Aumento de escala para a produção de Proteína verde fluorescente melhorada (Enhanced Green Fluorescent Protein - EGFP) a partir de Escherichia coli recombinante em biorreator convencional
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Data
2019-04-09
Autores
Sousa, Ana Paula Abuchain
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Universidade Estadual Paulista (Unesp)
Resumo
Avanços na biotecnologia proporcionaram possibilidades para o eficiente desempenho da produção em larga escala de diversas biomoléculas e consequentemente suas aplicações industriais. A Escherichia coli se destaca dentre a gama de microrganismos que agem como hospedeiros de genes, desempenhando a função de codificar a síntese proteica. Os vetores mais veiculados na produção de proteínas recombinantes em E. coli são baseados no operon lac, onde o isopropil β-D1-tio-galactopiranosídeo (IPTG), análogo a molécula de lactose, é utilizado para a indução da produção da proteína de interesse. Estudos descritos na literatura também observaram o bom desempenho da lactose como agente de indução da E. coli recombinante na expressão de proteína verde fluorescente melhorada (Enhanced Green Fluorescent Protein – EGFP), e suas vantagens quando comparada ao IPTG, como por exemplo menor custo e menor toxicidade. A EGFP se tornou promissora pelo fato de ser monomérica e não precisar de auxilio de quaisquer agentes adicionais para exibir atividade de fluorescência. Possui variadas utilidades no campo biológico como excelente biomarcador da expressão genica e biosensor. Em bioprocessos, operados em biorreatores convencionais é fundamental o estudo dos parâmetros interferentes nos cultivos para a otimização da expressão do produto desejado. A oxigenação em processos conduzidos de maneira aeróbica, também é uma tarefa desafiadora em biorreatores convencionais, sendo imprescindível o controle da concentração de oxigênio dissolvido no meio de cultura, o que pode ser limitante para o crescimento e para a expressão da proteína de interesse. Em processos de produção de proteínas heterólogas presume-se que a produtividade pode ser proporcional à densidade celular final deste microrganismo no cultivo. Sendo assim, o presente trabalho visou a melhora do processo de obtenção da proteína heteróloga EGFP em biorreator convencional no modo de operação batelada. Foram realizados experimentos testando diferentes concentrações de glicose, lactose e IPTG, onde se observou que a lactose se comportou de maneira mais eficiente e promissora como fonte de carbono e agente indutor da expressão proteica quando comparado ao IPTG. Através do planejamento experimental denominado Delineamento Central Composto (DCC) observou-se que dentre as variáveis estudadas (temperatura (X1), agitação (X2), preenchimento do frasco tipo Erlenmeyer (X3) e concentração de lactose (X4)) apenas temperatura foi significativamente estatística (nível de significância de 99%) para a expressão de EGFP no processo, de forma inversamente proporcional, enquanto que concentração de lactose e preenchimento do frasco se mostraram significativas para o crescimento celular de forma proporcional. Entre os experimentos realizados em biorreator convencional variando taxa de agitação de 400 rpm a 1000 rpm e taxa de aeração de 0,5 vvm a 0,75 vvm houve um aumento substancial da concentração de EGFP com o aumento da agitação, o que ocorreu devido a transferência de oxigênio ser melhorada em altas condições de agitação, ocorrendo aumento de kLa (coeficiente volumétrico de transferência de oxigênio (h-1)) e OTR (Oxygen Transfer Rate – taxa de transferência de oxigênio). A suplementação com 2,5 g/L de extrato de levedura ao meio utilizado nos experimento (composto de LB 25 g/L com lactose 40g/L) proporcionou o aumento do consumo da lactose de 20 g/L para 32 g/L aproximadamente. Observou-se também que as proteínas formadas ao final do processo em biorreator não estavam com ativação completa do cromóforo, que ocorre através da etapa de oxidação da molécula, pois em teste realizado submetendo a biomassa celular diluída, em vazão de oxigênio puro de 1 L/min aumentou em torno de 2,5 vezes a concentração de EGFP em relação a concentração inicial.
Advances in biotechnology have provided possibilities to the performance of large-scale of biomolecules and therefore of their industrial applications. Escherichia coli stands out among microorganisms that act as host genes, functioning as a synthetic protein. The most useful vectors for the production of recombinant proteins in E. coli are based on the operon lac, where isopropyl β-D1-thiogalactopyroside (IPTG), analogous to lactose, is used to induce the production of proteins of interest. Studies in the literature have also observed the good performance of lactose as an inducer of recombinant E. coli in the expression of Enhanced Green Fluorescent Protein (EGFP), and its advantages when compared to IPTG, such as lower cost and toxicity. EGFP becomes promising because it is monomeric and does not need to help the main agents for fluorescence activity. It has several uses in the biological field as an excellent biomarker of gene expression and biosensor. In bioprocesses, operated in conventional bioreactors, it is fundamental to study the interfering parameters in the cultures to optimize the expression of the desired product. Oxygenation in aerobically conducted processes is also a challenging task in conventional bioreactors, with control of the concentration of dissolved oxygen in the culture medium is essential, which may be limiting for growth and expression of the protein of interest. In processes of production of heterologous proteins, it is assumed that the productivity may be proportional to the final cell density of this microorganism in the culture. Thus, the present work aimed at improving the process of obtaining the heterologous protein EGFP in conventional bioreactor in the mode of batch operation. Experiments were performed testing different concentrations of glucose, lactose and IPTG, where it was observed that lactose behaved in a more efficient and promising way as a source of carbon and inducing agent of protein expression when compared to IPTG. Experimental design called the Central Compound Designation (CCD) showed that temperature (X1), agitation (X2), Erlenmeyer flask filling (X3) and lactose concentration (X4) (level of significance of 99%) for the expression of EGFP in the process, inversely proportional, whereas lactose concentration and filling of the flask were shown to be significant for the cellular growth proportionally. Among the experiments performed in conventional bioreactor, varying agitation rate from 400 rpm to 1000 rpm and aeration rate of 0.5 vvm to 0.75 vvm there was a substantial increase in EGFP concentration with increased agitation, which was due to oxygen transfer coefficient (h-1)) and OTR (Oxygen Transfer Rate) were observed. Supplementation with 2.5 g / L of yeast extract in the medium used in the experiment (composed of LB 25 g / L with lactose 40 g / L) provided an increase in lactose consumption from 20 g / L to approximately 32 g / L. It was also observed that the proteins formed at the end of the process in bioreactor were not with complete activation of the chromophore that occurs through the oxidation stage of the molecule, as in a test performed by subjecting the cellular biomass diluted, the pure oxygen flow rate of 1 L / min increased about 2.5 times the concentration of EGFP over the initial concentration.
Advances in biotechnology have provided possibilities to the performance of large-scale of biomolecules and therefore of their industrial applications. Escherichia coli stands out among microorganisms that act as host genes, functioning as a synthetic protein. The most useful vectors for the production of recombinant proteins in E. coli are based on the operon lac, where isopropyl β-D1-thiogalactopyroside (IPTG), analogous to lactose, is used to induce the production of proteins of interest. Studies in the literature have also observed the good performance of lactose as an inducer of recombinant E. coli in the expression of Enhanced Green Fluorescent Protein (EGFP), and its advantages when compared to IPTG, such as lower cost and toxicity. EGFP becomes promising because it is monomeric and does not need to help the main agents for fluorescence activity. It has several uses in the biological field as an excellent biomarker of gene expression and biosensor. In bioprocesses, operated in conventional bioreactors, it is fundamental to study the interfering parameters in the cultures to optimize the expression of the desired product. Oxygenation in aerobically conducted processes is also a challenging task in conventional bioreactors, with control of the concentration of dissolved oxygen in the culture medium is essential, which may be limiting for growth and expression of the protein of interest. In processes of production of heterologous proteins, it is assumed that the productivity may be proportional to the final cell density of this microorganism in the culture. Thus, the present work aimed at improving the process of obtaining the heterologous protein EGFP in conventional bioreactor in the mode of batch operation. Experiments were performed testing different concentrations of glucose, lactose and IPTG, where it was observed that lactose behaved in a more efficient and promising way as a source of carbon and inducing agent of protein expression when compared to IPTG. Experimental design called the Central Compound Designation (CCD) showed that temperature (X1), agitation (X2), Erlenmeyer flask filling (X3) and lactose concentration (X4) (level of significance of 99%) for the expression of EGFP in the process, inversely proportional, whereas lactose concentration and filling of the flask were shown to be significant for the cellular growth proportionally. Among the experiments performed in conventional bioreactor, varying agitation rate from 400 rpm to 1000 rpm and aeration rate of 0.5 vvm to 0.75 vvm there was a substantial increase in EGFP concentration with increased agitation, which was due to oxygen transfer coefficient (h-1)) and OTR (Oxygen Transfer Rate) were observed. Supplementation with 2.5 g / L of yeast extract in the medium used in the experiment (composed of LB 25 g / L with lactose 40 g / L) provided an increase in lactose consumption from 20 g / L to approximately 32 g / L. It was also observed that the proteins formed at the end of the process in bioreactor were not with complete activation of the chromophore that occurs through the oxidation stage of the molecule, as in a test performed by subjecting the cellular biomass diluted, the pure oxygen flow rate of 1 L / min increased about 2.5 times the concentration of EGFP over the initial concentration.
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Palavras-chave
Cultivo de E. coli recombinante, Biorreator convencional, Proteína verde fluorescente, Green Fluorescent Protein