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Id: biblio-974317
Autor: Zepeda, Andrea B; Pessoa Jr, Adalberto; Farías, Jorge G.
Título: Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast
Fonte: Braz. j. microbiol;49(supl.1):119-127, 2018. tab, graf.
Idioma: en.
Projeto: Comisión Nacional de Investigación Científica y Tecnológica de Chile; . AZP; . FAPESP.
Resumo: Abstract Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.
Descritores: Pichia/genética
Proteínas Recombinantes/biossíntese
Proteínas Recombinantes/genética
Carbono/metabolismo
Regiões Promotoras Genéticas
-Pichia/crescimento & desenvolvimento
Pichia/metabolismo
Temperatura Ambiente
Microbiologia Industrial
Responsável: BR1.1 - BIREME


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Id: biblio-974294
Autor: Zepeda, Andrea B; Figueroa, Carolina A; Pessoa, Adalberto; Farías, Jorge G.
Título: Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris
Fonte: Braz. j. microbiol;49(4):856-864, Oct.-Dec. 2018. graf.
Idioma: en.
Projeto: FAPESP.
Resumo: ABSTRACT The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 ºC + 1%M provided the most stable viability. By contrast, the temperature of 18 ºC + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.
Descritores: Pichia/metabolismo
Ácidos Graxos não Esterificados/metabolismo
-Pichia/crescimento & desenvolvimento
Pichia/genética
Temperatura Ambiente
Proteínas Recombinantes/genética
Meios de Cultura/metabolismo
Meios de Cultura/química
Metanol/metabolismo
Fermentação
Glicerol/metabolismo
Responsável: BR1.1 - BIREME


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Id: biblio-839389
Autor: Eissazadeh, Samira; Moeini, Hassan; Dezfouli, Maryam Ghandizadeh; Heidary, Somayyeh; Nelofer, Rubina; Abdullah, Mohd Puad.
Título: Production of recombinant human epidermal growth factor in Pichia pastoris
Fonte: Braz. j. microbiol;48(2):286-293, April.-June 2017. tab, graf.
Idioma: en.
Resumo: Abstract This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27 µg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60 h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.
Descritores: Pichia/metabolismo
Proteínas Recombinantes/metabolismo
Fator de Crescimento Epidérmico/metabolismo
-Pichia/genética
Proteínas Recombinantes/genética
Ensaio de Imunoadsorção Enzimática
Expressão Gênica
Clonagem Molecular
Meios de Cultura/química
Fator de Crescimento Epidérmico/genética
Fermentação
Concentração de Íons de Hidrogênio
Limites: Seres Humanos
Responsável: BR1.1 - BIREME


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Id: biblio-1015723
Autor: Wang, Jiayi; Lu, Lei; Feng, Fujuan.
Título: Combined strategies for improving production of a thermo-alkali stable laccase in Pichia pastoris
Fonte: Electron. j. biotechnol;28:7-13, July. 2017. tab, graf, ilus.
Idioma: en.
Projeto: National Natural Science Foundation of China; . Fundamental Research Funds for the Central Universities.
Resumo: Background: Laccases are copper-containing enzymes which have been used as green biocatalysts for many industrial processes. Although bacterial laccases have high stabilities which facilitate their application under harsh conditions, their activities and production yields are usually very low. In this work, we attempt to use a combinatorial strategy, including site-directed mutagenesis, codon and cultivation optimization, for improving the productivity of a thermo-alkali stable bacterial laccase in Pichia pastoris. Results: A D500G mutant of Bacillus licheniformis LS04 laccase, which was constructed by site-directed mutagenesis, demonstrated 2.1-fold higher activity when expressed in P. pastoris. The D500G variant retained similar catalytic characteristics to the wild-type laccase, and could efficiently decolorize synthetic dyes at alkaline conditions. Various cultivation factors such as medium components, pH and temperature were investigated for their effects on laccase expression. After cultivation optimization, a laccase activity of 347 ± 7 U/L was finally achieved for D500G after 3 d of induction, which was about 9.3 times higher than that of wild-type enzyme. The protein yield under the optimized conditions was about 59 mg/L for D500G. Conclusions: The productivity of the thermo-alkali stable laccase from B. licheniformis expressed in P. pastoris was significantly improved through the combination of site-directed mutagenesis and optimization of the cultivation process. The mutant enzyme retains good stability under high temperature and alkaline conditions, and is a good candidate for industrial application in dye decolorization.
Descritores: Pichia/metabolismo
Lacase/biossíntese
Lacase/genética
Bacillus licheniformis/enzimologia
-Temperatura Ambiente
Leveduras
Estabilidade Enzimática
Catálise
Mutagênese
Lacase/metabolismo
Corantes/metabolismo
Concentração de Íons de Hidrogênio
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-1022638
Autor: Wang, Junying; Li, Yu; Lu, Fuping.
Título: Molecular cloning and biochemical characterization of an α-amylase family from Aspergillus niger
Fonte: Electron. j. biotechnol;32:55-62, Mar. 2018. tab, ilus, graf.
Idioma: en.
Projeto: International Collaborative Project Supported by National Natural Science Foundation of China; . (NSFC); . National Research Foundation of South Africa; . National High Technology Research and Development Program; . Program for Changjiang Scholars and Innovative Research Team in University; . Tianjin Key Lab of Industrial Microbiology Tianjin University of Science & Technology.
Resumo: Background: α-Amylase is widely used in the starch processing, food and paper industries, hydrolyzing starch, glycogen and other polysaccharides into glucose, maltose and oligosaccharides. An α-amylase gene family from Aspergillus niger CBS513.88 encode eight putative α-amylases. The differences and similarities, biochemical properties and functional diversity among these eight α-amylases remain unknown. Results: The eight genes were cloned and expressed in Pichia pastoris GS115 by shaking-flask fermentation under the induction of methanol. The sequence alignment, biochemical characterizations and product analysis of starch hydrolysis by these α-amylases were investigated. It is found that the eight α-amylases belonged to three different groups with the typical structure of fungal α-amylase. They exhibited maximal activities at 30­40°C except AmyG and were all stable at acidic pH. Ca2+ and EDTA had no effects on the activities of α-amylases except AmyF and AmyH, indicating that the six amylases were Ca2+ independent. Two novel α-amylases of AmyE and AmyF were found. AmyE hydrolyzed starch into maltose, maltotriose and a small amount of glucose, while AmyF hydrolyzed starch into mainly glucose. The excellent physical and chemical properties including high acidic stability, Ca2+-independent and high maltotriose-forming capacity make AmyE suitable in food and sugar syrup industries. Conclusions: This study illustrates that a gene family can encode multiple enzymes members having remarkable differences in biochemical properties. It provides not only new insights into evolution and functional divergence among different members of an α-amylase family, but the development of new enzymes for industrial application.
Descritores: Aspergillus niger/enzimologia
alfa-Amilases/genética
alfa-Amilases/química
-Pichia/metabolismo
Amido
Temperatura Ambiente
Indústria Alimentícia
Clonagem Molecular
Fermentação
Concentração de Íons de Hidrogênio
Hidrólise
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-889144
Autor: Techaparin, Atiya; Thanonkeo, Pornthap; Klanrit, Preekamol.
Título: High-temperature ethanol production using thermotolerant yeast newly isolated from Greater Mekong Subregion
Fonte: Braz. j. microbiol;48(3):461-475, July-Sept. 2017. tab, graf.
Idioma: en.
Projeto: National Research University Project of Thailand.
Resumo: Abstract The application of high-potential thermotolerant yeasts is a key factor for successful ethanol production at high temperatures. Two hundred and thirty-four yeast isolates from Greater Mekong Subregion (GMS) countries, i.e., Thailand, The Lao People's Democratic Republic (Lao PDR) and Vietnam were obtained. Five thermotolerant yeasts, designated Saccharomyces cerevisiae KKU-VN8, KKU-VN20, and KKU-VN27, Pichia kudriavzevii KKU-TH33 and P. kudriavzevii KKU-TH43, demonstrated high temperature and ethanol tolerance levels up to 45 °C and 13% (v/v), respectively. All five strains produced higher ethanol concentrations and exhibited greater productivities and yields than the industrial strain S. cerevisiae TISTR5606 during high-temperature fermentation at 40 °C and 43 °C. S. cerevisiae KKU-VN8 demonstrated the best performance for ethanol production from glucose at 37 °C with an ethanol concentration of 72.69 g/L, a productivity of 1.59 g/L/h and a theoretical ethanol yield of 86.27%. The optimal conditions for ethanol production of S. cerevisiae KKU-VN8 from sweet sorghum juice (SSJ) at 40 °C were achieved using the Box-Behnken experimental design (BBD). The maximal ethanol concentration obtained during fermentation was 89.32 g/L, with a productivity of 2.48 g/L/h and a theoretical ethanol yield of 96.32%. Thus, the newly isolated thermotolerant S. cerevisiae KKU-VN8 exhibits a great potential for commercial-scale ethanol production in the future.
Descritores: Pichia/metabolismo
Saccharomyces cerevisiae/metabolismo
Etanol/metabolismo
-Pichia/isolamento & purificação
Pichia/genética
Pichia/química
Ásia Sudeste
Saccharomyces cerevisiae/isolamento & purificação
Saccharomyces cerevisiae/genética
Saccharomyces cerevisiae/química
Sorghum/metabolismo
Glucose/metabolismo
Temperatura Alta
Responsável: BR1.1 - BIREME


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Id: biblio-889135
Autor: Arias, Cesar Andres Diaz; Marques, Daniela de Araujo Viana; Malpiedi, Luciana Pellegrini; Maranhão, Andrea Queiroz; Parra, Dulcineia Abdalla Saes; Converti, Attilio; Pessoa Junior, Adalberto.
Título: Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source
Fonte: Braz. j. microbiol;48(3):419-426, July-Sept. 2017. tab, graf.
Idioma: en.
Projeto: CAPES.
Resumo: Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.
Descritores: Pichia/metabolismo
Microbiologia Industrial/métodos
Carbono/metabolismo
Anticorpos de Cadeia Única/biossíntese
Anticorpos/metabolismo
-Pichia/crescimento & desenvolvimento
Pichia/genética
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Meios de Cultura/metabolismo
Meios de Cultura/química
Anticorpos de Cadeia Única/genética
Fermentação
Glicerol/metabolismo
Lipoproteínas LDL/imunologia
Anticorpos/genética
Tipo de Publ: Estudos de Avaliação
Responsável: BR1.1 - BIREME


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Id: biblio-1024757
Autor: Biasoto, Henrique Pellin.
Título: Expressão da L-asparaginase II de Saccharomyces cerevisiae recombinante extracelular com glicosilação humanizada em Pichia pastoris / Extracellular expression of Saccharomyces cerevisiae's L-asparaginase II in Pichia pastoris with humanized glycosylation.
Fonte: São Paulo; s.n; 2019. 94 p. graf, tab.
Idioma: pt.
Tese: Apresentada a Universidade de São Paulo. Faculdade de Ciências Farmacêuticas para obtenção do grau de Mestre.
Resumo: L-asparaginase é um inibidor eficiente do crescimento tumoral, usado em sessões de quimioterapia contra a Leucemia Linfoblástica Aguda (LLA), resultando na remissão completa da doença em 90% dos pacientes tratados. A L-asparaginase II de Saccharomyces cerevisiae (ScASNaseII) tem alto potencial de superar os efeitos adversos da L-asparaginase de bactéria, porém sua produção endógena resulta em uma proteína hipermanosilada e, consequentemente, imunogênica. A cepa de Pichia pastoris Glycoswitch tem a maquinaria para expressar e secretar altas quantidades de enzima com glicosilação humanizada. Nesse trabalho, descrevemos o processo genético para expressar a ScASNaseII no meio extracelular pela P. pastoris Glycoswitch, e também os parâmetros bioquímicos, perfil cinético, citotoxicidade contra células leucêmicas e a interferência da glicosilação na atividade da enzima obtida. Nossos dados mostram que a cepa aplicada foi capaz de expressar ScASNaseII no meio extracelular passível de purificação de proteínas contaminantes com apenas um passo cromatográfico. A atividade específica para asparagina foi 218,2 UI/mg e a atividade glutaminásica representou 3,1% da atividade asparaginásica. Os parâmetros cinéticos foram KM = 120,5 µM e a eficiência catalítica de 3,8 x 105 M-1s-1. Análises por meio de gel nativo sugerem uma conformação tetramérica de aproximadamente 150 kDa. Essa é uma nova estratégia de produzir essa enzima de forma extracelular, com mais facilidade de purificação e com melhores propriedades biotecnológicas

L-asparaginase is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cell; its use results in 90% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to overcome the side effects of bacteria L-asparaginase, but the endogenous production of it results in hypermannosylated immunogenic enzyme. However, Pichia pastoris Glycoswitch strain has the machinery to express and secrete high quantity of the enzyme and with humanized glycosylation. Here we describe the genetic process to acquire the ScASNaseII in the extracellular medium expressed by P. pastoris Glycoswitch, and the biochemical properties of the resultant enzyme, kinetic profile, cytotoxicity against ALL cell line and the interference of glycosylation in its activity. Our data show that the strain employed is able to express extracellular asparaginase active and possible to be purified of contaminant proteins using a single chromatographic step. The specific activity using asparagine was 218.2 IU.mg-1 and the glutaminase activity represents 3.1% of its asparaginase activity. The kinetics parameters were KM=120.5 µM and a catalytic efficiency of 3.8x105 M-1s-1. The Native-PAGE suggested a tetrameric protein conformation, with approximately 150 kDa. This is a novel strategy to produce this enzyme extracellularly, easier to purify and with better biotechnological properties
Descritores: Pichia/isolamento & purificação
Asparaginase/análise
Saccharomyces cerevisiae/isolamento & purificação
-Glicosilação
Proteínas Recombinantes
Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico
Responsável: BR40.1 - DBD - Divisão de Biblioteca e Documentacão do Conjunto das Químicas
BR40.1; T620.8, B579e. 30100022628-F


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Id: biblio-1008418
Autor: Martínez, Duniesky; Menéndez, Carmen; Hernández, Lázaro; Sobrino, Alina; Trujillo, Luis E; Rodríguez, Ivan; Pérez, Enrique R.
Título: Scaling-up batch conditions for efficient sucrose hydrolysis catalyzed by an immobilized recombinant Pichia pastoris cells in a stirrer tank reactor
Fonte: Electron. j. biotechnol;25:39-42, ene. 2017. tab, graf.
Idioma: en.
Resumo: Background: Invert sugar is used greatly in food and pharmaceutical industries. This paper describes scaling-up batch conditions for sucrose inversion catalyzed by the recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase entrapped in calcium alginate beads. For the first time, we describe the application of a kinetic model to predict the fractional conversion expected during sucrose hydrolysis reaction in both, a model and a prototype bioreactor with 0.5- and 5-L working volume, respectively. Results: Different scaled-up criteria used to operate the 0.5-L bioreactor were analyzed to explore the invert sugar large scale production. After model inversion studies, a 5-L scaled-up reaction system was performed in a 7-L stirred reactor. Both scaled-up criteria, immobilized biocatalyst dosage and stirring speed, were analyzed in each type of bioreactors and the collected data were used to ensure an efficient scale-up of this biocatalyst. Conclusions: To date, there is not enough information to describe the large-scale production of invert sugar using different scaled-up criteria such as dose of immobilized biocatalyst and stirring speed effect on mass transfer. The present study results constitute a valuable tool to successfully carry out this type of high-scale operation for industrial purposes.
Descritores: Pichia/metabolismo
Sacarose/metabolismo
Biotecnologia/métodos
-Pichia/citologia
Sacarose/química
Cinética
Reatores Biológicos
Thermotoga maritima/enzimologia
Alginatos
Enzimas Imobilizadas
Biocatálise
Hidrólise
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-1015357
Autor: Barros, Tácita Borges.
Título: Expressão e caracterização da glicoproteína D do HSV-1 geneticamente fusionada às oncoproteínas E6 e E7 do HPV-16 e HPV-18 (gDE7E6) em células de mamífero / Expression and characterization of the genetically fused HSV-1 glycoprotein D to E6 and E7 oncoproteins HPV-16 e HPV-18 (gDE7E6) in mammalian cells.
Fonte: São Paulo; s.n; 2019. 88 p. graf.
Idioma: pt.
Tese: Apresentada a Universidade de São Paulo. Faculdade de Ciências Farmacêuticas para obtenção do grau de Mestre.
Resumo: O câncer cervical é um dos tipos de câncer mais comuns entre as mulheres, e a infecção persistente pelos HPV-16 e HPV-18 é responsável por 70% dos casos. As vacinas profiláticas disponíveis possuem alta eficácia na prevenção da infecção pelos tipos mais prevalentes de HPV. No entanto, este tipo de abordagem não beneficia mulheres que já apresentam lesões precursoras ou tumores cervicais avançados, e a busca por abordagens terapêuticas para esse tipo de câncer é considerada uma necessidade. A qualidade do antígeno representa um aspecto fundamental para o sucesso de vacinas terapêuticas baseadas em proteínas recombinantes. Neste sentido, os sistemas de expressão em células eucarióticas, como leveduras e células de mamíferos são considerados adequados para a produção de proteínas com aplicação biotecnológica. O objetivo principal deste trabalho contemplou a expressão das proteínas de fusão gDE7E6 do HPV-16 e do HPV-18 e a oncoproteína E7 do HPV-16 em células da levedura Pichia pastoris e expressão da gDE7E6 do HPV-16 e do HPV-18 em células de mamífero HEK293T e CHODG-44 para obtenção de antígenos purificados com futura aplicação em vacinas terapêuticas contra tumores associados ao HPV-16 e HPV-18. Os genes que codificam as proteínas gDE7E6 dos HPV-16 e HPV-18 e da E7 do HPV-16 foram clonados no vetor pPIC9K, os quais foram linearizados por digestão enzimática e utilizados na transformação da P. pastoris. A expressão das proteínas foi analisada nos tempos de 24, 48, 72 e 96 horas, no entanto, não foi observada a produção das proteínas no sobrenadante e nem no lisado celular. Diante desta constatação, iniciamos a expressão das proteínas gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células de mamíferos HEK293T e CHODG-44. As sequências genéticas das proteínas gDE7E6 do HPV-16 e do HPV-18 foram clonadas no vetor de expressão pNU1 e analisadas por digestão enzimática. Análises de SDS-PAGE e western blot demonstraram a expressão das proteínas gDE7E6 do HPV-16 e do HPV-18 em até 96 horas em células HEK293T. Em paralelo, realizamos a transfecção estável dos plasmídeos contendo as sequencias da gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células CHO-DG44. Com o intuito de aumentar a expressão das proteínas de interesse na população mista de CHODG-44, realizamos amplificação genômica com metotrexato (MTX), sendo possível observar aumento da expressão das proteínas, conforme aumento gradativo nas concentrações de MTX. Posteriormente, foram feitas tentativas para isolar um clone produtor das proteínas gDE7E6 HPV-16 e HPV-18, através de clonagem por diluição limitante e sistema automatizado, sendo possível isolar um clone para cada construção através de matriz semisólida, confirmado por western blot e citometria de fluxo. Apesar de demonstrar a expressão das proteínas de interesse em sistema de expressão baseado em células de mamífero, o rendimento obtido após a purificação por afinidade ao níquel foi extremamente baixo, o que dificulta a obtenção dos antígenos para fins vacinais

Cervical cancer is one of the most common cancers among women, and persistent infection with HPV-16 and HPV-18 accounts for 70% of the cases. Available prophylactic vaccines are highly effective in preventing infection by the most prevalent types of HPV. However, this type of approach does not benefit women who already have precursor lesions or advanced cervical tumors, and the search for therapeutic approaches to this type of cancer is considered a necessity. Antigen quality represents a key aspect for the success of therapeutic vaccines based on recombinant proteins. In this sense, expression systems based in eukaryotic cells such as yeast and mammalian cells are considered suitable for the production of proteins with biotechnological applications. The main objective of this work was to express the gDE7E6 fusion proteins HPV-16 and HPV-18 and the E7 oncoprotein HPV-16 in Pichia pastoris and expression of gDE7E6 HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44 to obtain purified antigens with future applications in therapeutic vaccines against HPV-16 and HPV-18 associated tumors. The genes encoding the gDE7E6 proteins HPV-16 and HPV-18 and E7 HPV-16 were cloned into the pPIC9K vector, which were linearized by enzymatic digestion and used in the transformation of P. pastoris. Expression of the proteins was analyzed at 24, 48, 72 and 96 hours, however, the production of the proteins in the supernatant and in the cell lysate was not observed. In light of this finding, we initiated the expression of gDE7E6 proteins HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44. The genetic sequences of gDE7E6 proteins HPV-16 and HPV-18 were cloned into the pNU1 expression vector and analyzed by enzymatic digestion. SDSPAGE and western blot analyzes demonstrated expression of gDE7E6 proteins HPV-16 and HPV-18 within 96 hours in HEK293T cells. In parallel, we performed stable transfection of plasmids containing gDE7E6 HPV-16 and HPV-18 sequences into CHODG44 cells. In order to increase the expression of the proteins in the mixed population of CHODG-44, we performed genomic amplification with methotrexate (MTX), and it was possible to observe an increase in protein expression, as a gradual increase in MTX concentrations. Therefore, attempts were made to isolate a clone producing gDE7E6 proteins HPV-16 and HPV-18 by limiting dilution and automated system, being possible to isolate one clone for each construct through a semisolid matrix, confirmed by western blot and flow cytometry. Despite observing protein expression in mammalian cell-based expression system, the yield obtained after nickel affinity purification was extremely low, which makes it difficult to obtain the antigens for vaccine purposes
Descritores: Proteínas Oncogênicas/classificação
Papillomavirus Humano 16
Papillomavirus Humano 18
-Pichia
Neoplasias do Colo do Útero/fisiopatologia
Herpesvirus Humano 1
Eucariotos
Antígenos/análise
Responsável: BR40.1 - DBD - Divisão de Biblioteca e Documentacão do Conjunto das Químicas



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BIREME/OPAS/OMS - Centro Latino-Americano e do Caribe de Informação em Ciências da Saúde