Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo
Autor(a) principal: | |
---|---|
Data de Publicação: | 2018 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/9858 |
Resumo: | One of the main challenges in the therapeutic proteins production using Escherichia coli is to obtain a high purity product. Among the main contaminants (nucleic acids, polysaccharides and contaminating proteins) lipopolysaccharides deserve special attention, since its inflammatory action can lead to septic shock in mammals and for this reason its removal is crucial. Recently a E. coli genetically modified strain capable of producing recombinant proteins with low endotoxic response became commercially available and it is known as ClearColi. In this context, the purpose of the present study was to evaluate the pneumococcal surface protein A fragment (PspA) production in ClearColi as well as the growth and the physiological responses under different growth and induction conditions. The experiments were performed on shaker using a chemically defined medium (HDF) with glycerol as the main carbon source and lactose and IPTG, as inducers, at 37, 32 and 27 oC, using the factorial 22 design method. From the samples were determined the concentrations of biomass (DO600nm), nucleic acids (Abs260nm), polysaccharides (phenol-sulfuric method), lipopolysaccharides (KDO method) and proteins such as PspA and the contaminants (Bradford and electrophoresis followed by densitometry). Similar experiments were performed with a conventional production platform of this recombinant protein, based on E. coli BL21 (DE3), for comparison. In addition, an experiment using a stirred bioreactor was performed in the best conditions identified in the preliminary experiments on shaker. Thus, as results of the shaker experiments, ClearColi maximum specific growth rate was found to be about 25% smaller than the achieve with conventional E. coli. The highest PspA specific production was achieved using the inducer mixture at 32 ° C, being 165 ± 3 mg/gDCW for ClearColi and 221 ± 13 mg/gDCW for E. coli BL21. It was also found that the production of contaminating proteins was not influenced by any of the conditions evaluated for ClearColi, whereas for E. coli BL21 only at 32oC it was lower. The production of nucleic acids, polysaccharides and lipopolysaccharides occurred mainly during the exponential phase, for most of the studied conditions, therefore, their minimization depends on the choice of culture conditions during the exponential phase. From the bioreactor culture data a biomass yield of 0.63 gDCW/Lh and a protein yield of 81.2 mgPspA/Lh were estimated. The yields achieved with ClearColi were about 64% lower than those obtained with BL21, due to the longer cultivation time required with ClearColi. However, the detoxified strain has the advantage of achieving high PspA specific production values, as well as a low production of contaminating proteins when compared to the conventional strain. In addition, the detoxified strain produces a modified LPS, which does not cause significant pyrogenic reaction. Thus, the present study demonstrated that the endotoxin-free strain use as a platform for the production of recombinant proteins in industrial scale is technically possible and presents itself as a very interesting alternative for therapeutic proteins. |
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Paredes, Sheyla Alexandra HidalgoZangirolami, Teresa Cristinahttp://lattes.cnpq.br/4546701843297248Gonçalves, Viviane Maimonihttp://lattes.cnpq.br/6520430298584189http://lattes.cnpq.br/2465175082064817730a9fbb-708d-433d-8f2b-66314b068b172018-05-03T13:18:32Z2018-05-03T13:18:32Z2018-03-02PAREDES, Sheyla Alexandra Hidalgo. Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo. 2018. Dissertação (Mestrado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2018. Disponível em: https://repositorio.ufscar.br/handle/ufscar/9858.https://repositorio.ufscar.br/handle/ufscar/9858One of the main challenges in the therapeutic proteins production using Escherichia coli is to obtain a high purity product. Among the main contaminants (nucleic acids, polysaccharides and contaminating proteins) lipopolysaccharides deserve special attention, since its inflammatory action can lead to septic shock in mammals and for this reason its removal is crucial. Recently a E. coli genetically modified strain capable of producing recombinant proteins with low endotoxic response became commercially available and it is known as ClearColi. In this context, the purpose of the present study was to evaluate the pneumococcal surface protein A fragment (PspA) production in ClearColi as well as the growth and the physiological responses under different growth and induction conditions. The experiments were performed on shaker using a chemically defined medium (HDF) with glycerol as the main carbon source and lactose and IPTG, as inducers, at 37, 32 and 27 oC, using the factorial 22 design method. From the samples were determined the concentrations of biomass (DO600nm), nucleic acids (Abs260nm), polysaccharides (phenol-sulfuric method), lipopolysaccharides (KDO method) and proteins such as PspA and the contaminants (Bradford and electrophoresis followed by densitometry). Similar experiments were performed with a conventional production platform of this recombinant protein, based on E. coli BL21 (DE3), for comparison. In addition, an experiment using a stirred bioreactor was performed in the best conditions identified in the preliminary experiments on shaker. Thus, as results of the shaker experiments, ClearColi maximum specific growth rate was found to be about 25% smaller than the achieve with conventional E. coli. The highest PspA specific production was achieved using the inducer mixture at 32 ° C, being 165 ± 3 mg/gDCW for ClearColi and 221 ± 13 mg/gDCW for E. coli BL21. It was also found that the production of contaminating proteins was not influenced by any of the conditions evaluated for ClearColi, whereas for E. coli BL21 only at 32oC it was lower. The production of nucleic acids, polysaccharides and lipopolysaccharides occurred mainly during the exponential phase, for most of the studied conditions, therefore, their minimization depends on the choice of culture conditions during the exponential phase. From the bioreactor culture data a biomass yield of 0.63 gDCW/Lh and a protein yield of 81.2 mgPspA/Lh were estimated. The yields achieved with ClearColi were about 64% lower than those obtained with BL21, due to the longer cultivation time required with ClearColi. However, the detoxified strain has the advantage of achieving high PspA specific production values, as well as a low production of contaminating proteins when compared to the conventional strain. In addition, the detoxified strain produces a modified LPS, which does not cause significant pyrogenic reaction. Thus, the present study demonstrated that the endotoxin-free strain use as a platform for the production of recombinant proteins in industrial scale is technically possible and presents itself as a very interesting alternative for therapeutic proteins.Um dos principais desafios na produção de proteínas terapêuticas por Escherichia coli é a obtenção de um produto de alta pureza. Dentre os principais contaminantes (ácidos nucléicos, polissacarídeos e proteínas contaminantes) os lipopolissacarídeos merecem especial atenção, uma vez que possuem ação inflamatória e podem levar ao choque séptico em mamíferos, por esta razão a sua remoção é crucial. Recentemente tornou-se disponível comercialmente a ClearColi, linhagem de E. coli capaz de produzir proteínas recombinantes com reduzida atividade endotóxica. Neste contexto, o objetivo do presente trabalho foi avaliar a produção do fragmento de proteína A de superfície do pneumococo (PspA) em ClearColi, o crescimento e as respostas fisiológicas dessas células, sob diferentes condições de crescimento e indução. Experimentos em shaker foram realizados com meio definido (HDF) contendo glicerol como fonte de carbono, utilizou-se como indutores lactose e IPTG, nas temperaturas de 37, 32 e 27 oC, de acordo com a metodologia de planejamento fatorial 22. Foram determinadas as concentrações de biomassa (DO600nm), ácidos nucléicos (Abs260nm), polissacarídeos (método fenol-sulfúrico), lipopolissacarídeos (método KDO) e proteínas, tanto a PspA quanto as contaminantes (Bradford e eletroforese seguida de densitometria). Experimentos semelhantes foram realizados com a plataforma convencional de produção dessa proteína recombinante, baseada em E. coli BL21 (DE3), para comparação. Também foi realizado um cultivo com ClearColi em biorreator tipo tanque agitado, em batelada, empregando a melhor condição identificada nos experimentos em shaker. Por meio dos experimentos em shaker verificou-se que a velocidade específica máxima de crescimento da ClearColi foi cerca de 25 % menor do que a observada para a E. coli convencional. A maior produção específica de PspA foi alcançada na temperatura de 32 oC com a mistura de IPTG e lactose, como indutores, sendo 165 ± 3 mg/gMS com ClearColi e 221 ± 13 mg/gMS com E. coli BL21. Verificou-se também que a produção de proteínas contaminantes não foi influenciada por nenhuma das condições avaliadas para ClearColi, enquanto para E. coli BL21 foi menor a 32oC. Em relação à formação de ácidos nucléicos, polissacarídeos e lipopolissacarídeos, observou-se que são produzidos majoritariamente durante a fase exponencial, para a maioria das condições estudadas, portanto, a sua minimização depende da escolha de condições de cultivo durante a fase exponencial. A partir dos dados do biorreator, obteve-se a produtividade em biomassa de 0,63 gMS/Lh e em proteína de interesse de 81,2 mgPspA/Lh. As produtividades alcançadas com a ClearColi são cerca de 64 % inferiores às obtidas com a BL21 devido ao maior tempo requerido para o cultivo com ClearColi. No entanto, a linhagem detoxificada apresentou como vantagem altos valores de produção específica de PspA e uma baixa produção de proteínas contaminantes, quando comparada a linhagem convencional. Além disso a ClearColi produz LPS modificados que não provocam reação pirogênica significativa. Assim, o presente estudo demonstrou que o emprego da cepa detoxificada como plataforma de produção de proteínas recombinantes em escala industrial é tecnicamente possível e apresenta-se como uma alternativa bastante interessante para a produção de proteínas terapêuticas.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP: 2015/10.291-8porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Engenharia Química - PPGEQUFSCarClearColiE. coli detoxificadaPspAProteína contaminanteLipopolissacarídeosInduçãoTemperaturaEndotoxin-free E. coliLipopolysaccharidesInductionENGENHARIAS::ENGENHARIA QUIMICAEscherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococoEndotoxin-free Escherichia coli as production platform of pneumococcal surface recombinant protein Ainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisOnline4c81169f-86ab-4df0-8284-9cb6516960a4info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARLICENSElicense.txtlicense.txttext/plain; charset=utf-81957https://repositorio.ufscar.br/bitstream/ufscar/9858/4/license.txtae0398b6f8b235e40ad82cba6c50031dMD54ORIGINALPAREDES_Sheyla_2018.pdfPAREDES_Sheyla_2018.pdfapplication/pdf2297157https://repositorio.ufscar.br/bitstream/ufscar/9858/5/PAREDES_Sheyla_2018.pdfc9066c2059f27983d81a65db638c9ad4MD55TEXTPAREDES_Sheyla_2018.pdf.txtPAREDES_Sheyla_2018.pdf.txtExtracted texttext/plain185647https://repositorio.ufscar.br/bitstream/ufscar/9858/6/PAREDES_Sheyla_2018.pdf.txtae034968b609228191f7abbf02121dd1MD56THUMBNAILPAREDES_Sheyla_2018.pdf.jpgPAREDES_Sheyla_2018.pdf.jpgIM Thumbnailimage/jpeg7386https://repositorio.ufscar.br/bitstream/ufscar/9858/7/PAREDES_Sheyla_2018.pdf.jpg770abbf2852a65d4359fb50a38a17f1eMD57ufscar/98582023-09-18 18:31:14.087oai:repositorio.ufscar.br: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Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:14Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo |
dc.title.alternative.eng.fl_str_mv |
Endotoxin-free Escherichia coli as production platform of pneumococcal surface recombinant protein A |
title |
Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo |
spellingShingle |
Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo Paredes, Sheyla Alexandra Hidalgo ClearColi E. coli detoxificada PspA Proteína contaminante Lipopolissacarídeos Indução Temperatura Endotoxin-free E. coli Lipopolysaccharides Induction ENGENHARIAS::ENGENHARIA QUIMICA |
title_short |
Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo |
title_full |
Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo |
title_fullStr |
Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo |
title_full_unstemmed |
Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo |
title_sort |
Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo |
author |
Paredes, Sheyla Alexandra Hidalgo |
author_facet |
Paredes, Sheyla Alexandra Hidalgo |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/2465175082064817 |
dc.contributor.author.fl_str_mv |
Paredes, Sheyla Alexandra Hidalgo |
dc.contributor.advisor1.fl_str_mv |
Zangirolami, Teresa Cristina |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/4546701843297248 |
dc.contributor.advisor-co1.fl_str_mv |
Gonçalves, Viviane Maimoni |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/6520430298584189 |
dc.contributor.authorID.fl_str_mv |
730a9fbb-708d-433d-8f2b-66314b068b17 |
contributor_str_mv |
Zangirolami, Teresa Cristina Gonçalves, Viviane Maimoni |
dc.subject.por.fl_str_mv |
ClearColi E. coli detoxificada PspA Proteína contaminante Lipopolissacarídeos Indução Temperatura |
topic |
ClearColi E. coli detoxificada PspA Proteína contaminante Lipopolissacarídeos Indução Temperatura Endotoxin-free E. coli Lipopolysaccharides Induction ENGENHARIAS::ENGENHARIA QUIMICA |
dc.subject.eng.fl_str_mv |
Endotoxin-free E. coli Lipopolysaccharides Induction |
dc.subject.cnpq.fl_str_mv |
ENGENHARIAS::ENGENHARIA QUIMICA |
description |
One of the main challenges in the therapeutic proteins production using Escherichia coli is to obtain a high purity product. Among the main contaminants (nucleic acids, polysaccharides and contaminating proteins) lipopolysaccharides deserve special attention, since its inflammatory action can lead to septic shock in mammals and for this reason its removal is crucial. Recently a E. coli genetically modified strain capable of producing recombinant proteins with low endotoxic response became commercially available and it is known as ClearColi. In this context, the purpose of the present study was to evaluate the pneumococcal surface protein A fragment (PspA) production in ClearColi as well as the growth and the physiological responses under different growth and induction conditions. The experiments were performed on shaker using a chemically defined medium (HDF) with glycerol as the main carbon source and lactose and IPTG, as inducers, at 37, 32 and 27 oC, using the factorial 22 design method. From the samples were determined the concentrations of biomass (DO600nm), nucleic acids (Abs260nm), polysaccharides (phenol-sulfuric method), lipopolysaccharides (KDO method) and proteins such as PspA and the contaminants (Bradford and electrophoresis followed by densitometry). Similar experiments were performed with a conventional production platform of this recombinant protein, based on E. coli BL21 (DE3), for comparison. In addition, an experiment using a stirred bioreactor was performed in the best conditions identified in the preliminary experiments on shaker. Thus, as results of the shaker experiments, ClearColi maximum specific growth rate was found to be about 25% smaller than the achieve with conventional E. coli. The highest PspA specific production was achieved using the inducer mixture at 32 ° C, being 165 ± 3 mg/gDCW for ClearColi and 221 ± 13 mg/gDCW for E. coli BL21. It was also found that the production of contaminating proteins was not influenced by any of the conditions evaluated for ClearColi, whereas for E. coli BL21 only at 32oC it was lower. The production of nucleic acids, polysaccharides and lipopolysaccharides occurred mainly during the exponential phase, for most of the studied conditions, therefore, their minimization depends on the choice of culture conditions during the exponential phase. From the bioreactor culture data a biomass yield of 0.63 gDCW/Lh and a protein yield of 81.2 mgPspA/Lh were estimated. The yields achieved with ClearColi were about 64% lower than those obtained with BL21, due to the longer cultivation time required with ClearColi. However, the detoxified strain has the advantage of achieving high PspA specific production values, as well as a low production of contaminating proteins when compared to the conventional strain. In addition, the detoxified strain produces a modified LPS, which does not cause significant pyrogenic reaction. Thus, the present study demonstrated that the endotoxin-free strain use as a platform for the production of recombinant proteins in industrial scale is technically possible and presents itself as a very interesting alternative for therapeutic proteins. |
publishDate |
2018 |
dc.date.accessioned.fl_str_mv |
2018-05-03T13:18:32Z |
dc.date.available.fl_str_mv |
2018-05-03T13:18:32Z |
dc.date.issued.fl_str_mv |
2018-03-02 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
PAREDES, Sheyla Alexandra Hidalgo. Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo. 2018. Dissertação (Mestrado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2018. Disponível em: https://repositorio.ufscar.br/handle/ufscar/9858. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/9858 |
identifier_str_mv |
PAREDES, Sheyla Alexandra Hidalgo. Escherichia coli detoxificada como plataforma para produção de proteína recombinante A de superfície de pneumococo. 2018. Dissertação (Mestrado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2018. Disponível em: https://repositorio.ufscar.br/handle/ufscar/9858. |
url |
https://repositorio.ufscar.br/handle/ufscar/9858 |
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por |
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info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Universidade Federal de São Carlos Câmpus São Carlos |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Engenharia Química - PPGEQ |
dc.publisher.initials.fl_str_mv |
UFSCar |
publisher.none.fl_str_mv |
Universidade Federal de São Carlos Câmpus São Carlos |
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