Da produção da proteína verde fluorescente à sua extração utilizando sistemas aquosos bifáficos
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Data
2018-01-26
Autores
Lopes, Camila
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Universidade Estadual Paulista (Unesp)
Resumo
A proteína verde fluorescente (GFP, do inglês Green Fluorescent Protein) é um biomarcador utilizado na produção de proteínas de fusão, amplamente empregado in vivo e in vitro. Este trabalho avaliou a produção da GFP expressa em Escherichia coli BL21 (DE3) [pLysS; pET28(a)] em mesa incubadora rotativa e biorreator, e sua posterior extração utilizando Sistemas Micelares de Duas Fases Aquosas (SMDFA). Na etapa de produção em mesa incubadora rotativa, estudou-se a influência da taxa de agitação, tempo de indução e concentração do composto indutor, isopropil-β-1-D-tiogalactopiranosídeo (IPTG), sobre a produção da GFP, e apenas a variação da taxa de agitação e o tempo de indução alteraram a produção de GFP. A melhor produção de GFP (314,59 mg/L) foi obtida a 30°C após 22 h de cultivo a uma agitação de 150 rpm, induzindo com 0,5 mM de IPTG realizada 10 h após o início do cultivo (meio da fase exponencial de crescimento). Posteriormente, avaliou-se menores concentrações de IPTG (0,25; 0,125 mM) na produção de GFP e observou-se que os níveis de produção foram mantidos. Estes estudos iniciais, foram a base para as condições empregadas em biorreator tanque agitado, que operou no modo batelada a uma agitação de 200 rpm, aeração de 2 vvm (volume de ar/ volume de meio) e indução com 0,125 mM de IPTG após 6 h de cultivo. Nas condições citadas, obteve-se apenas uma produção de 128,13 mg/L de GFP, desse modo, apesar da potencialidade da ampliação de escala na produção desta biomolécula, estudos posteriores são ainda necessários. Após os estudos de produção, realizou-se o rompimento das células bacterianas por ciclos continuados de congelamento/ descongelamento. A GFP do lisado celular foi então submetida a estudos de sua estabilidade perante diferentes pHs (3 - 12) e temperaturas (25º, 37º e 50ºC), mostrando-se estável entre os pHs 7 a 11 e em temperaturas de 25°C e 37°C, mas não estável a uma temperatura de 50°C. Nos estudos de extração foram utilizados os SMDFA compostos por tampão McIlvaine pH 7, Triton X-114, e o líquido iônico [P6,6,6,14]Cl como adjuvante. O melhor coeficiente de partição (K = 30,91) foi obtido no sistema formado por 2,5 % (m/m) de Triton X-114; 0,5% (m/m) de [P6,6,6,14]Cl. Os resultados obtidos demonstraram que uma escolha apropriada das melhores condições de cultivo permitem um aumento da produção de GFP utilizando a cepa de E. coli BL21 (DE3) [pLysS; pET28(a)]. Além disso, os estudos com SMDFA mostraram alta influência dos seus componentes na partição da GFP, demonstrando a potencialidade de sistemas aquosos na purificação da GFP.
Green Fluorescent Protein (GFP) is a in vivo and in vitro biomarker widely used in the production of fusion proteins. This work evaluated the production of GFP expressed in Escherichia coli BL21 (DE3) [pLysS; pET28 (a)] in shaker and bioreactor, and its subsequent extraction using Aqueous Micellar Two-Phase Systems (AMTPS). The influence of the agitation rate, induction time and concentration of the inductor, isopropyl-β-1-D-thiogalactopyranoside (IPTG), on GFP production, was studied using a shaker. The results shown that only the agitation rate and the induction time have affect the GFP production. The best GFP production (314.59 mg/L) was obtained at 30°C after 22 h of culture at 150 rpm, induced with 0.5 mM IPTG after 10 h after of the start of cultivation (at half of exponential growth). Subsequently, lower IPTG concentrations (0.25, 0.125 mM) were evaluated for the GFP production, being maintained the production levels. From these conditions, a cultivation using agitated tank bioreactor was then carried out. The bioreactor operated in batch mode at 200 rpm with a 2 vvm aeration (air volume / volume of medium), being the GFP production induced with 0.125 mM IPTG after 6 h of microorganism’ growth. Under that conditions, a production of 128.13 mg/L of GFP was obtained. Thus, despite the potential of scale-up in the production of this biomolecule, further studies are still required. After the GFP production studies, the bacterial cells were disrupted using continuous freezing / thawing cycles. GFP recovered from the cell lysate was then used to perform stability studies at different pHs (3 - 12) and temperatures (25°, 37° and 50°C). The stability studies shown that GFP was stable between pHs 7 to 11 and at temperatures of 25°C and 37°C, but not stable at a temperature of 50°C. Afterwards, liquid-liquid extraction studies using AMTPS composed of McIlvaine buffer (pH 7), Triton X-114, and the ionic liquid [P6,6,6,14]Cl as an adjuvant, were performed. The best partitioning coefficient (K = 30.91) was obtained with the system composed of 2.5% (w/w) of Triton X-114 and 0.5% (w/w) of [P6,6,6,14]Cl. The set of results have demonstrated that a proper choice of the cultivation conditions allows the increase of GFP production using the E. coli BL21 (DE3) [pLysS; pET28(a)] strain. Furthermore, the partitioning studies using AMTPS have shown a high influence of their phase-forming components in the GFP partitioning, demonstrating the potentiality of AMTPS in the GFP extraction and purification.
Green Fluorescent Protein (GFP) is a in vivo and in vitro biomarker widely used in the production of fusion proteins. This work evaluated the production of GFP expressed in Escherichia coli BL21 (DE3) [pLysS; pET28 (a)] in shaker and bioreactor, and its subsequent extraction using Aqueous Micellar Two-Phase Systems (AMTPS). The influence of the agitation rate, induction time and concentration of the inductor, isopropyl-β-1-D-thiogalactopyranoside (IPTG), on GFP production, was studied using a shaker. The results shown that only the agitation rate and the induction time have affect the GFP production. The best GFP production (314.59 mg/L) was obtained at 30°C after 22 h of culture at 150 rpm, induced with 0.5 mM IPTG after 10 h after of the start of cultivation (at half of exponential growth). Subsequently, lower IPTG concentrations (0.25, 0.125 mM) were evaluated for the GFP production, being maintained the production levels. From these conditions, a cultivation using agitated tank bioreactor was then carried out. The bioreactor operated in batch mode at 200 rpm with a 2 vvm aeration (air volume / volume of medium), being the GFP production induced with 0.125 mM IPTG after 6 h of microorganism’ growth. Under that conditions, a production of 128.13 mg/L of GFP was obtained. Thus, despite the potential of scale-up in the production of this biomolecule, further studies are still required. After the GFP production studies, the bacterial cells were disrupted using continuous freezing / thawing cycles. GFP recovered from the cell lysate was then used to perform stability studies at different pHs (3 - 12) and temperatures (25°, 37° and 50°C). The stability studies shown that GFP was stable between pHs 7 to 11 and at temperatures of 25°C and 37°C, but not stable at a temperature of 50°C. Afterwards, liquid-liquid extraction studies using AMTPS composed of McIlvaine buffer (pH 7), Triton X-114, and the ionic liquid [P6,6,6,14]Cl as an adjuvant, were performed. The best partitioning coefficient (K = 30.91) was obtained with the system composed of 2.5% (w/w) of Triton X-114 and 0.5% (w/w) of [P6,6,6,14]Cl. The set of results have demonstrated that a proper choice of the cultivation conditions allows the increase of GFP production using the E. coli BL21 (DE3) [pLysS; pET28(a)] strain. Furthermore, the partitioning studies using AMTPS have shown a high influence of their phase-forming components in the GFP partitioning, demonstrating the potentiality of AMTPS in the GFP extraction and purification.
Descrição
Palavras-chave
proteína verde fluorescente, biomarcador, produção, extração, sistemas micelares de duas fases aquosas, green fluorescent protein, biomarker, production, extraction, aqueous micellar two-phase systems