecular aparente de 17 kDa, sugerindo a presenÃa do peptÃdeo de ligaÃÃo e da cauda de histidina.<br>Frutalin, the Artocarpus incise alfa-D-galactose binding seed lectin was expressed and processed in a heterologous system for protein expression, using the metilotrophic yeast Pichia pastoris. In order to obtain the proposed objectives, three strategies were followed: identify the frutalin gene from the genomic DNA, obtained from the leaves; obtain, from the GenScript Corporation, the synthetic frutalin polypeptide chains gene, optimized for expression in Pichia system; and isolate frutalin gene, from mRNA present in fresh seeds in order to obtain the cDNA, using the RT-PCR technique. The obtained genes were inserted in the Pichia genome, in order to evaluate the lectin expression. While in the first strategy only the beta chain gene was obtained, in the other two the complete frutalin gene was obtained. The best results were obtained from the third strategy. The expression process was temperature dependent, with the optimum at 18 οC and the recombinant frutalin showed an apparent molecular mass of 17 kDa, which suggest the presence of both the linker peptide and the histidine tag.
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Aizemberg, Raquel. "Produção de glicerol quinase em Pichia pastoris /." Araraquara : [s.n.], 2011. http://hdl.handle.net/11449/88349.
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Orientador: Edwil Aparecida de Lucca Gattás<br>Banca: Eleonora Cano Carmona<br>Banca: Rubens Monti<br>Resumo: A levedura Pichia pastoris vem sendo largamente utilizada como um eficiente sistema de expressão para a produção de proteínas heterólogas, pois é um sistema seguro, fácil e mais barato que sistemas de expressão de outros eucariotos. Neste trabalho, a enzima de interesse é a glicerol quinase (GK), que cataliza a transferência do fosfato terminal do ATP para o glicerol originando glicerol-3-fosfato e ADP. Esta reação pode ser utilizada na determinação da concentração de glicerol, subproduto da fermentação alcoólica. A leitura do consumo de glicerol é realizada pela determinação espectrofotométrica do NADH gerado na reação de oxido-redução catalizada pela enzima glicerol-3-fosfato desidrogenase. Este estudo de indução foi realizado em diferentes condições de crescimento da levedura Pichia pastoris. Os resultados mostraram a seleção do melhor clone da levedura Pichia pastoris para a expressão extracelular da enzima glicerol quinase, e a determinação das melhores condições do meio de cultura para a produção da enzima de interesse foram: concentração do meio de cultura BMMY (20 vezes), densidade inicial de célula (0,1 mg/mL), concentração de metanol na fase de indução (1%), natureza do tampão (fosfato de potássio), pH (6,0), suplementação de glicerol no meio BMMY (1%), peptona (marca Difco), sem adição de sulfato de amônio, caseína e glicina, uso do meio BMMY e liofilização do mesmo. Estudos de parâmetros cinéticos foram realizados e a atividade máxima da GK foi obtida em pH 9,8, a 50ºC e 2,5 μM de substrato, por metodologia clássica, além da presença de sulfato de magnésio e diluição da enzima de 30 vezes. A enzima apresentou alta estabilidade térmica ― a atividade foi completamente... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: The yeast Pichia pastoris has been widely used as an efficient expression system for production of heterologous proteins because it is a safe, easy and cheaper than expression systems in other eukaryotes.In this studie, the enzyme of interest is glycerol kinase (GK), which catalizes the transfer of terminal phosphate from ATP to glycerol resulting glycerol-3-phosphate and ADP. This reaction can be used in determining the concentration of glycerol, a byproduct of fermentation. The reading of the consumption of glycerol is carried out by spectrophotometric determination of NADH generated in the redox reaction catalyzed by the enzyme glycerol-3-phosphate dehydrogenase. This study of induction was performed in different conditions of growth of the yeast Pichia pastoris. The results show that selecting the best clone of the yeast Pichia pastoris for the expression of extracellular enzyme glycerol kinase, and determining the best conditions of the culture medium for producing the enzyme of interest were: concentration of the culture medium BMMY (20 times), initial cell density (0.1 mg/mL), methanol concentration in the induction phase (1%), nature of buffer (potassium phosphate), pH (6.0), glycerol supplementation in BMMY medium (1%), peptone (Difco), without addition of ammonium sulfate, casein and glycine in BMMY and lyophilized medium. Studies of kinetic parameters were conducted and the GK maximum activity was obtained at pH 9.8 at 50°C and 2.5 μM substrate by conventional method, besides the presence of magnesium sulfate and diluting the enzyme 30 times. The enzyme showed high thermal stability - the activity was fully maintained up to 50°C for one hour - and at pH 7.0 for 7 days and kept under refrigeration, freeze-dried extract showed a decrease in enzymatic activity. Calculated by... (Complete abstract click electronic access below)<br>Mestre
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Felix, Wagner Pereira. "Produção heteróloga de frutalina em Pichia pastoris." reponame:Repositório Institucional da UFC, 2008. http://www.repositorio.ufc.br/handle/riufc/18810.
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FELIX, Wagner Pereira. Produção heteróloga de frutalina em Pichia pastoris. 2008. 113 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2008.<br>Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-07-28T15:13:10Z
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Previous issue date: 2008<br>Frutalin, the Artocarpus incise alfa-D-galactose binding seed lectin was expressed and processed in a heterologous system for protein expression, using the metilotrophic yeast Pichia pastoris. In order to obtain the proposed objectives, three strategies were followed: identify the frutalin gene from the genomic DNA, obtained from the leaves; obtain, from the GenScript Corporation, the synthetic frutalin polypeptide chains gene, optimized for expression in Pichia system; and isolate frutalin gene, from mRNA present in fresh seeds in order to obtain the cDNA, using the RT-PCR technique. The obtained genes were inserted in the Pichia genome, in order to evaluate the lectin expression. While in the first strategy only the beta chain gene was obtained, in the other two the complete frutalin gene was obtained. The best results were obtained from the third strategy. The expression process was temperature dependent, with the optimum at 18 οC and the recombinant frutalin showed an apparent molecular mass of 17 kDa, which suggest the presence of both the linker peptide and the histidine tag.<br>A frutalina, lectina α-D-galactose ligante, foi expressa e processada num sistema heterólogo para expressão de proteínas utilizando a levedura metilotrófica Pichia pastoris. Para atingir os objetivos esperados foram desenvolvidas três estratégias: identificar o gene que codifica para a frutalina a partir do DNA genômico obtido de folhas de A. incisa; sintetizar, por uma empresa especializada, o gene que codifica para as cadeias polipeptídicas da frutalina, otimizando-o para a expressão em P. pastoris e isolar o gene que codifica para a frutalina, a partir do mRNA presente em sementes maduras e frescas, para obtenção do cDNA utilizando a técnica da RT-PCR. Enquanto na primeira estratégia, foi obtido apenas o gen da cadeia beta, nas duas outras estratégias o gen completo foi obtido. No entretanto, a estratégia que mostrou melhor expressão da frutalina recombinante em P. pastoris foi a da obtenção do gene que codifica para a frutalina, a partir do mRNA. A expresão foi tempertura dependente e a frutalina recombinante apresentou uma massa molecular aparente de 17 kDa, sugerindo a presença do peptídeo de ligação e da cauda de histidina.
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DeMille, Janet. "Mapping the cytotoxic T-lymphocyte epitopes of Pichinde virus." Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/6495.
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One of the aims of this study was to determine whether the glycoproteins of Pichinde virus harbour any cytotoxic T-lymphocyte (CTL) epitopes on the murine haplotypes, H-2$\sp{\rm b}$ and H-2$\sp{\rm d}$ since, on neither of these MHC backgrounds are there any CTL epitopes on the nucleoprotein of this virus. Using a vaccinia virus recombinant, vvGPC, which expresses the full-length glycoprotein precursor (GPC) of Pichinde virus, standard chromium release CTL assays were performed. Three independent assays are shown for each of the haplotypes. In each of these assays for both of the haplotypes, it was observed that CTL derived against Pichinde virus did not recognize vvGPC-infected target cells nor did CTL derived against vvGPc recognize Pichinde-infected target cells. This indicates that no CTL were generated with either virus that might recognize the glycoproteins of Pichinde virus and, therefore, that the glycoproteins do not contain CTL epitopes on these murine MHC backgrounds. A second aim of this work was to compare the CTL epitopes of Pichinde wild-type virus with two temperature sensitive mutants derived from it. Both these mutants have been shown to be defective in their glycoprotein processing. The H-2$\sp{\rm d}$-restricted epitopes appear to be disrupted in TS13 as it is not recognized by Pichinde-specific CTL derived on this background. TS908 is not recognized by Pichinde-specific CTL on either haplotype suggesting that the wild-type virus' epitopes were probably disrupted during the derivation of this mutant. (Abstract shortened by UMI.)
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Nguyen, Jack. "Development of a high throughput reporter system using β-Galactosidase in the yeast : Pichia Pastoris". Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/623.
Full textAbstract:
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.
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Cochran, Keith Jacob. "Combined fermentation and recovery using expanded bed chromatography." Worcester, Mass. : Worcester Polytechnic Institute, 2006. http://www.wpi.edu/Pubs/ETD/Available/etd-081806-184321/.
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Silva, Aline Ferreira. "Avaliação do potencial de leveduras fermentadoras de pentoses na produção de etanol 2G, a partir de bagaço de sorgo /." Jaboticabal, 2019. http://hdl.handle.net/11449/183183.
Full textAbstract:
Orientador: Márcia Justino Rossini Mutton<br>Resumo: A utilização de biomassa lignocelulósica para produzir biocombustíveis, como o etanol, pode fornecer uma alternativa sustentável aos combustíveis fósseis. Os bagaços de sorgo sacarino e sorgo biomassa são considerados matérias-primas lignocelulósicas potenciais para a produção de etanol por meio de pré-tratamento, hidrólise enzimática e fermentação. Um dos entraves do processo é a utilização de microrganismos capazes de metabolizar pentoses e fermentarem em altas temperaturas. Neste contexto, este trabalho teve como objetivo avaliar as condições de adaptação e o desempenho fermentativo das estirpes de leveduras (LJ04 e Pichia kudriavzevii LJ03), em temperaturas de 37Cº e 40ºC, para produção de etanol de segunda geração, a partir de hidrolisados de bagaço de sorgo sacarino (Malibu J53 e Malibu A1001) e sorgo biomassa (Palo Alto) pré-tratados com explosão à vapor catalisada com ácido. Os bagaços dos genótipos foram avaliados quanto a composição química e rendimentos dos açúcares recuperáveis. Testes fermentativos foram realizadas com as leveduras Pichia kudriavzevii LJ03 e LJ04, nas temperaturas de 37ºC e 40ºC para avaliar três meios de cultivo com diferentes teores de hidrolisado (Meio 1 – 0%; Meio 2 – 33%; Meio 3 - 50%) durante 120 horas de fermentação. A partir do melhor meio de cultivo, fermentações em bioerreatores foram realizadas com as condições estabelecidas, em 96 horas de fermentação. Os resultados evidenciaram a eficiência do pré-tratamento e da hidrólise enzimática... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Lignocellulosic biomass to produce biofuels such as ethanol may provide a sustainable alternative to fossil fuels. Sweet sorghum bagasse is considered a potential lignocellulosic raw material for the production of fuel ethanol by means of pretreatment, enzymatic hydrolysis and fermentation. One of the difficult to the process is the use of microrganisms capable of metabolizing pentoses and fermenting at high temperatures. In this context, this work aimed to evaluate the adaptation conditions and fermentative performance of two yeast strains (LJ04 and Pichia kudriavzevii LJ03), at temperatures of 37º and 40ºC, for production of second generation ethanol from hydrolysates of Sweet sorghum bagasse (Malibu J53 and Malibu A1001) and Sorghum biomass (Palo Alto) pretreated with acid catalyzed steam explosion. Bagasse from those genotypes was evaluated for chemical composition and recoverable sugar yields. Fermentation tests were performed with Pichia kudriavzevii LJ03 and LJ04 yeast strain at 37ºC and 40ºC to evaluate three culture media with different hydrolyzed liquor contents (Medium 1 - 0%; Medium 2 - 33%; Medium 3 - 50%) during 120 hours of fermentation. After that, the best culture medium was chosen, fermentation in bioreactors were performed under the established conditions, in 96 hours of fermentation. The results showed the efficiency of pretreatment and enzymatic hydrolysis and the recovery of sugars without the formation of inhibitors. Strain LJ04 was classified as Pichia... (Complete abstract click electronic access below)<br>Doutor
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Gunduz, Burcu. "Recombinant Transglutaminase Production By Metabolically Engineered Pichia Pastoris." Master's thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614761/index.pdf.
Full textAbstract:
Transglutaminases (EC 2.3.2.13) are enzymes that catalyze an acyl
transfer reaction between a &gamma<br>-carboxyamide group of a peptide bound
glutaminyl residue (acyl donor) and a variety of primary amines (acyl
acceptors), including the amino group lysine. Transglutaminase has a potential
in obtaining proteins with novel properties, improving nutritional quality of
foods with the addition of essential amino acids, preparing heat stable gels,
developing rheological properties and mechanical strength of foods and
reducing the applications of food additives.
The aim of this study is to develop intracellular and extracellular
microbial protransglutaminase (pro-MTG) producing recombinant Pichia
pastoris strains by using genetic engineering techniques. In this context first,protransglutaminase gene (pro-mtg) from Streptomyces mobaraensis was
amplified by PCR both for intracellular and extracellular constructs using
proper primers then they were cloned into the pPICZ&alpha<br>-A expression vectors,
separately. Both intracellular (pPICZ&alpha<br>A::pro-mtgintra) and extracellular
(pPICZ&alpha<br>A::pro-mtgextra) constructs were prepared with strong alcohol oxidase
1 promoter which is induced by methanol. Pichia pastoris X33 cells were
transfected by linear pPICZ&alpha<br>A::pro-mtgintra and pPICZ&alpha<br>A::pro-mtgextra,
separately and plasmids were integrated into the Pichia pastoris X33 genome at
AOX1 locus. After constructing the recombinant P. pastoris strains, batch
shaker bioreactor experiments were performed for each recombinant cell and
the best producing strains were selected according to Dot blot and SDS-PAGE
analyses. The selected recombinant P. pastoris strains, carrying pPICZ&alpha<br>A::promtgextra
gene and pPICZ&alpha<br>A::pro-mtgintra gene in their genome were named as
E8 and I1, respectively.
Afterwards, a controlled pilot scale bioreactor experiment in a
working volume of 1 L was performed with E8 clone and produced pro-MTG
was activated by Dispase I. The variations in the recombinant MTG activity, cell
concentration, total protease activity, AOX activity and organic acid
concentrations throughout the bioprocess were analyzed and specific growth
rates, specific consumption rates and yield coefficients were calculated
regarding to measured data. Maximum MTG activity was obtained as 4448 U L-
1 and the maximum cell concentration was measured as 74.1 g L-1 at t=36 h of
the bioprocess. In this study, an active transglutaminase enzyme was
produced extracellularly by P. pastoris for the first time and the third highest
extracellular MTG activity was achieved with E8 clone.
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Den, Haan Riaan. "Engineering of Pichia stipitis for enhanced xylan utilization." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53409.
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Thesis (PhD)--Stellenbosch University, 2003.<br>ENGLISH ABSTRACT: Plant biomass, the most abundant renewable resource in nature, consists of matrices of
mainly lignin, cellulose, hemicellulose as well as inorganic components. Xylan, the
major hemicellulose component in plant cell walls, is the most abundant polysaccharide
after cellulose. This makes the main constituent sugar of xylan, D-xylose, the second
most abundant renewable monosaccharide in nature. Very few hemicelluloses are either
homopolymeric or entirely linear. Therefore, the variety of enzymes involved in their
hydrolysis is more complex than the enzyme group responsible for the hydrolysis of
cellulose. Although the ability to degrade xylan is common among bacteria and
filamentous fungi, this trait is relatively rare among yeasts. However, some strains of the
yeast Pichia stipitis are, amongst others, able to degrade xylan. As P. stipitis is also one
of the best D-xylose fermenting yeasts thus far described, this yeast has the potential of
fermenting polymeric xylan directly to ethanol. However, it was shown that the natural
xylanolytic ability of this yeast is very weak.
In this study, xylanolytic genes were expressed in P. stipitis to test the ability of the yeast
to produce heterologous proteins, and to determine the enhancement of xylan utilisation
by the recombinant strain. The native xylose reductase gene (XYLl) and transketolase
gene (TKL) and the heterologous Saccharomyces cerevisiae phosphoglycerate kinase
(PGKl) gene promoter were cloned into P. stipitis transformation vectors and used to
express the Trichoderma reesei ~-xylanase encoding gene (xyn2) as reporter gene. It was
shown that the XYLl promoter was induced in the presence of D-xylose and that the TKL
promoter was constitutively expressed. The PGKl promoter of S. cerevisiae did not
function in P. stipitis .
When the T reesei xyn2 gene and the Aspergillus kawachii ~-xylanase encoding gene
(xynC) were expressed under control of the XYLl promoter, extracellular ~-xylanase
activity of up to 136 nkat/ml and 171 nkatlml was observed, respectively. This activity
declined over time due to the presence of extracellular proteases, secreted by P. stipitis.
Growing the cultures in a fermentor and controlling the pH level to pH 6 did not alleviate the reduction of heterologous l3-xylanase activity. When the Aspergillus niger
l3-xylosidase encoding gene (xlnD) was expressed as a fusion gene (designated XL02)
with the S. cerevisiae mating factor secretion signal (MFal) under control of the
P. stipitis TKL promoter, extracellular l3-xylosidase activity of 0.132 nkatlml was
observed. Co-expression of the xyn2 and XL02 genes led to B-xylanase and l3-xylosidase
activities of 128 nkatlml and 0.113 nkat/ml, respectively. Co-expression of the xynC and
XL02 genes led to l3-xylanase and l3-xylosidase activities of 165 nkat/ml and 0.124
nkatlml, respectively.
The expression of the fungal xylanolytic genes in P. stipitis also led to an increased
biomass yield when the recombinant strains were cultured on birchwood xylan as sole
carbon source. The strain co-expressing the A. kawachii l3-xylanase and A. niger
l3-xylosidase encoding genes was the most successful, yielding a 3.2-fold higher biomass
level than the control strain. Biomass levels of the recombinant strains were further
improved on average by 85% by growing them in a fermentor under conditions of high
oxygenation. The strains were also tested for direct conversion of xylan to ethanol and
the strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding
genes produced 1.35 giL ethanol, which represents a 3.6-fold increase in ethanol yield
over the reference strain. These strains represent a step towards the efficient degradation
and utilisation of hemicellulosic materials by ethanol-producing yeasts.<br>AFRIKAANSE OPSOMMING: Plant biomassa, die volopste hernubare koolstotbron in die natuur, bestaan uit matrikse
van lignien, sellulose en hemisellulose. Xilaan, die hoof hemisellulose komponent in
plantselwande, is na sellulose die volopste polisakkaried. Gevolglik is die hoof
suikerkomponent van xilaan, naamlik D-xilose, die tweede volopste hernubare
monosakkaried in die natuur. Baie min hemisellulose molekules is homopolimere of
heeltemal linieêr. Daarom is die ensieme betrokke by die atbraak van hemiselluloses
meer kompleks as die ensieme betrokke by die atbraak van sellulose. Bakterieë en
filamentagtige fungi wat oor die vermoë om xilaan af te breek beskik, kom wydversprei
voor maar relatief min giste kan xilaan benut. Sommige rasse van die gisspesie Pichia
stipitis het egter beperkte vermoë om xilaan af te breek. P. stipitis is ook een van die
beste D-xilose fermenterende giste wat tot dusver beskryf is en het dus die potensiaalom
etanol vanafpolimeriese xilaan te produseer.
In hierdie studie is gene wat kodeer vir xilaanatbrekende ensieme in P. stipitis uitgedruk
om die vermoë van die gis as heteroloë uitdrukking sisteem te evalueer. Verder is die
effek van die heteroloë xilaanatbrekende ensieme tydens groei op xilaan as enigste
koolstotbron getoets. Die promoters van die xilosereduktasegeen (XYLl), die
transketolasegeen (TKL) van P. stipitis en die fosfogliseraatkinasegeen (PGKl) van
Saccharomyces cerevisiae is in P. stipitis transformasie vektore gekloneer en gebruik om
die Trichoderma reesei ~-xilanasegeen (xyn2) as verklikkergeen uit te druk. Dit het
bewys dat die XYLI promotor induseerbaar is in die teenwoordigheid van D-xilose terwyl
die TKL geen konstant uitgedruk was. Die PGKI promotor van S. cerevisiae was nie
funksioneel in P. stipitis nie.
Ekstrasellulêre ~-xilanase aktiwiteit van onderskeidelik 136 nkatlml en 171 nkatlml kon
waargeneem word wanneer die T reesei xyn2 geen of die Aspergillus kawachii
~-xilanasegeen (xynC) onder beheer van die XYLI promotor uitgedruk is. Hierdie
aktiwiteit het afgeneem na gelang van tyd a.g.v. die teenwoordigheid van ekstrasellulêre
proteases wat deur P. stipitis uitgeskei word. Die afname van ekstrasellulêre ~-xilanase aktiwiteit kon nie voorkom word deur die kulture in 'n fermentor te groei en die pH vlak
tot pH 6 te beheer nie. Tydens uitdrukking van die Aspergillus niger ~-xilosidase geen
(xlnD) as 'n fusiegeen (genoem XL02) met die paringsfaktor sekresiesein (MFal) van
S. cerevisiae onder transkripsionele beheer van die P. stipitis TKL promotor, kon
ekstrasellulêre ~-xilosidase aktiwiteit van 0.132 nkatlml waargeneem word.
Gesamentlike uitdrukking van die xyn2 en XL02 gene het gelei tot ~-xilanase en
~-xilosidase aktiwiteite van 128 nkatlml and 0.113 nkat/ml, onderskeidelik.
Gesamentlike uitdrukking van die xynC en XL02 gene het gelei tot ~-xilanase en
~-xilosidase aktiwiteite van 165 nkatlml and 0.124 nkatlml, onderskeidelik.
Die uitdrukking van xilaanatbrekende ensieme III P. stipitis het verhoogbe
biomassaproduksie teweeg gebring wanneer die rekombinante gisrasse op birchwood
xilaan as enigste koolstotbron gegroei het. Die rekombinante ras wat die A. kawachii
~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitdruk, was die mees
suksesvolle ras en het 3.2-voudig hoër biomassa as die kontrole ras opgelewer. Die
biomassa van die rekombinante rasse tydens groei op xilaan as enigste koolstotbron kon
gemiddeld met 85% verhoog word deur die giste onder hoë suurstotkonsentrase in 'n
fermentor te kweek. Die rekombinante rasse is verder ook getoets vir hul vermoë om
xilaan direk tot etanol om te skakel. Die rekombinante ras wat die A. kawachii
~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitgedruk het, het 'n 3.6-
voudige verhoging in etanolproduksie getoon en 1.35 gIL ethanol gelewer. Hierdie
rekombinante gisrasse verteenwoordig 'n stap nader aan die doeltreffende atbraak en
benutting van hemisellulose deur etanolproduserende giste.
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Araújo, Diana Filipa Vieira. "Production of chitin-glucan complex by Pichia pastoris." Master's thesis, Faculdade de Ciencias e Tecnologia, 2013. http://hdl.handle.net/10362/10897.
Full textAbstract:
Dissertation for the Degree of Master in Biotechnology<br>The yeast Pichia pastoris produces chitin-glucan complex (CGC), as a cell wall component. CGC is composed of two types of biopolymers, chitin and β-glucans, which confer it great potential for use in the food, cosmetic and pharmaceutical industries. CGC hydrolysis, allows obtaining chitin/chitosan and glucans individually. The chitin and chitosan obtained from CGC have the considerable advantage of being of non-animal origin, which further extends their applications. In last year’s, the production of CGC by Pichia pastoris was realized with glycerol as sole carbon source, achieving high cell density.
In this study, different substrates were tested for cultivation of P. pastoris and CGC production. In the first part, mixtures of glucose and xylose, in varying proportions, were tested. Since glucose and xylose are two of the main sugar components of lignocellulosic wastes, the ability of P. pastoris to use them as carbon sources would allow their valorization into value-added products. In the second part, several wastes and byproducts generated by different industries were tested for their suitability as substrates for P. pastoris cultivation.
In study of the glucose/xylose mixtures, the best performance was achieved in batch bioreactor experiments with 25% xylose in the medium (20 gL-1 of xylose and 60 gL-1 of glucose), where 35.25 gL-1 biomass was obtained in 64 hours of cultivation. CGC content in the cell wall reached 15% with a volumetric productivity of 0.085 gL-1.h-1. The molar ratio of chitin:β-glucan in the extracted biopolymer was 47:53, higher than obtained with crude glycerol (16:84).
In the second part of study, several wastes and byproducts (used cooking oil, sugarcane molasses, cheese whey, waste paper and spent coffee grounds) were tested. The results show that P. pastoris presented low biomass concentration using any of these substrates. Nevertheless, in batch bioreactor experiments the best results were achieved with sugarcane molasses, where 17.78 gL-1 biomass were obtained with a CGC content of 17%.
Among the tested substrates, the mixtures of glucose/xylose appear to be the most promising due the good CGC production obtained and the high glucosamine molar fraction in produced polymer. This study opens the hypothesis of utilization of lignocellulosic materials with xylose percentages up to 50%.
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Fonseca, Marisa Cristina da. "Produção de estreptavidina recombinante pela levedura Pichia pastoris." Universidade Federal de Viçosa, 2006. http://locus.ufv.br/handle/123456789/5386.
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Previous issue date: 2006-02-20<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>Streptavidin has been exploited as affinity tag to isolate protein in biotinilated columns. Recombinant Pichia pastoris KM71/Stp strains containing the streptavidin core gene were cultured in fed-batch generating 150 g L-1 biomass. This biomass was achieved with a simpler and shorter process that has never been reported. The yeast was pre-cultured into 50 mL minimum medium with glycerol as only carbon source at 25ºC and 250 rpm. After 12 hours incubation, the culture was transferred to a bioreactor with 200 mL of the same initial A600 0,2. After 24 hours incubation the culture was fed-batch with glycerol and basal salts medium to reach 400 mL. The glycerol concentration of 2.0 moles. L-1 combined with a flow of 0.11 mL. min-1 and aeration by air injection dispersed with a porous stone and magnetic stirring of 500 rpm were the set of conditions to yield maximum biomass. The streptavidin concentration at the supernatant of the free cell culture at 96 hours, the maximum induction period, has achieved 4.0 g. L-1, reducing to 3.2 and 0.87 g. L-1 with two reutilizations. At the same time period the immobilized culture yield 75 %, 50 % and 80% less at the first, second and third culture utilization, respectively. The immobilization and recycling of recombinant P. pastoris biomass can prove to be a potential strategy to improve volumetric productivity.<br>Com o intuito de utilizar estreptavidina como alvo para isolar proteínas de interesse numa coluna biotinilada, P. pastoris KM71 recombinante contendo o gene do core da estreptavidina, foi cultivada em regime de batelada alimentada na fase de produção de biomassa, alcançando uma concentração de 150 g L-1. Essa biomassa foi alcançada com um novo protocolo, que além de reduzir os passos, introduziu um aparato simples, mas eficiente de dispersão de ar. Um reator com capacidade para 1 L, com 200 mL de meio mínimo com glicerol, foi inoculado com uma pré-cultura para uma A600 inicial de 0,2. Após 24 horas de incubação, a 25 ºC, 500 rpm e injeção e dispersão de ar através de pedra porosa, a alimentação foi iniciada com meio de sais basais e glicerol até atingir um volume de 400 mL. A concentração de 2,0 moles L-1 de glicerol e fluxo de 0,11 mL min-1 utilizados na alimentação, permitiram obter máxima biomassa. Na fase de indução de estreptavidina, foi estudada a reutilização da biomassa na produção de estreptavidina em duas condições: livres em suspensão e imobilizadas em partículas de alginato de cálcio. Em ambos os casos a proteína produzida apresentou-se biologicamente funcional, exibindo ligação esperada à biotina. A concentração de estreptavidina no sobrenadante da cultura de células livres no período de máxima indução (96 horas) atingiu 4,0 g L-1, reduzindo para 3,2 e 0,87 g L-1 respectivamente em duas reutilizações. Quando comparada às concentrações de estreptavidina obtidas pelas células livres em cada utilização, a imobilização resultou na produção de 75% na primeira utilização, 50% na segunda, mas alcançando quase 80% na terceira utilização. A imobilização e a reutilização da biomassa de P. pastoris recombinante ainda não haviam sido reportadas e a produção de estreptavidina nessas condições demonstrou ser uma técnica em potencial.
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Thor, Der. "Cloning and characterization of MET2 in Pichia pastoris." Scholarly Commons, 2002. https://scholarlycommons.pacific.edu/uop_etds/570.
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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.
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Aw, Rochelle. "Factors affecting the specific productivity of Pichia pastoris." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/11047.
Full textAbstract:
Pichia pastoris has become one of the most popular recombinant protein expression platforms, despite the lack of understanding into the fundamentals of protein expression. Whilst P. pastoris exhibits high volumetric productivity, it has a low specific productivity, which can be further reduced by protein-specific problems. This thesis employs several strategies commonly used to increase specific productivity, and assesses their impact on the productivity and cell biology of P. pastoris. Gene dosage has been reported to increase titre; therefore multiple copies of human serum albumin were integrated into P. pastoris to assess the correlation between recombinant protein productivity and copy number. Post-transformational vector amplification was used to generate clones containing up to five copies of HSA. However it was not possible to correlate copy number and yield as 15 L bioreactor cultures showed significant genetic instability. The mean final copy number was 2.6 ± 1.0. Further work was undertaken to evaluate possible ways to prevent instability, such as different selection methods, mutation of RAD51 and RAD52 which are both possible RecA homologs and whether the locus of vector integration plays a part. Integration into the rDNA locus resulted in increased stability with a five copy clone averaging 3.8 ± 1.6. Furthermore, no clones showed complete loss of the integrated vector as observed with integration into the AOX1 locus. Additionally, the little understood phenomenon of clonal variation was investigated which has been reported to affect specific productivity. Nine clones, with a range of productivity, were chosen for transcriptomic analysis. Variation between different clones was not uniform, even within the high, mid and low secretor groups. However, the ER associated degradation pathway was consistently upregulated in the high secretors which could be exploited in the future for strain development and selection.
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Lange, Stefan. "Etablierung von neuen Methoden zur Herstellung rekombinanter Antikörper und zur spezifischen Selektion von Antikörpervarianten im hohen Durchsatz." [S.l. : s.n.], 2002. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10073673.
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Ostendorp, Thorsten. "Structure and function of the metal-binding protein S100B and its interaction with the receptor for advanced glycation end products." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-23752.
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Grove, Heather Lee. "Cloning and characterization of the Pichia Pastoris PMR1 gene." Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/613.
Full textAbstract:
Pichia pastoris, a popular protein expression system, is limited in its ability to secrete heterologous proteins. The PMR1 gene, the disruption of which is known to improve the secretion of prochymosin, human prourokinase, and human tissue plasminogen activator in Saccharomyces cerevisiae, was cloned from P. pastoris. The pmr 1 mutant in S. cerevisiae also displayed a slow growth phenotype when grown on low Ca2+ medium. The putative P. pastoris PMR1 gene, encoding for a 924 amino acid P-type Ca2+ ATPase, was disrupted in P. pastoris and the secretion of horseradish peroxidase (HRP) and β-galactosidase (β-gal) analyzed. Secreted HRP activity was determined using 3,3',5,5' tetramethylbenzidine (TMB) colorimetric assay and western analysis. β-gal expression and secretion was determined by western analysis. Secretion in P. pastorius Δpmr1 for both heterologous proteins showed no appreciable difference compared to wild type, nor did P. pastoris Δpmr1 display the slow growth phenotype seen in S. cerevisiae Δpmr1 (Rudolph H. et al., 1989).
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Nepomuceno, Denise Rocha. "ExpressÃo em Escherichia coli e Pichia pastoris de uma quitinase antifÃngica da famÃlia 19 das glicosÃdeo hidrolases de Chromobacterium violaceum." Universidade Federal do CearÃ, 2012. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=9171.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico<br>Chromobacterium violaceum à uma bactÃria Gram-negativa, saprÃfita, nÃo patogÃnica e de vida livre. A anotaÃÃo do genoma dessa espÃcie revelou muitos genes codificando produtos com potenciais aplicaÃÃes em diversas Ãreas. Dentre esses produtos estÃo vÃrias quitinases, enzimas capazes de degradar quitina, um polissacarÃdeo de N-acetil-β-D-glucosamina presente na parede celular de muitos fungos e no exoesqueleto de insetos e crustÃceos. As quitinases sÃo de grande interesse biotecnolÃgico, podendo ser utilizadas para converter quitina em derivados Ãteis, e tambÃm para o controle de fungos e insetos que causam danos em culturas agrÃcolas. O presente trabalho teve como objetivo realizar a caracterizaÃÃo bioquÃmica, estrutural e biolÃgica de uma quitinase recombinante (CV1897), da famÃlia 19 das glicosil hidrolases, de C. violaceum ATCC 12472. A sequÃncia completa da ORF CV1897 (codificando CV1897PS+, com o peptÃdeo sinal nativo) foi amplificada por reaÃÃo em cadeia da polimerase (PCR) e clonada nos vetores pET302/NT-His e pPICZαA para expressÃo em Escherichia coli e Pichia pastoris, respectivamente. A sequÃncia parcial da ORF (rCV1897PS-, sem o peptÃdeo sinal nativo) foi expressa apenas em P. pastoris. Em E. coli, a rCV1897PS+ foi produzida principalmente em corpos de inclusÃo, de forma insolÃvel e inativa. Em P. pastoris, as proteÃnas (rCV1897PS+ e rCV1897PS-) foram secretadas para o meio de cultura de forma solÃvel e funcional. As enzimas foram purificadas por cromatografia de afinidade em nÃquel imobilizado, com rendimentos de 1,8 mg/L (rCV1897PS+) e 2,1 mg/L (rCV1897PS-), sendo detectadas por eletroforese em gel de poliacrilamida na presenÃa de SDS (SDS-PAGE) como duas isoformas com massas moleculares diferentes (43,2 kDa e 51,4 kDa; 44 e 50 kDa, respectivamente). A quitinase recombinante (CV1897PS+) mostrou atividade Ãtima em Ph 5,0 e foi ativa quando tratada a temperaturas de atà 40 ÂC por 30 min. A Rcv1897 apresentou atividade quitinÃsica frente a quitina coloidal e glicol-quitina (em gel SDS-PAGE). Os espectros de dicroÃsmo circular revelaram que a estrutura da proteÃna possui predominantemente estrutura secundÃria do tipo α-hÃlice (37%), com 26% de fitas-β e 38% de estrutura desordenada. Os espectros de fluorescÃncia revelaram que a proteÃna foi produzida na sua conformaÃÃo enovelada. A rCV1897PS+ inibiu a germinaÃÃo de conÃdios e o crescimento micelial dos fungos fitopatogÃnicos Penicillium herquei e Colletotrichum lindemuthianum. As quitinases rCV1897PS+ e rCV1897PS- em associaÃÃo com o fluconazol atuaram de forma sinÃrgica contra diferentes cepas da levedura patogÃnica Candida tropicalis. Os estudos bioquÃmicos, estruturais e biolÃgicos da rCV1897 poderÃo ser Ãteis para aplicaÃÃo biotecnolÃgica dessa enzima.<br>Chromobacterium violaceum is a Gram-negative, saprophytic, non-pathogenic and free living bacterium. The annotation of the genome of this species revealed many genes encoding products with potential applications in many areas. Among these products are several chitinases, which are enzymes capable of degrading chitin, a polysaccharide of N-acetyl-β-D-glucosamine. Chitin is found in the cell wall of many fungi and in the exoskeleton of insects and crustaceans. Chitinases are of great biotechnological interest because these enzymes can be used to produce useful derivatives from chitin, and also to be exploited for the control of fungi and insects that cause damages in crops. This study aimed to carry out the biochemical, biological and structural characterization of a recombinant chitinase (CV1897) belonging to family GH19 from C. violaceum ATCC 12472. The complete sequence of the ORF CV1897 (encoding CV1897SP+, with the native signal peptide) was amplified by polymerase chain reaction (PCR) and cloned into the vectors pET302/NT-His and pPICZαA, for expression in Escherichia coli and Pichia pastoris, respectively. The partial sequence of the ORF CV1897 (encoding CV1897SP-, without the native signal peptide) was expressed only in P. pastoris. In E. coli the rCV1897SP+ was mainly produced as insoluble inclusion bodies. In P. pastoris, the two versions of the protein (rCV1897PS+ and rCV1897PS-) were secreted into the culture medium, in their soluble and functional form. The enzymes were purified by affinity chromatography on immobilized nickel ions, with yields of 1.8 mg/L (rCV1897PS+) and 2.1 mg/L (rCV1897PS-), being detected by electrophoresis as two isoforms with different molecular weights. The recombinant chitinase (rCV1897PS+) showed optimal hydrolytic activity at pH 5.0 and was active after treatement at temperatures up to 40 ÂC for 30 min. The rCV1897 showed chitinolytic activity against colloidal chitin and glycol-chitin on SDS-PAGE. Circular dichroism spectra showed that the protein has predominantly α-helical arrangements (37%), also having 26% of β-strands and 38% disordered structure. The fluorescence spectra revealed that the protein was produced in their folded conformation. The rCV1897PS+ inhibited the conidial germination and mycelial growth of the phytopathogenic fungi Penicillium herquei and Colletotrichum lindemuthianum. Both rCV1897PS+ and rCV1897PS- in combination with fluconazole acted synergistically against different strains of the human pathogenic yeast Candida tropicalis. The biochemical, structural and biological studies of rCV1897 may be useful for biotechnological application of this enzyme.
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Santana, Ana Carolina. "Expressão e caracterização das quimases recombinantes específicas de mastócitos de camundongos (mMCP4 e 5)." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-18122014-150712/.
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Os mastócitos, em associação com outras células inflamatórias se acumulam em locais de tumor. Entretanto, pouco é conhecido sobre o papel exato dos mastócitos e de seus mediadores na progressão tumoral e angiogênese. Resultados recentes do nosso laboratório mostraram que a expressão das triptases específicas de mastócitos está correlacionada com a progressão tumoral (de Souza, Jr, PLosOne 7(7): e40790). Além do mais, existe uma associação entre mastócitos e a angiogênese tumoral. Então, tornou-se interessante investigar o papel das quimases específicas de mastócitos (mMCP-4 e -5) neste processo. Visto que estas quimases não são disponíveis comercialmente, a primeira etapa deste estudo foi produzir as quimases mMCP-4 e -5 recombinantes. Assim, as sequências dos genes respectivos para mMCP-4 e -5, além das sequências para seis resíduos de His e do sítio suscetível a enteroquinase foram clonadas no vetor de expressão pPIC9. A cepa GS115 de Pichia pastoris foi transformada com o respectivo vetor para mMCP-4 ou -5. Os clones transformados foram crescidos em meio BMMY e induzidos com várias concentrações de metanol para a produção de mMCP-4 ou -5 recombinantes. Depois de 96 horas, o meio foi centrifugado e as quimases mMCP-4 ou -5 foram purificadas do sobrenadante usando resina de níquel. A identidade das proteínas foi confirmada por Western Blotting usando o anticorpo anti-his, assim como os anticorpos anti-mMCP-4 e -5. As proteases foram ativadas pela clivagem do sítio suscetível a enteroquinase por cinco horas, a 22°C. A ativação das enzimas foi confirmada através da degradação de seus substratos específicos. Estes resultados mostram que P. pastoris é um organismo eficiente para a expressão de ambas as proteases. Estas proteases recombinantes se constituem em ferramentas importantes para se elucidar o papel da mMCP-4 ou -5 na angiogênese.<br>Mast cells, in association with other inflammatory cells, are known to accumulate at tumor sites. However, little is known about the exact role that mast cells and their mediators play in tumor progression and angiogenesis. Recent results from our laboratory have shown that expression of mast cell specific tryptases correlates with tumor progression (de Souza, Jr, PLosOne 7(7): e40790). Furthermore, there is an association between mast cells and tumor angiogenesis. It then became of interest to investigate the role of mast cell specific chymases (mMCP-4 and -5) in this process. Since these chymases are not commercially available, the first step in this study was to produce recombinant mMCP-4 and -5. The gene sequences for these chymases along with the sequence for six His residues and an enterokinase susceptible peptide were cloned into the expression vector pPIC9. The GS115 strain of Pichia pastoris was transformed with the respective vector for either rmMCP-4 or -5. The transformed clones were grown in BMMY media and induced to produce rmMCP-4 or -5 with various concentrations of methanol. After 96 hours, the medium was centrifuged and rmMCP-4 or -5 was purified from the supernatant using nickel-nitrilotriacetic acid agarose beads. The identity of the proteins was confirmed by Western blotting using an anti-his antibody as well as anti-mMCP-4 and -5. These results show that P. pastoris is an efficient organism in which to express both proteases.
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Mbawala, Augustin. "Contribution à l'étude chimique et structurale des phosphopeptidomannanes parietaux de la levure pichia pastoris IFP 206 cultivée en présence de sources carbonées méthanol (levure très floculante) et glucose (levure peu floculante)." Nancy 1, 1990. http://www.theses.fr/1990NAN10036.
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La levure pichia pastoris IFP 206 a été cultivée dans un milieu à base de sels minéraux contenant comme source de carbone soit du méthanol (levure très floculante) soit du glucose (levure peu floculante). L'analyse chimique des parois et des phosphopeptidomannanes (PPM) isolés de cellules entières de cette levure a montré des différences significatives notamment dans leur teneur en acides aminés qui augmente avec le degré de floculation et inversement en ce qui concerne celle des hexosamines. Nous n'avons pas observé une forte élévation de la teneur en mannose et en phosphore des parois ou des PPM parallèlement avec le degré de floculation, ce qui laisse supposer que chez la levure pichia pastoris IFP 206, ce phénomène biologique se fait plutôt par le biais d'un facteur parietal caractéristique. En effet, l'étude structurale des oligomannosides composant les PPM a montré qu'ils renferment des liaisons de type alpha (1-2) et/ou beta (1-2) et que le mannopentaose, nettement plus important dans les mannanes de la levure floculante que ceux de la levure peu floculante, pourrait être considéré comme l'un des facteurs responsables de la floculation de la levure pichia pastoris IFP 206
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Kongsaeree, Puapong Kelly Christine. "Optimization of recombinant ligninolytic enzyme production in Pichia pastoris." Related electronic resource: Current Research at SU : database of SU dissertations, recent titles available full text, 2004. http://wwwlib.umi.com/cr/syr/main.
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Solà, i. Rodrigo Aina. "Estudi del metabolisme central del carboni de Pichia pastoris." Doctoral thesis, Universitat Autònoma de Barcelona, 2004. http://hdl.handle.net/10803/5303.
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Aquesta tesi s'emmarca en els àmbits la biologia de sistemes i l'enginyeria metabòlica, és a dir, en l'anàlisi quantitatiu de sistemes biològics complexes. En aquest estudi s'ha utilitzat el llevat metilotròfic Pichia pastoris com a organisme model, el qual ha adquirit durant els darrers anys una importància creixent en l'àmbit de la biotecnologia, mostrant una gran capacitat com a sistema de producció de proteïnes recombinants. L'objectiu principal de l'estudi ha estat establir tècniques i metodologies experimentals i computacionals d'anàlisi quantitativa de la fisiologia cel·lular al sistema de P. pastoris. L'anàlisi quantitativa (i modelització matemàtica) dels processos cel·lulars és una eina molt valuosa per al redisseny racional del metabolisme cel·lular amb l'objectiu d'optimitzar els sistemes vius per a determinades aplicacions biotecnològiques (enginyeria metabòlica), així com per al disseny i optimització racional de processos de cultiu i expressió de proteïnes recombinants. Aquests objectius depenen de l'elucidació de la xarxa de bioreaccions, concretament de la recopilació de dades experimentals disponibles sobre els components de la xarxa, una estimació dels fluxos in vivo i la descripció teòrica (modelització) de la xarxa. <br/>Aquest treball ha estat centrat en l'estudi sistemàtic in vivo del metabolisme central del carboni de P. pastoris. El creixement del llevat sobre el sucre glucosa o d'altres compostos com la glicerina o metanol, substrats àmpliament utilitzats en processos de cultiu de P. pastoris, implica a un conjunt o xarxa de reaccions bioquímiques. La comparació de les variacions de fluxos metabòlics in vivo d'aquesta xarxa en cèl·lules de llevat creixent sota diferents condicions ambientals proporciona valuosa informació sobre el seu comportament i regulació. <br/>En aquesta tesi, s'han realitzat una sèrie de cultius en bioreactors operant en continu (quimiostat), els quals permeten mantenir les cèl·lules en condicions fisiològiques controlades i constants (estat metabòlic estacionari). Es varen realitzar estudis metabòlics de cèl·lules de llevat a diferents estats fisiològics, canviant el tipus de substrat disponible per a les cèl·lules o bé utilitzant barreges dels mateixos en diferents proporcions, i/o variant la velocitat específica de creixement cel·lular, per aleshores analitzar les variacions en la topologia de la xarxa metabòlica (rutes metabòliques actives) i les variacions en les fraccions de fluxos metabòlics. <br/>L'estimació de les variacions de fluxos metabòlics es realitzà experimentalment mitjançant tècniques de marcatge isotòpic dels substrats amb 13C, combinades amb tècniques de Ressonància Magnètica Nuclear (RMN). En aquest treball, s'ha adaptat una metodologia prèviament desenvolupada per a l'anàlisi de metabolisme de bactèries, i posteriorment estesa a l'anàlisi d'organismes eucariotes utilitzant el llevat S. cerevisiae com a model. Aquesta metodologia permet determinar quines rutes metabòliques del metabolisme central del carboni estan actives i quantificar els fluxos de metabòlits a través d'aquestes. <br/>Els resultats obtinguts han mostrat en primer lloc que les rutes metabòliques per a la biosíntesi d'aminoàcids en Pichia pastoris són essencialment les mateixes que les descrites al llevat model Saccharomyces cerevisiae. A més, s'han detectat diferències significatives a nivell del cicle dels àcids tricarboxílics i de les reaccions anapleròtiques al comparar el creixement cel·lular a les diferents condicions del cultiu (diferents substrats i diferents velocitats específiques de creixement).<br/>Aquesta metodologia pot ser emprada en un futur de manera sistemàtica en l'estudi d'altres paràmetres clau dels processos de cultiu i producció de proteïnes recombinants en llevats com Pichia pastoris, tant genètics, com ambientals, així com la caracterització dels fluxos metabòlics per a estats d'estrès fisiològics de rellevància en el disseny del procés de cultiu/expressió.<br>The present thesis can be framed within the Systems Biology and Metabolic Engineering research areas, i.e. in the quantitative analysis of complex biological systems. The model biological system used in this work has been the methylotrophic yeast Pichia pastoris. This organism is becoming increasingly employed as a host system for the production of a wide variety of heterologous proteins. <br/>The quantitative analysis and the mathematic modelling of cellular processes has been shown to be a very valuable tool in the area of Biotechnology and Bioengineering, both for the rational design of cellular metabolism (metabolic engineering) and for the rational design and optimization of culture processes and expression of foreign proteins.<br/>The present work has been focused on the in vivo study of the Pichia pastoris' central carbon metabolism. Growth of this yeast on glucose and other carbon sources such as glycerol or methanol involves a network of biochemical reactions (metabolic pathways) that take place in the cytoplasm and/or in different subcellular compartments (organelles).<br/>The comparison between the in vivo variations of the topology (active metabolic pathways) and the in vivo variations of the metabolic fluxes (metabolic reaction rates) of this network under different environmental conditions and different genotypes, generate important information about its behaviour and regulation.<br/>In this work, a series of chemostat cultures have been performed at different growth conditions. Chemostat cultivations allow maintaining the cells at a controlled and constant physiological state (stationary metabolic state). In this way, it was possible to study different cellular metabolic steady states corresponding to different physiological states. These were achieved by growing cells under different environmental conditions, namely different carbon sources at growth rates. <br/>The measurement of the metabolic flux variations was done experimentally using isotopic tracer techniques involving substrates labelling with 13C, combined with Nuclear Magnetic Resonance (NMR) techniques. The methodology used in this study had been previously developed for the analysis of bacterial metabolism and subsequently extended to the analysis of eukaryotic organisms using Saccharomyces cerevisiae as a model. Such methodology has been adapted to P. pastoris cells growing on glycerol and/or methanol as carbon sources, thus allowing the identification of the active metabolic pathways of the central carbon metabolism of this organism and the quantification of the flux metabolites through them.<br/>The results obtained show, first of all, that the metabolic pathways for the amino acid biosynthesis in Pichia pastoris are essentially the same as the ones described for the model yeast S. cerevisiae. In addition, significant differences were detected at the level of the tricarboxylic acids cycle (TCA) and the anaplerotic reactions when comparing the cellular growth at different culture conditions (i.e. different carbon sources and different specific growth rates).<br/>The methodology employed in this work can be useful in the future for the systematic study of the influence of other key culture parameters (genetic or environmental) to the heterologous protein production with P. pastoris. It can also be useful for the characterization of the metabolic fluxes under different physiological stress states relevant for heterologous protein expression and overall process culture design.
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Tomàs, Gamisans Màrius. "Developing strategies for systems metabolic engineering of Pichia pastoris." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/458538.
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Pichia pastoris s’ha convertit en una de les plataformes cel·lulars més utilitzades per a la producció de proteïnes recombinants i metabòlits d’alt valor afegit. En els darrers anys s’han aconseguit fites importants en l’anàlisi quantitativa a nivell de sistemes de la seva fisiologia. Aquesta gran quantitat d’informació ha permès desenvolupar models metabòlics a escala genòmica, que permeten el desenvolupament de noves estratègies per l’enginyeria de soques i de bioprocés. Amb anterioritat a aquest estudi s’havien publicat tres models metabòlics a escala genòmica per a P. pastoris. No obstant això, aquests models presentaven algunes inconsistències en algunes vies metabòliques, en la nomenclatura de metabòlits i reaccions, així com les anotacions associades a certes vies. A més, algunes de les rutes metabòliques o característiques específiques de P. pastoris eren representades de forma errònia o incompleta. És per això que en aquest estudi es desenvolupa un model metabòlic a escala genòmica consens, que integra els models anteriors. A més, també es fa una revisió exhaustiva de diverses rutes metabòliques i nombroses vies es corregeixen i actualitzen d’acord amb les publicacions disponibles. Com a resultat, el nou model, iMT1026, pot reproduir amb més precisió els paràmetres de creixement experimentals de cèl·lules creixent en glucosa i diferents nivells de disponibilitat d’oxigen. Amb la voluntat d’expandir les capacitats del model, es generen noves dades fisiològiques fent servir dos dels substrats més importants per aquesta factoria cel·lular. Es realitzen unes series de cultius en continu per a la caracterització del perfil fisiològic de P. pastoris creixent en glicerol i metanol com a fonts úniques de substrat. A més, també es caracteritza la composició macromolecular de la biomassa. Posteriorment, s’incorporen en el model noves equacions de biomassa específiques per a cada font de carboni. Aquestes noves dades experimentals han permès estimar els paràmetres energètics associats a les fonts de carboni i validar el model (iMT1026 v3.0) per aquestes condicions de creixement.
Tot i la validació de iMT1026 v3.0 en un rang més ampli de condicions, en aquest treball es prova en dues aplicacions diferents: en l’anàlisi de fluxos metabòlics basat en 13C i com a eina de suport per a la interpretació de resultats en soques amb modificades en el metabolisme redox. Tot i que hi ha un únic estudi on s’analitza la relació entre fluxos metabòlics en cèl·lules creixent en glicerol, no es té constància de cap estudi d’anàlisi de fluxos metabòlics en aquesta font de carboni. Així doncs, es redueix el model metabòlic a escala genòmica a un model del metabolisme central i es fa servir per a l’anàlisi de fluxos metabòlics basats en 13C en cèl·lules creixent amb glicerol a diferents velocitats de creixement. Els resultats obtinguts són molt consistents amb els cultius previs en glicerol. També s’utilitza iMT1026v3.0 com a suport per a la interpretació del perfil fisiològic obtingut en soques amb el metabolisme redox modificat. Una soca que expressa un fragment d’anticòs es modifica genèticament mitjançant l’expressió d’una NADH quinasa, de manera que el balanç de cofactors redox queda pertorbat. Les soques generades mostren una producció de proteïna recombinant més elevada i una alteració en el perfil macroscòpic de creixement. Mitjançant l’anàlisi in silico dels perfils fisiològics resultants, es prediuen possibles canvis metabòlics associats a l’alteració del balanç de cofactors que estan d’acord amb el perfil macroscòpic observat. Així doncs, en línies generals, en aquest treball es desenvolupa una eina precisa per a l’enginyeria de sistemes metabòlics. A més, és validada en condicions vàries i s’utilitza en dues aplicacions diferents que demostren la seva fiabilitat.<br>Pichia pastoris has become one of the most extensively used platform cell factories for recombinant protein and high-value added metabolite production. In the past recent years, important breakthroughs in the systems-level quantitative analysis of its physiology have been achieved. This wealth of information has allowed the development of genome-scale metabolic models, which make new approaches possible for host cell and bioprocess engineering. Previous to this work, three different genome-scale metabolic models were available for P. pastoris. Nevertheless, these models showed some inconsistencies regarding certain pathways, including the terminology for both metabolites and reactions and annotations. Furthermore, some P. pastoris specific metabolic traits were misrepresented. Therefore, in this study, a consensus genome-scale metabolic model has been developed, thereby integrating the prior models. In addition, a comprehensive revision of metabolic pathways was performed and several pathways were curated and updated according to the currently available literature. As a result, the new model, iMT1026, is able to more accurately reproduce experimental growth parameters using glucose as carbon source and different oxygen availability conditions. In order to expand the capabilities of the consensus model, new physiological datasets of cells growing on two of the most relevant substrates for this cell factory were generated. Specifically, a series of chemostat cultivations were performed to characterise the physiologic profile and macromolecular biomass composition of P. pastoris growing on glycerol and methanol as sole carbon sources. Also, macromolecular biomass composition was analysed, allowing us to incorporate new carbon-source specific stoichiometric biomass equations into the model, as well as to estimate the associated energetic parameters. Overall, a new version of the model (iMT1026 v3.0) was validated for these growing conditions.
In addition to the validation of iMT1026 v3.0 for a wider range of carbon sources and growth conditions, we have further tested its performance in two different applications, namely, the generation of reduced metabolic models suitable for 13C-based metabolic flux analysis and, assisting the interpretation of physiological growth parameters of redox-cofactor engineered strains. In particular, the genome-scale metabolic model has been reduced into a core model and used for 13C-based metabolic flux analysis of cells growing on glycerol at different growth rates. To our knowledge, this is the first study ever reported of 13C-MFA using glycerol as sole carbon source. Notably, flux analyses are highly consistent with pioneering 13C-based metabolic profiling studies of P. pastoris growing on glycerol. iMT1026 v3.0 was also employed for assisting the interpretation of the physiological profiles obtained for redox-cofactor engineered strains. A recombinant strain producing an antibody fragment was engineered to overexpress a heterologous NADH kinase, aiming at increased NADPH regeneration rates. Notably, the redox-engineered strains showed an increase in recombinant protein production and altered macroscopic growing profiles. In silico analysis of the impact of NADH kinase overexpression using the iMT1026 model predicted possible metabolic changes associated to the redox cofactor imbalance that were in agreement with the observed physiological phenotypes.
Overall, a refined tool for systems metabolic engineering is provided in the present study. Moreover, such tool has been validated for a wide range of environmental conditions and employed in two different applications, confirming its reliability.
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Orman, Mehmet Ali. "Extracellular Recombinant Human Growth Hormone Production By Pichia Pastoris." Master's thesis, METU, 2007. http://etd.lib.metu.edu.tr/upload/3/12608616/index.pdf.
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In this study, the effects of bioprocess operation parameters on recombinant human growth hormone (rhGH) production by P. pastoris were systematically investigated. In this frame, first, for the extracellular expression and purification of human growth hormone by recombinant P. pastoris the cDNA of hGH, fused with a polyhistidine tag and also fused with a target site for the Factor Xa protease in which cleavage produces a mature N- and C- termini of rhGH, was cloned into pPICZ&<br>#945<br>A plasmid and the constructed system within the plasmid, pPICZ&<br>#945<br>A::hGH, was integrated to AOX1 locus of P. pastoris and expressed under alcohol oxidase promoter which is induced by methanol. With dot-blot analysis, the appropriate two strains producing human growth hormone at high levels and having different methanol utilization phenotype (Mut+ and Muts) were chosen among the other transformants. Then, the effects of methanol concentrations on the expression of rhGH and cell growth were analyzed and both of the phenotypes were compared in defined and complex media in laboratory scale air filtered shake bioreactors. The highest rhGH concentration for Mut+ and MutS, was found as 0.052 kg m-3 and 0.16 kg m-3, respectively, at 2 %(v/v) methanol concentration in complex medium. When methanol was used as the sole carbon source in defined medium, Muts phenotype had very low specific growth rate on methanol due to the intrinsic characteristics of it, therefore detectable rhGH was not observed, on the other hand, optimum rhGH concentration produced by Mut+ strain was found as 0.032 kg m-3 at 3% (v/v) methanol concentration in defined medium. In mixed system (glycerol/methanol) which is also defined, when the optimum glycerol concentration, 30 kg m-3, was used, Muts produced the highest rhGH, 0.110 kg m-3, at 1% (v/v) methanol concentration and any increase in methanol concentration resulted in lower rhGH production, on the other hand, Mut+ strain produced 0.060 kg m-3 rhGH at 4% (v/v) methanol concentration, which indicated that higher rhGH production capacity of Mut+ strain was obtained at high methanol concentrations.
Using the designed defined medium for Mut+ phenotype where methanol was used as the sole carbon source with an optimum concentration of 3% (v/v), the effects of oxygen transfer on rhGH production, by-product formation, and cell growth, oxygen transfer and fermentation characteristics were investigated by using pilot scale bioreactor. Oxygen transfer effects on rhGH production were investigated at QO/VR=0.5 vvm<br>N=250, 500, 625, 750 min-1 conditions. The variations in rhGH , cell, amino acid and organic acid concentrations with the cell cultivation time, specific cell growth rate, the oxygen uptake rate, the liquid phase coefficient by using the dynamic method, maintenance coefficient for oxygen and yield coefficients were determined. The highest rhGH concentration was obtained at 0.5 vvm, 500 min-1 condition as 0.023 kg m-3 with 5.37 kg m-3 cell density.
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Čiplys, Evaldas. "Žmogaus virusų paviršiaus glikoproteinų ekspresijos tyrimas mielėse Pichia pastoris." Master's thesis, Lithuanian Academic Libraries Network (LABT), 2010. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2007~D_20101125_183218-56493.
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Vienas pagrindinių biomedicininės paskirties baltymų gamybos iššūkių yra pigių ir saugių ekspresijos sistemų, tinkamų glikoproteinų sintezei, paieška bei esamų sistemų tobulinimas. Vaistai, sukurti baltymų pagrindu, sudaro apie ketvirtadalį naujai patvirtinamų vaistų rinkos, o apie 60% jų sudaryti iš glikoproteinų. Dabar glikoproteinų sintezei naudojamos žinduolių kultūros turi keletą trūkumų. Jose gaunamų rekombinantinių baltymų kaina yra didelė, ribotas tūrinis našumas, ląstelės lėtai dauginasi ir auga, būna užkrėstos retrovirusais, gaunamas heterogeniškas produktas ir užima daug laiko sukurti stabilią ląstelių liniją. Itin intensyviai vykstantis tinkamų ekspresijos sistemų kūrimas kol kas nedavė norimų rezultatų. Mielių, kaip ir augalų bei vabzdžių, ekspresijos sistemos, dėl keletos priežasčių yra įvardijamos kaip vienos pagrindinių kandidatų užimti šią vietą. Visų pirma, mielės yra pripažintos kaip saugus organzimas, jų gentika, biochemija ir fiziologija yra gerai ištirta, be to, sėkmingai pradėti kurti rekombinantiniai mielių kamienai su sudėtingu žinduolių tipo N-glikozilinimu. Vis tik mielėse susintetintų glikoproteinų, tinkamų farmacijos pramonei, skaičius yra labai nedidelis, dažniausiai glikoproteinai nebūna tinkamai suvynioti ir modifikuoti, o esmininės priežastys, paaiškinančios mielių trūkumus sintetinant tokio tipo baltymus, nėra išaiškintos. Eukariotų genų inžinerijos laboratorijoje jau yra sukaupta nemaža patirties sintetinant mielėse virusinius glikoproteinus... [toliau žr. visą tekstą]<br>Growing market of the glycoprotein based drugs increases demands of safe, cheap and effective expression systems for production of glycoproteins. Mammalian cell cultures, which are being used for this purpose, are very expensive and ineffective. Yeasts are rising as one of the best alternatives. Well known genetics, biochemistry and physiology are only few advantages. Yeasts are also considered to be safe and easy to manipulate organism. Still, despite introducing humanized glycosylation pathways, yeast based expression systems are not able to produce glycoproteins for pharmaceutics, with a very few exceptions. So, further researches in adapting yeast for glycoproteins synthesis must be made. This work is directed for this purpose. In this work mumps, measles and influenza virus surface glycoproteins were expressed in yeast Pichia pastoris. Results show, that mumps virus hemagliutinin-neuraminidase is not synthesized in P.pastoris. Synthesis of measles virus (MeV) hemagliutinin (H) glycoprotein was not effective, with recombinant protein not possessing characteristics of native analogue. MeV-H was found in two forms: unglycosylated polypeptide precursor and glycosylated form, both aggregated and insoluble in non-ionic detergent. Increase in MeV-H expression level resulted in extensive accumulation of unglycosylated MeV-H protein precursors in the cytoplasm of yeast cells. Addition of S.cerevisiae α-factor secretion signal sequence to globular part of MeV-H made... [to full text]
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Strauss, Colin Earl, and University of Lethbridge Faculty of Arts and Science. "Development of Pichia pastoris as a ruminal escape vehicle." Thesis, Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2000, 2000. http://hdl.handle.net/10133/148.
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The yeast expression system Pichia pastoris was investigated as an encapsulation technology capable of serving as a rumen escape vehicle. Cellularly encapsulated protein is protected from the ruminal environment so long as the cell membrane, which surrounds and isolates the intracellular protein is physically intact. Intracellular expression of Green Fluorescent Protein (GFP) allows for the monitoring of cellular integrity as necessary for the protection of encapsulated protein from ruminal proteases. Upon cellular lysis GFP is exposed to extracellular proteases which result in both the proteolytic degradation of the protein-based GFP chromophore and its associated fluorescence. Visualization of rumen fluid under epifluorescent microscopy revealed a high level of background autofluorescence owing to the fluorescent plant particles, microbes, and fluorescent compounds therein. Visualization of GFP in rumen fluid can be optimized through GFP variant selection, filter set design, and light source selection based on bulb emission spectra. Incubation of intracellular GFP expressing P. pastoris in batch culture ruminal in vitro simulations demonstrated that 93%, 97%, and 25% of the P. pastoris inoculum maintained cellular integrity in clarified rumen fluid, bacterial fraction of rumen fluid, and whole rumen fluid, respectively, when incubated over 36 to 48 h. Continuous fermentation in vitro rumen simulations (Rusitec) demonstrated a P. pastoris escape rate of 19% when added daily to fully adapted Rusitec vessels having a dilution rate of 0.75d-1. Abomasal in vitro simulations demonstrated that 84% of the P. pastoris inoculum was lysed within 12 h, as necessary for the release of encapsulated protein. P.pastoris may be an effective post-fuminal delivery vehicle, provided that similar results are obtained in vivo.<br>xiv, 120 leaves : ill. ; 28 cm.
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Emberson, Louise. "Characterisation of soluble human CD33 expressed in Pichia pastoris." Thesis, University of Kent, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396398.
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Feitosa, Valker Araujo. "Produção de fragmento recombinante de anticorpo em Pichia pastoris." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/9/9134/tde-10062014-084638/.
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Foram estudados a composição e o pH do meio de cultivo para a produção do fragmento de anticorpo (scFv) anti-LDL(-), expresso em Pichia pastoris recombinante. Os experimentos que definiram a composição e pH do meio assim como a concentração inicial de células na fase de indução foram realizados em agitador orbital a 250 rpm, com temperatura de 30 ºC na fase de crescimento e 20 ºC na fase de indução, durante 72 horas, com adição diária de 1% (v/v) de metanol. Para modificação do meio foi realizado um planejamento experimental empregando como variáveis independentes: extrato de soja, casaminoácidos e ureia, os quais substituíram o YNB e a biotina presentes no meio padrão (BMMY). Apesar de haver maior produção no meio com extrato de soja, o meio contendo 10 g.L-1 de casaminoácidos foi selecionado, uma vez que este favoreceu a etapa de purificação. A partir da faixa de pH estudada entre 3,0 e 8,0, determinou-se que o pH 8,0 no início da fase de indução favorece a maior produção. Finalmente, o meio BMMY-CA (pH 8,0) foi utilizado para cultivo em biorreator com volume de trabalho de 10L e a partir deste cultivo foram calculados os parâmetro cinéticos (velocidades de crescimento, de consumo de substrato e de produção, bem como produtividade). Diante do conjunto de experimentos realizados, foi possível otimizar a composição do meio de cultivo e as condições operacionais, que possibilitaram um aumento do rendimento bem como aumento do volume de produção do scFv anti-LDL(-) em biorreator.<br>Composition and pH culture medium for the production of anti- LDL(-) antibody fragment (scFv) were studied in recombinant yeast Pichia pastoris. The experiments that defined medium composition, pH and the initial cell concentration in the induction phase were carried out in baffled shaker flasks at 250 rpm, with temperature at 30°C for growth phase and 20°C for induction phase, during 72 hours with daily addition of 1% (v/v) methanol. A design of experiments employing for medium modification with independent variables: soy extract, casamino acids and urea, which replaced the YNB and biotin present in the standard medium (BMMY) was conducted for medium formulation. Even though, there was an increase in medium production with soy extract, the medium containing 10 g.L-1 casamino acids was selected since it favors the purification step. Through a pH range study (3.0 to 8.0), it was determined that pH 8.0 during induction phase offered higher levels. Then, the best results from shaker flask cultivation (BMMY-CA with casamino acids and pH 8.0) were carried out in 1L bioreactor, showing a biomass and a scFv production increase compared to the standard, BMMY (pH 6.0). Finally, the medium BMMY-CA (pH 8,0) was submitted to a volume scale-up into a 10L bioreactor, which was also used to evaluate kinetic parameters (rates of growth, substrate consumption and production as well as productivity). In conclusion, by optimizing culture medium and operating conditions it was possible to increase yield and scale-up the production of scFv anti-LDL(-) in bioreactor.
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Coleman, Ellen(Ellen M. ). "Establishment of a novel Pichia Pastoris host production platform." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/126948.
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Thesis: M.B.A., Massachusetts Institute of Technology, Sloan School of Management, in conjunction with the Leaders for Global Operations Program at MIT, May, 2020<br>Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, in conjunction with the Leaders for Global Operations Program at MIT, May, 2020<br>Cataloged from the official PDF of thesis.<br>Includes bibliographical references (pages 61-62).<br>The Cell Line Development group at Amgen is responsible for manufacturing and optimizing the cell lines utilized in production of Amgen's biologic drug portfolio. Traditionally, these cell lines are produced from mammalian host organisms, primarily Chinese Hamster Ovary (CHO) cells, due to their unique ability to secrete human-like glycosylated proteins. The CHO platform has undergone significant optimization throughout the industry over the past 30 years, however, productivity and efficiency improvements are now becoming harder to realize. Alternative hosts offer a unique opportunity to drive significant cost of goods improvements throughout the biologic drug manufacturing process. Microbial hosts benefit from low genomic complexity, fast doubling times, and can grow to high cell densities in low-cost media. The yeast strain, Pichia pastoris, combines these advantages with the ability to secrete glycosylated products at equivalent product quality levels as CHO-based processes.<br>The Alternative Host Consortium, an MIT-industry partnership, is focused on the advancement of Pichia and other alternative hosts to eventually drive broader commercial utilization and help curb the rising cost of biologic medicines. This project aimed to quantify the strategic advantage of the Pichia host in Amgen's pipeline, and determine when, why and how such a product would be manufactured. The first segment of the work presented here includes various bioprocess development experiments performed to establish proof-of-concept protein production data in Pichia. The results show successful production of two relatively simple proteins at concentrations similar to existing published results. Additionally, chemically defined media and controlled fed-batch fermentation experiments were run to better mimic manufacturing scale operations. The second segment of the project focused on quantifying the strategic cost advantage of the Pichia platform compared with CHO.<br>The business case analysis centered on potential raw material and plant time savings to determine the critical Pichia process features required to be cost competitive.<br>by Ellen Coleman.<br>M.B.A.<br>S.M.<br>M.B.A. Massachusetts Institute of Technology, Sloan School of Management<br>S.M. Massachusetts Institute of Technology, Department of Mechanical Engineering
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Johnson, Sabrina D. "Characterization of the Pichia pastoris alcohol oxidase I promoter." Scholarly Commons, 2003. https://scholarlycommons.pacific.edu/uop_etds/575.
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The methylotrophic yeast, Pichia past oris, is one of the most respected and widely used systems today. The ability of this yeast to produce large masses of protein and metabolize methanol as a sole source of carbon and energy is attributed to the highly induceable Alcohol Oxidase I promoter (AOXI). Despite of the disperse popularity and use of this promoter over the last 15 years, little is known about the transcription controls at a molecular level. A 5'>3' deletion analysis of the AOXI promoter was perrormed to gain understanding of the promoter's regulation and provided insight to the approximate locations of the important regulatory regions. A total of 10 truncations were made unveiling two areas ofhigh activity located between positions, -257 to-235, and, -235 to -188. In addition, a 14-base pair internal deletion was made between positions, -215 to -201. This region was shown to be necessary for transcriptional activation by deletion analysis. Sufficiency studies suggested that this 14-base pair element could serve as an activator sequence in both glucose and methanol.
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Perry, Evan T. "Expression of Human Neutrophil Cathepsin G in Pichia pastoris." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/honors/225.
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Cathepsin G (CatG), a serine protease found in the azurophil granules of neutrophils, participates in killing engulfed microorganisms. CatG is a poorly understood enzyme, in part because it can only be obtained as mature enzyme purified from human blood, and because it seems to have dual specificity for chymotrypsin-like and trypsin-like substrates. Therefore, yeast Pichia pastoris was used to express immature recombinant human CatG to provide a source of the enzyme free of biohazards and to allow study of its dual specificity and C-terminal processing. To avoid potential cleavage by a yeast kexin protease, the amino sequence was modified without altering CatG’s biological activity to remove one glycosylation site and eight dibasic sites. An N-terminal 6-His-cytochrome B5 (CytB5) heme binding fusion domain was linked to the modified human CatG by an enteropeptidase cleavage site for activation. The DNA for this construct was codon-optimized and placed in the pPICzα secretion vector. After transforming P. pastoris strain X-33, 48 Zeocin-resistant clones were screened for relative levels of CatG activity following activation by recombinant human enteropeptidase. Recombinant human CatG was partially purified from fermentation medium by nickel affinity chromatography and its activity was confirmed by assays using the substrate Succinyl-Ala-Ala-Pro-Phe-SBzl. Supported by a Student Faculty Collaborative Grant from the ETSU Honors College and ETSU Office of Research and Sponsored Programs and by NHLB grant R15HL091770.
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Zeferino, Isabela Alves de Melo. "Identificação de compostos inibidores presentes nos hidrolisados hemicelulósicos de biomassa vegetal e seus efeitos sobre a produção de etanol por Pichia stipitis." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/97/97131/tde-14092016-162722/.
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O presente estudo teve como objetivo identificar os principais compostos inibidores presentes nos hidrolisados hemicelulósicos de palha de arroz (HHPA) e bagaço de malte (HHBM), assim como avaliar o perfil fermentativo de Pichia stipitis em meio semissintético contendo os compostos previamente identificados. Foi também objetivo do trabalho avaliar a fermentação da levedura em HHPA e HHBM não destoxificados, de forma a se obter informações sobre o grau de toxicidade destes meios. Após identificação dos inibidores, incluindo ácido acético, furfural, 5-hidroximetilfurfural, vanilina, ácido ferúlico e ácido p-cumárico, foram realizados ensaios fermentativos em meio semissintético variando-se a concentração de cada composto. Os resultados mostraram que, para todos os níveis de ácido acético avaliados (1,5 a 4,5 g.L-1), o fator de conversão de substrato em etanol (YP/S) foi menos afetado do que a produtividade volumétrica em etanol (QP), sendo que para os meios contendo o maior nível deste ácido os valores de YP/S e QP foram reduzidos em 25 e 77%, respectivamente, em relação ao controle. Dentre os furanos (furfural, de 12 a 84 mg.L-1 e hidroximetilfurfural, de 218 a 553 mg.L-1), o hidroximetilfurfural na concentração de 553 mg.L-1 foi o que apresentou a maior redução nos valores de YP/S (_30%) e QP (_50%) em relação ao controle. Para os fenólicos (vanilina, siringaldeído, ácido ferúlico e ácido pcumárico), o maior impacto sobre o metabolismo fermentativo de P stipitis foi observado com o siringaldeído visto que, em todos os níveis avaliados (90 a 620 mg.L-1), os parâmetros fermentativos foram afetados. Os resultados também demonstraram que, com exceção do ácido p-cumárico, a levedura foi capaz de assimilar todos os inibidores presentes no meio e que o perfil de assimilação variou com o tipo e concentração do composto. A vanilina e os furanos foram completamente assimilados em todos os níveis estudados, enquanto que o ácido acético foi totalmente assimilado pela levedura apenas na menor concentração. Nas fermentações realizadas em HHPA e HHBM não destoxificados, os resultados mostraram que a produção de etanol foi similar ao meio semissintético sem inibidores, porém em tempos mais elevados. Além disso, em HHBM o valor de YP/S foi ligeiramente superior (_10%) ao observado no HHPA, o que pode ser atribuído às variações na composição nutricional de cada hidrolisado. O perfil fermentativo da levedura em ambos os hidrolisados mostrou uma cinética mais lenta quando comparado aos meios semissintéticos mimetizando a composição destes, o que sugere a existência de outras substâncias tóxicas nos hidrolisados além das avaliadas neste estudo. Com base nos resultados obtidos no presente estudo pode-se concluir que, dentre os inibidores identificados nos hidrolisados, o ácido acético foi o composto de maior potencial inibitório, seguido pelos furanos e fenólicos, sendo o efeito destes compostos mais pronunciado na produtividade volumétrica em etanol do que no fator de conversão de substrato em etanol. Estas informações são de fundamental importância na elaboração de estratégias de destoxificação e/ou condução do processo visando à melhoria da fermentação em hidrolisados.<br>The aim of this study was to identify the major inhibitory compounds present in rice straw hemicellulose hydrolysate (RSHH) and brewer\'s spent grain hemicellulose hydrolysate (BSGHH), as well as evaluate Pichia stipitis fermentation profile in semi-synthetic medium containing the previously identified compounds. It was also purpose of this study to evaluate the fermentation of RSHH and BSGHH without detoxification, in order to obtain information about the degree of toxicity of these media. After inhibitors identification, including acetic acid, furfural, 5- hydroxymethylfurfural, vanillin, ferulic acid and p-coumaric acid, fermentation assays were carried out on semi-synthetic medium by varying the concentration of each compound. The results showed that, for all evaluated levels of acetic acid (from 1.5 to 4.5 g.L-1), the ethanol yield (YP/S) was less affected than the ethanol volumetric productivity (QP), and for the media containing the higher evaluated level, the values of YP/S and QP were reduced by 25 and 77%, respectively, compared to control. Among furans (furfural, from 12 to 84 mg.L-1 and hydroxymethylfurfural, from 218 to 553 mg.L-1), the hydroxymethylfurfural at 553 mg.L-1 showed the highest reduction in YP/S (~30%) and QP (~50%) compared to control. For the phenolic compounds (vanillin, syringaldehyde, ferulic acid and pcoumaric acid), the greatest impact on the Pichia stipitis fermentative metabolism was observed with syringaldehyde since the fermentative parameters were affected at all evaluated levels (from 90 to 620 mg.L-1). The results also showed that, except for the p-coumaric acid, the yeast was able to assimilate all inhibitory compunds in the medium and that the assimilation profile varied with the type and concentration of these compounds. Vanillin and furans were completely assimilated at all studied levels, whereas acetic acid was completely assimilated only at the lowest concentration. The ethanol production in RSHH and BSGHH was similar to that observed in semi-synthetic medium without inhibitors, but in a longer period of time. In addition, the YP/S value obtained in BSGHH was slightly higher (~10%) than that observed in RSHH, which can be attributed to variations in the nutritional composition of each hydrolysate. The yeast fermentation profile in both hydrolysates showed slower kinetics when compared to semi-synthetic medium containing the same inhibitors, which suggests the existence of other toxic substances in the hydrolysate in addition to those evaluated in this study. Based on the results obtained in this study, it can be concluded that, among the inhibitors identified in hydrolysates, acetic acid was the main inhibitory compound, followed by furans and phenolics. The most pronounced effect of these compounds was in ethanol volumetric productivity rather than in the ethanol yield. This information is of fundamental importance in the development of detoxification and/or process conduction strategies aimed at improving the fermentation in hemicellulosic hydrolysates.
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Neto, AntÃnio Viana Lopes. "Atividade fungistÃtica de uma quitinase recombinante do feijÃo de corda [Vigna unguiculata (L.) (Walp.)] contra Lasiodiplodia theobromae Pat. (Griff. e Maubl.), agente causal da resinose do cajueiro (Anacardium occidentale L.)." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=13310.
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CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior<br>O objetivo deste trabalho foi avaliar a atividade biolÃgica de uma quitinase recombinante (rVuChi) de feijÃo-caupi (Vigna unguiculata) contra o fungo fitopatogÃnico Lasiodiplodia theobromae. A proteÃna recombinante foi expressa em Pichia pastoris, coletada e purificada apÃs 72h de induÃÃo, utilizando cromatografia de afinidade em matriz de quitina. A quitinase foi eluÃda a partir da cromatografia de afinidade com Ãcido acÃtico a 0,1 M. Ensaio enzimÃtico foi realizado contra o substrato sintÃtico (quitina coloidal), a fim de determinar a atividade da proteÃna recombinante purificada. A quitinase apresentou atividade especÃfica de 5.637,32 U/mg de proteÃna. Testes biolÃgicos foram realizados. Nestes testes trÃs diferentes isolados de L. theobromae, identificados como CNPAT CCJ-127, CNPAT CCJ-166 e CNPAT CCJ-184, foram utilizados e os experimentos foram realizados em triplicata. Os isolados fÃngicos foram obtidos da coleÃÃo de trabalho do LaboratÃrio de Fitopatologia da Embrapa AgroindÃstria Tropical (Fortaleza-CE, Brasil). Em todos os ensaios biolÃgicos o fungicida Carbomax 500 SC (Carbendazim), a uma concentraÃÃo de 2 mL/L, e Ãgua destilada estÃril foram utilizados como controles positivos e negativos, respectivamente. Um total de 50, 100 e 300 Âg de quitinase recombinante (rVuChi) foi utilizado em todos os testes. O primeiro ensaio foi baseado na metodologia de difusÃo em disco de papel de filtro em que foram investigados os efeitos da proteÃna sobre o crescimento do micÃlio, bem como a formaÃÃo de halo de inibiÃÃo sobre o crescimento micelial do fungo. O segundo ensaio foi baseado no ensaio de difusÃo em Ãgar. Fotografias foram usadas para registrar as observaÃÃes. A quitinase rVuChi mostrou efeito fungistÃtico variando de moderado a forte sobre o crescimento micelial de todos os isolados de L. theobromae, particularmente quando usada nas doses de 100 e 300 Âg, no ensaio de difusÃo em disco. CNPAT CCJ-127 foi o isolado mais resistente à aÃÃo fungistÃtica de rVuChi, como observado pelo menor impacto da proteÃna em seu crescimento micelial. No teste de difusÃo em Ãgar a quantidade de 300 Âg foi a mais efetiva, da mesma forma como observado para o de difusÃo em disco de papel de filtro. AlÃm disso, o efeito da proteÃna foi mais pronunciado nos isolados CNPAT CCJ-166 e CNPAT CCJ-184 e menos impactante no isolado CNPAT CCJ-127. A quitinase recombinante rVuCHi mostrou ser um inibidor do crescimento micelial de trÃs diferentes isolados de L. theobromae. Os efeitos fungistÃticos da proteÃna aqui descritos podem ser devido à sua capacidade de degradar quitina, um biopolÃmero estrutural que faz parte da parede celular de vÃrios fungos fitopatogÃnicos, incluindo L. theobromae. Entretanto, mais estudos precisam ser conduzidos para revelar os possÃveis mecanismos de aÃÃo de rVuChi sobre L. theobromae.<br>The aim of this work was to evaluate the biological activity of a recombinant chitinase (rVuChi) from cowpea (Vigna unguiculata) against the phytopathogenic fungus Lasiodiplodia theobromae. The recombinant protein was expressed in Pichia pastoris, collected and purified after 72h of induction, using a chitin affinity chromatography. The chitinase was eluted from the affinity chromatography using 0.1 M acetic acid. Enzymatic assay was performed against the synthetic substrate (colloidal chitin) in order to determine the activity of the purified recombinant protein. The chitinase displayed a specific activity of 5,637.32 U/mg of protein. Biological tests were performed. In these tests three different isolates of L. theobromae, identified as CNPAT CCJ-127, CNPAT CCJ-166 and CNPAT CCJ-184, were used and the experiments were performed on triplicate. The fungal isolates were obtained from the collection of work from the laboratory of plant pathology from the Embrapa AgroindÃstria Tropical (Fortaleza-CE, Brasil). In all biological assays the fungicide Carbomax 500 SC (Carbendazim) at a concentration of 2 mL/L and sterile distilled water were used as positive and negative controls, respectively. A total of 50, 100 and 300 Âg of recombinant chitinase (rVuChi) was used in all tests. The first test was based on the disk diffusion methodology using filter paper in which the effects of the protein on the mycelium growth, as well as the formation of an inhibition zone on the fungal hyphae were investigated. The second test was based on the diffusion assay in agar. Photographs were used to register the observations. The rVuChi showed moderate to strong fungistatic activities on the mycelial growth of all L. theobromae isolates when used at 100 and 300 Âg in the disk diffusion assay. CNPAT CCJ-127 was the most resistant specimen to the rVuChi fungistatic action, as observed by the lower impact of the protein on it is mycelial growth. In the agar diffusion test the amount of 300 Âg was the most effective, as observed in the disk diffusion test. In addition, the effect of the protein was most pronounced on the isolates CNPAT CCJ-166 and CNPAT CCJ-184 and less impacting on CNPAT CCJ-127. The recombinant chitinase rVuCHi showed to be an inhibitor of the mycelial growth of three L. theobromae isolates. The fungistatic effects of the protein described here may be due to its ability to degrade chitin, a structural biopolymer that makes part of the cell wall of several phytopathogenic fungi, including L. theobromae. Once this is only a scientific speculation, more studies need to be made to definitely reveal the mechanism of action of rVuChi on L. theobromae.
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Branco, Juliana Inês. "Avaliação da imunogenicidade de diferentes formas alélicas da proteína recombinante PvAMA-1expressa em Pichia pastoris: impacto da diversidade antigênica." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/9/9142/tde-12112018-145334/.
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A malária é um problema de saúde pública no Brasil e no mundo. Em 2016, o número de casos estimado pela Organização Mundial de Saúde foi de 216 milhões. Plasmodium falciparum é a espécie mais prevalente e responsável pelo maior número de mortes no mundo, sobretudo no continente africano. Por outro lado, o Plasmodium vivax é conhecido por sua ampla distribuição geográfica, sendo a espécie que predomina nas Américas, incluindo o Brasil. Nos últimos 20 anos, nosso grupo tem gerado e caracterizado diversas proteínas recombinantes baseadas em antígenos imunodominantes de P. vivax que podem servir como base para o desenvolvimento de uma vacina contra malária. Entre os antígenos de merozoítas, uma das principais proteínas em estudo pelo nosso grupo é o Antígeno 1 de Membrana Apical de P. vivax (PvAMA-1), caracterizado previamente como altamente imunogênico em infecções naturais e em camundongos imunizados, na presença de diferentes adjuvantes. O objetivo do presente estudo foi investigar o efeito da diversidade antigênica dessa proteína no reconhecimento por anticorpos específicos e na indução de imunidade contra o parasita. Para isso, foram geradas seis novas proteínas representando diferentes alelos descritos na natureza: PvAMA-1-Belem, PvAMA-1-Sal-I, PvAMA-1-Chesson-I, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX e PvAMA-1-PNG_62_MU. As proteínas recombinantes foram expressas em leveduras Pichia pastoris e purificadas em duas etapas cromatográficas. Em seguida, as imunizações em camundongos C57BL/6 foram realizadas com as proteínas administradas de forma isolada, ou em combinação, na presença do adjuvante agonista de TLR3 (Poly I:C). Por ELISA, observamos que todas as formulações foram capazes de induzir anticorpos IgG contra as proteínas homólogas e heterólogas, o que sugere que a diversidade antigênica entre as formas alélicas não compromete o reconhecimento. Os dados gerados no presente trabalho sugerem que uma formulação contendo mistura de diferentes alelos representando a proteína AMA-1 pode ser explorada para o desenvolvimento de uma vacina de ampla cobertura contra o P. vivax.<br>Malaria is a public health problem in Brazil and throughout the world. In 2016, the World Health Organization estimated there were 216 million cases of malaria. Plasmodium falciparum is the most prevalent species and is responsible for the largest number of deaths, especially in the African continent. However, Plasmodium vivax is known for its wide geographic distribution, being the species that prevails in the Americas, including Brazil. In the last 20 years, our group has generated and characterized several recombinant proteins based on immunodominant antigens of P. vivax that can serve as a basis for the development of a malaria vaccine. Among the merozoite antigens, one of the main proteins studied by our group is P. vivax apical membrane antigen-1 (PvAMA-1), previously characterized as highly immunogenic in natural infections and immunized mice, in the presence of different adjuvants. The objective of this study was to investigate the effect of antigenic diversity of this protein in the recognition of specific antibodies and the induction of immunity against the parasite. For this, six new proteins were generated representing different alleles described in nature: PvAMA-1-Belem, PvAMA-1-Sal-i, PvAMA-1-Chesson-i, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX, and PvAMA-1-PNG_62_MU. Recombinant proteins were expressed in Pichia pastoris yeast and purified by two chromatographic stages. Then, C57BL/6 mice were immunized with these proteins administered in isolation or in combination, in the presence of the TLR3 agonist adjuvant, Poly I:C. Using an enzyme-linked immunosorbent assay, we observed that all formulations induced IgG antibodies against homologous and heterologous proteins. This indicates that antigenic diversity between allele forms does not compromise recognition. This finding suggests that a formulation containing a mixture of different alleles representing the PvAMA-1 protein can be exploited for developing of a wide coverage vaccine against P. vivax.
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STORCH, Otávio Brod. "Avaliação como probióticos para frangos de corte de Pichia pastoris e Pichia pastoris recombinante contendo o gene da fosfolipase C de Clostridium perfringens." Universidade Federal de Pelotas, 2008. http://repositorio.ufpel.edu.br/handle/ri/2527.
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Previous issue date: 2008-11-17<br>Antibiotics used for the last decades as growth promoters for animals, were banned
in several countries because of the risk of inducing the appearance of antibiotic
resistant bacteria. Probiotics, live microorganisms that benefit health promoting the
stability of the gut microbiota, are their most promising substitutes. The objective of
this research was to determine the probiotic properties of Pichia pastoris and a
recombinant P. pastoris containing the Clostridium perfringens fosfolipase C gene.
Ross® P8 female chicks were randomly distributed in four groups of ten each, in
three repeats, and fed with a commercial food devoid of antibacterials. Group 1 was
fed with un-supplemented food; group 2 was supplemented with 1x106 viable P.
pastoris strain KM71H gr-1; group 3 with 1x106 viable recombinant P. pastoris
containing the C. perfringens fosfolipase C gene gr-1 and group 4 with 1x106 viable
spores of Bacillus cereus var. Toyoi gr-1. Water and food were supplied ad libitum.
Weight gain of each animal and food conversion of each group at 49 days of age,
were estimated. Individual seroconversions against C. perfringens α toxin at days 1,
10, 20, 30 and 49 were estimated. At the end of the experiment the animals were
slaughtered and samples from different organs examined histologically. Mean live
weights at 49 days of age were 2.172, 2.228, 2.410 and 2.333 Kg for groups 1, 2, 3
and 4, respectively, different at P<0.05. Mean food conversions were 2.58, 2.41,
2.35 and 2.50 for groups 1, 2, 3 and 4, respectively. Seroconversions at 49 days of
age were 1.1, 1.4, 1.5 and 1.3 for groups 1, 2, 3 and 4, respectively, different at
P<0.05. Histological alterations were not detected. It was concluded that P. pastoris
and recombinant P. pastoris may be used as probiotics for broilers due to their
capacity of increasing food efficiency and seroconversion without undesirable
effects.<br>Os antibióticos utilizados como promotores de crescimento nas últimas décadas
foram banidos em vários países devido ao risco de induzir o surgimento de
bactérias resistentes. Entre as alternativas para sua substituição, os probióticos,
suplementos alimentares compostos de microrganismos vivos que beneficiam a
saúde do hospedeiro através do equilíbrio da microbiota intestinal, aparecem como
a mais plausível. Este trabalho objetivou determinar as propriedades como
probiótico da levedura Pichia pastoris e sua variante recombinante contendo o
gene da fosfolipase C de Clostridium perfringens. Frangas de um dia de idade da
linhagem Ross® P8 foram distribuídas aleatoriamente em quatro grupos de dez
animais cada, em três repetições, e alimentadas com ração comercial isenta de
antibacterianos. O grupo 1 (Controle) recebeu ração não suplementada, o grupo 2
recebeu a mesma ração suplementada com 1x106 células viáveis gr-1 de P. pastoris
cepa KM71H, o grupo 3 ração suplementada com 1x106 gr-1 de células viáveis de
P. pastoris recombinante contendo o gene da toxina α de C. perfringens, e o grupo
4 ração suplementada com 1x106 gr-1 de esporos viáveis de Bacillus cereus var.
Toyoi. Ração e água foram oferecidos ad libitum. Estimou-se o ganho de peso de
cada animal aos 49 dias de idade, e a conversão alimentar de cada grupo.
Determinou-se a soroconversão individual por ELISA, utilizando como antígeno a
toxina α padrão de C. perfringens, a partir de amostras de sangue coletadas aos 1,
10, 20, 30 e 49 dias de idade. Ao término do experimento os animais foram
abatidos e amostras de órgãos submetidos a análise histopatológica. No dia 49, os
pesos vivos médios foram 2,172, 2,228, 2,410 e 2,333 kg, significativamente
diferentes (P<0,05) ao grupo controle, para os grupos 1, 2, 3 e 4, respectivamente.
As conversões alimentares médias foram 2,58, 2,41, 2,35 e 2,5 para os grupos 1,
2, 3 e 4, respectivamente. As soroconversões ao dia 49 foram 1,1, 1,4, 1,5 e 1,3
para os grupos 1, 2, 3 e 4, respectivamente, significativamente diferentes (P<0,05)
ao grupo controle. Nos estudos histopatológicos não foram encontradas alterações.
Concluiu-se que P. pastoris e P. pastoris recombinante podem ser utilizadas como
probiótico em frangos de corte por aumentar a eficiência alimentar e a
soroconversão, sem apresentar contraindicações.
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Tomailla, Tenazoa Juan Víctor. "Estudio comparativo de la diversidad de peces en áreas forestadas y deforestadas en quebradas del alto Río Pichis, Oxapampa (Pasco)." Bachelor's thesis, Universidad Nacional Mayor de San Marcos, 2010. https://hdl.handle.net/20.500.12672/891.
Full textAbstract:
Las áreas deforestadas influyen sobre el clima, sistema hídrico y en el caudal de los ríos, además la erosión por la pérdida de la cobertura vegetal, ocasiona mayor carga de sedimentos en los ríos originan inundaciones más acentuadas en época lluviosa y niveles más bajos en época seca. También, la deforestación de zonas ribereñas influye sobre el número y tipo de organismos que habitan en los arroyos adyacentes, sin embargo, existe poca información. Con la finalidad de comparar la diversidad de peces en áreas naturales y deforestadas se realizó un estudio en tres quebradas del alto Río Pichis, Oxapampa, Pasco, con dos estaciones por quebrada (con y sin vegetación ribereña). Se realizaron dos muestreos: octubre 2005 y mayo 2006. Se registraron parámetros físico-químicos y características de hábitat, fotografías y se realizó pesca con redes de arrastre hacia la orilla. Se capturaron en total 3653 peces, provenientes de 12 muestras y seis estaciones de muestreo. Se identificaron 40 especies, en 12 familias y cinco órdenes. La mayor abundancia fue registrada en las estaciones deforestadas al igual que los mayores registros de riqueza. La diversidad no mostró variaciones marcadas entre los dos tipos de estaciones. El orden Characiformes fue el más abundante y registró la mayor riqueza de peces en todas las estaciones, destacando la familia Characidae. Los gráficos de la ordenación NMDS y los dendogramas formulados a partir de los índices de Similitud de Bray-Curtis, muestran una mayor similitud de cada tipo de hábitat que entre ellos. El mayor número de peces y especies en los tramos evaluados de los lugares deforestados con bancos de herbáceas en las orillas podría deberse a la presencia de una mayor heterogeneidad de hábitats, asimismo la remoción de bosque ribereño produjo algunos cambios en la estructura comunitaria de peces.
-- Palabras Claves: Amazonía peruana, estructura comunitaria, diversidad de especies, deforestación ribereña, peces<br>-- The deforested areas could modified the weather and the hydrographic system, and the quantity of water of the rivers, then follow the increase of the erosion as result of the vegetation removed and showing a major amount of the sediments in the rivers producing higher flooding during the rainy season and lowest levels during the dry season. Besides, the deforestation of the reverie zones has direct effect on the number and type of the organisms which are living along the related streams. However, there is a lack of quantitative information about the deforestation related to the aquatic fauna affected. With the objective to compare the fish diversity in natural areas and deforested areas one study was carried out along the three streams at the Alto Río Pichis, Oxapampa, Pasco, sampling in two stations by stream (with or without reverie vegetation). The sampling was made in: october 2005 and may 2006. It was recorded the phisic-chemical parameters and habitat characteristics, photography and the fishing was made with seines to the borders. It was collected 3653 fishes, from 12 samples and six sampling stations. Were identified 40 species, belonging 12 families and five orders. The major abundant was recorded at the deforested stations and the same the higher values of richness. The diversity did not show remarked variations between two kinds of stations. The order Characiforms was the most abundant and recorded the higher value of richness en every station, especially Characidae family. The graphics of ordination NMDS and dendograms formulated since the Similarity of Bray-Curtis index, show us a major similarity of every kind of habitat than between both of them. The major number of fishes and species in the evaluated sectors of deforested areas with grasses banks could be related to a major heterogeneity of habitats, it selves to shore wood remotion made several changes in the community structure of fishes.
-- Key Words: Peruvian Amazon, community structure, fishes, riparian deforestation, species diversity.<br>Tesis
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Moy, Allison. "Comparison of zeocin and G418 resistance markers in Pichia pastoris." Scholarly Commons, 2009. https://scholarlycommons.pacific.edu/uop_etds/736.
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Wanas, Essam A. "Cloning and expression of the glycoproteins of pichinde virus by vaccinia virus." Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/6489.
Full textAbstract:
Pichinde virus (Pic), like the other arenaviruses, possesses two glycoproteins, GP1 and GP2, that are derived by proteolytic cleavage from a precursor molecule, GPC. Within the arenaviruses, GP1 is the most heterogeneous protein, and GP1 of Pic differs from that of the other arenaviruses in having twice as many potential N-linked glycosylation sites, most of which appear to be utilized. In order to examine the effects of this heavy glycosylation on Pic GP1 structure and inmunogenicity, GPC of Pic was cloned and expressed in vaccinia virus. The recombinant vaccinia (vvGPC) expresses authentic Pic GPC as demonstrated by immunoprecipitation with MAb and several polyclonal anti-Pic sera. GPC expressed in vaccinia is fully glycosylated as it comigrates with Pic GPC. At the same time, sequence analysis of cDNA shows both nucleotide and amino acid changes compared to published sequences for Pic GPC, indicating that variation in the same strain of this virus occurs as virus is passaged in separate laboratories. Experiments to assess the ability of Pic GPC expressed in vaccinia to elicit anti-Pic antibody show that rabbit anti-vvGPC detects authentic Pic GPC and GP1. Site-directed mutagenesis was employed to remove a potential N-linked glycosylation site (aa 181-183) in Pic GPC. Attempts were made to produce recombinant vaccinia, vvGPC-183, harbouring the mutant Pic GPC.
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Maassen, Nicole. "Gewinnung von Gärungsmutanten der Hefe Pichia stipitis durch zufällige Integrationsmutagenese." [S.l.] : [s.n.], 2007. http://deposit.ddb.de/cgi-bin/dokserv?idn=984566716.
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Monforte, Mercado Sergi. "Systems metabolic engineering for recombinant protein production in Pichia pastoris." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/669385.
Full textAbstract:
El llevat metilotròfic Pichia pastoris (Komagataella sp.) és un dels sistemes d’expressió més atractius per a la producció de proteïna recombinant, mercat contínuament en expansió. El fort promotor del gen de l’alcohol oxidasa 1 (PAOX1), induït per metanol però reprimit per glucosa, glicerol o etanol, és un dels més emprats per aquest propòsit. No obstant, existeixen encara diversos colls d’ampolla fisiològics que limitant el procés.
En aquest context, diferents estratègies han estat proposades i provades per tal de millorar la producció heteròloga de molts tipus diferents de proteïna. Les aproximacions més habituals inclouen increment en el nombre de còpies de gen heteròleg, enginyeria de promotors i modificació dels mecanismes de plegament i secreció. L’objectiu d’aquesta tesi ha estat el desenvolupament de noves estratègies per incrementar la producció de proteïna recombinant, emprant la lipasa de Rhizopus oryzae (Rol) com a proteïna model en el sistema d’expressió basat en el PAOX1.
Primerament, els gens del factors de transcripció de PAOX1, MXR1 i MIT1, es van sobreexpressar constitutivament per tal de millorar la transcripció de ROL. Això es va confirmar degut a una millora en la capacitat assimilatòria de metanol i un increment en els nivells relatius de mRNA de ROL i varis gens relacionats amb el metabolisme del metanol, i.e. revertint l’efecte de titulació causat per la transcripció de múltiples cassettes d’expressió de ROL. Tot i aquestes millores, els nivells extracel·lulars d’activitat lipàsica no van augmentar de forma significativa en cultius en quimiòstat, apuntant a colls d’ampolla addicionals limitant la producció de Rol.
En segon lloc, es van explorar possibles dianes d’enginyeria metabòlica en el metabolisme cel·lular de P. pastoris emprant el model metabòlic a escola genoma (GEM) consens iMT1026 v3.0. Aquest pas in silico va proporcionar diversos knock-outs prometedors que serien experimentalment testats fent servir el sistema d’edició genòmica CRISPR/Cas9. Les simulacions apuntaven a la disponibilitat de NADPH i una limitada aportació de determinats aminoàcids (serina i cisteïna) com a potencials factors limitants de la producció de Rol. Una reducció en el fitnes cel·lular que afecta a la viabilitat de les soques que es buscaven obtenir va impedir la verificació de la majoria dels knock-outs proposats.
Finalment, donat que les nostres anàlisis i estudis prèviament publicats identificaven el NADPH com un cofactor important limitant la producció de proteïna recombinant, els nostres esforços es van dirigir a incrementar la seva disponibilitat a través d’estratègies de knock-in de gens. Específicament, vam sobreexpressar dos gens que codificaven per enzims redox, una NADH quinasa i una NADH oxidasa, amb l’objectiu de pertorbar directament l’equilibri redox de la cèl·lula. A més, es va comprovar l’efecte fisiològic d’aquests enzims fent servir diferents mescles co-substrat/metanol com a font de carboni. En resum, vam observar un increment en la producció de proteïna recombinant amb diferents graus de millora depenent de la font de carboni provada. També vam realitzar anàlisis transcriptòmiques i una avaluació in silico dels nostres resultats per tal de presentar una interpretació millor de l’estat fisiològic de la cèl·lula. Dins del nostre coneixement, aquest és el primer estudi dirigit a incrementar la generació de NADPH en un sistema d’expressió basat en PAOX1, en condicions de creixement en metanol.
A grans trets, noves estratègies d’enginyeria de soques han estat proposades i provades durant l’execució d’aquest estudi. A més a més, s’han aplicat GEMs i aproximacions relacionades amb biologia de sistemes, demostrant que són eines potents i prometedores per al disseny racional d’organismes industrials.<br>The methylotrophic yeast Pichia pastoris (Komagataella sp.) is one of the most attractive expression systems for heterologous protein production, which constitutes a continuously expanding market. The strong alcohol oxidase gene 1 promoter (PAOX1), induced by methanol but repressed by glucose, glycerol or ethanol, is one of the most used for this purpose. Nevertheless, there still exist several physiological bottlenecks limiting the process.
In this context, several strategies have been proposed and tested in order to improve the heterologous production of many different types of proteins. Common approaches include increasing heterologous gene copy number, promoter engineering and modification of the folding and secretory mechanisms. The aim of this thesis has been the development of new strategies to increase recombinant protein yields, using the Rhizopus oryzae lipase (Rol) as model protein in a PAOX1-based expression system.
Firstly, the PAOX1 transcription factor genes MXR1 and MIT1 were constitutively overexpressed aiming at improving ROL transcription. This was confirmed by an improved methanol assimilation capacity and an increase in relative mRNA levels of ROL and several genes related with methanol metabolism, i.e. reverting the titration effect caused by the transcription of multiple ROL expression cassettes. Despite such improvements, extracellular lipase activity levels did not increase significantly in chemostat cultures, pointing out to additional bottlenecks limiting Rol production.
Second, possible metabolic engineering targets in P. pastoris’ cell metabolism were explored using the consensus genome-scale metabolic model (GEM) iMT1026 v3.0. This in silico step provided several promising knock-outs which were going to be experimentally tested using the CRISPR/Cas9 genome editing system. The simulations pointed to NADPH availability and limited supply of some amino acids (serine and cysteine) as potential Rol production limiting factors. A reduction in cell fitness affecting the viability of the obtained strains impeded to verify most of the proposed knock-outs.
Finally, since our in silico analyses and previously published studies identified NADPH as an important limiting cofactor in recombinant protein production, our efforts were geared towards increasing its availability through gene knock-in strategies. Specifically, we overexpressed two genes encoding redox enzymes, a NADH kinase and a NADH oxidase, with the aim to directly perturb the cell’s redox balance. Further, we tested the physiological effect of these enzymes using different co-substrate/methanol mixtures as carbon source. In short, we observed an increase in recombinant protein production with different degrees of improvement depending on the carbon source(s) tested. We also performed a transcriptomic analysis and an in silico evaluation of our results in order to provide a better interpretation of the cell physiological state. To our knowledge, this is the first study aiming to increase NADPH generation in the PAOX1-based expression system, under methanol growth conditions.
Overall, novel strain engineering strategies have been proposed and tested during the execution of this study. Furthermore, GEMs and related systems biology approaches were applied, proving to be promising powerful tools for rational engineering of industrial microorganisms.
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Cornelissen, Gesine. "Integrierte Bioprozessentwicklung zur Herstellung pharmakologischer wirksamer Proteine mit Pichia pastoris /." Düsseldorf : VDI-Verl, 2004. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=012990697&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Panagiotou, Vasiliki. "Clonal selection and characterization of epigenetic variation in Pichia pastoris." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/59881.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2010.<br>Page 71 blank. Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 67-70).<br>Recombinant proteins produced by different host organisms have been broadly used as therapeutics. Considering the demand for large quantities of protein drugs, methods are needed to increase reactor titers in a timely and cost-effective manner. We used random chemical mutagenesis to modify a wild-type strain of the heterologous protein production host Pichia pastoris, which resulted in overall improvement of the secretion rate of the mutated population. More than 4000 single-cells were simultaneously screened for high secretion of a human Fc fragment using microengraving and the top-producing clones were retrieved. Future characterization of these improved clones by transcript profiling should yield information about networks of genes central in heterologous protein secretion in the yeast P. pastoris.<br>by Vasiliki Panagiotou.<br>S.M.
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Thor, Der. "Cloning and characterization of MET2 in Pichia pastoris : a thesis." Scholarly Commons, 2001. https://scholarlycommons.pacific.edu/uop_etds/570.
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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.