Desenvolvimento de plataformas sustentáveis para estabilização e purificação da Proteína Verde Fluorescente recombinante produzida por Escherichia coli
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Data
2020-11-26
Autores
Santos, Nathalia Vieira dos
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Universidade Estadual Paulista (Unesp)
Resumo
A biotecnologia permitiu o desenvolvimento de novas biomoléculas para aplicações médicas e industriais. No entanto, o uso de muitos produtos biológicos notáveis ainda é limitado em grande escala devido às suas baixas estabilidades (que dificultam distribuição, armazenamento e manuseio) e preços muito elevados (principalmente devidos aos processos complexos e caros de produção e purificação). Um melhor entendimento das propriedades e estabilidade das biomoléculas, aliados ao desenvolvimento de plataformas mais simples, baratas e sustentáveis para estabilização e purificação de produtos biotecnológicos, pode ajudar a superar essas limitações e expandir seus usos comerciais. Assim, o objetivo do trabalho foi melhorar as aplicações em larga escala de biomoléculas usando a Proteína Verde Fluorescente Melhorada (EGFP, do inglês Enhanced Green Fluorescent Protein, importante biomarcador e biossensor) como uma prova de conceito. Nesse sentido, a pesquisadora projetou estudos aprofundados da estrutura de EGFP e suas propriedades de fluorescência, avaliou sua estabilidade na presença de diferentes solventes ou sob condições de estresse, e desenvolveu novos sistemas sustentáveis para a estabilização e purificação da EGFP usando Líquidos Iônicos (LIs), sais orgânicos e polímeros compatíveis com biomoléculas. Nos estudos de propriedades da EGFP, foi possível detectar um pico de fluorescência adicional e nunca relatado para esta proteína, resolvendo uma controvérsia científica sobre as conformações e atividade da EGFP. Além disso, nos estudos de estabilidade em pH, foi possível demonstrar a relação entre a fluorescência da EGFP e pH, assim como elucidar os impactos do pH ácido na estrutura da proteína. Essas informações podem ajudar no desenvolvimento de novos biossensores de pH neutro-a-ácido. Os estudos da atividade da EGFP na presença de LIs e sob condições de estresse mostraram que LIs podem ter diferentes impactos na estabilidade de proteínas, dependendo de sua natureza, concentração e comprimento da cadeia alquílica. Por exemplo, alguns LIs como cloreto de colina, octanoato de colina e decanoato de colina protegeram a fluorescência de EGFP, enquanto LIs baseados em imidazólios com maior comprimento de cadeia alquílica reduziram sua atividade. Além disso, foi possível encontrar LIs que triplicaram a preservação a longo prazo da atividade da EGFP em temperatura ambiente, que podem potencialmente ser aplicados como estabilizadores de proteínas em formulações biofarmacêuticas. Na etapa de purificação, uma plataforma sustentável foi desenvolvida associando sistemas bifásicos aquosos (compostos por polímeros e sais) e ultrafiltração, obtendo EGFP > 97 % pura e também recuperando mais de 60% dos solventes utilizados no processo. Em conclusão, os estudos da atividade e estabilidade da EGFP, aliados ao desenvolvimento de técnicas alternativas sustentáveis de purificação de baixo custo, podem ampliar seu uso comercial e tem aplicação potencial para outras biomoléculas.
Biotechnology allowed the development of novel biomolecules for medical and industrial applications. However, there are still limitations to the use of many outstanding biological products at large-scale due to their low stability (which hinders their distribution, storage, and handling) and very high prices (mostly caused by their intricate and costly production and purification processes). A better understanding of the properties and stability of biomolecules, allied with the development of simpler, cheaper, and more sustainable stabilization and purification platforms for biotechnological products, can help to overcome these limitations and expand their commercial use. Hence, the goal of this work was to improve the large-scale applications of biomolecules using the Enhanced Green Fluorescent Protein (EGFP, an important biomarker and biosensor) as a proof of concept. In this sense, the researcher designed in-depth studies of EGFP structure and fluorescence properties, evaluated its stability in the presence of different solvents or under stress conditions, and developed novel sustainable stabilization and purification systems for EGFP using Ionic Liquids (ILs), organic salts, and polymers compatible with biomolecules. In the studies of EGFP properties and structure, it was possible to detect an additional and never reported fluorescence peak for this protein and solve a scientific controversy regarding EGFP conformations and activity. Additionally, on the pH stability studies, it was possible to demonstrate the pH-dependence of the fluorescence of EGFP and elucidate the impacts of acidic pH on the structure of the protein, which can aid in the development of novel neutral-to-acidic pH-biosensors. The studies of EGFP activity in the presence of ILs and under stress conditions showed ILs have different impacts on the stability of proteins, depending on their nature, concentration, and alkyl chain length. For example, some ILs like cholinium chloride, cholinium octanoate, and cholinium decanoate protected EGFP fluorescence, while imidazolium-based ILs with longer alkyl chain length reduced the activity of the protein. Furthermore, it was possible to find ILs that tripled the long-term preservation of EGFP activity at room temperature, which could potentially act as protein stabilizers in biopharmaceutical formulations. For the purification stage, it was possible to develop a sustainable purification platform associating aqueous biphasic systems (composed of polymers and salts) and ultrafiltration, obtaining > 97 % pure EGFP while recovering 60 % of the solvents used in the process. In conclusion, the study of EGFP activity and stability, allied with the development of sustainable alternative low-cost purification techniques, can broaden its commercial use and have potential applications for other biomolecules.
Biotechnology allowed the development of novel biomolecules for medical and industrial applications. However, there are still limitations to the use of many outstanding biological products at large-scale due to their low stability (which hinders their distribution, storage, and handling) and very high prices (mostly caused by their intricate and costly production and purification processes). A better understanding of the properties and stability of biomolecules, allied with the development of simpler, cheaper, and more sustainable stabilization and purification platforms for biotechnological products, can help to overcome these limitations and expand their commercial use. Hence, the goal of this work was to improve the large-scale applications of biomolecules using the Enhanced Green Fluorescent Protein (EGFP, an important biomarker and biosensor) as a proof of concept. In this sense, the researcher designed in-depth studies of EGFP structure and fluorescence properties, evaluated its stability in the presence of different solvents or under stress conditions, and developed novel sustainable stabilization and purification systems for EGFP using Ionic Liquids (ILs), organic salts, and polymers compatible with biomolecules. In the studies of EGFP properties and structure, it was possible to detect an additional and never reported fluorescence peak for this protein and solve a scientific controversy regarding EGFP conformations and activity. Additionally, on the pH stability studies, it was possible to demonstrate the pH-dependence of the fluorescence of EGFP and elucidate the impacts of acidic pH on the structure of the protein, which can aid in the development of novel neutral-to-acidic pH-biosensors. The studies of EGFP activity in the presence of ILs and under stress conditions showed ILs have different impacts on the stability of proteins, depending on their nature, concentration, and alkyl chain length. For example, some ILs like cholinium chloride, cholinium octanoate, and cholinium decanoate protected EGFP fluorescence, while imidazolium-based ILs with longer alkyl chain length reduced the activity of the protein. Furthermore, it was possible to find ILs that tripled the long-term preservation of EGFP activity at room temperature, which could potentially act as protein stabilizers in biopharmaceutical formulations. For the purification stage, it was possible to develop a sustainable purification platform associating aqueous biphasic systems (composed of polymers and salts) and ultrafiltration, obtaining > 97 % pure EGFP while recovering 60 % of the solvents used in the process. In conclusion, the study of EGFP activity and stability, allied with the development of sustainable alternative low-cost purification techniques, can broaden its commercial use and have potential applications for other biomolecules.
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Palavras-chave
Proteína Verde Fluorescente, Purificação de Proteínas, Estabilidade de Proteínas, Líquidos Iônicos, Sistemas Aquosos Bifásicos, Biossensores, Green Fluorescent Protein, Protein Purification, Protein Stability, Ionic Liquids, Aqueous Biphasic Systems, Biosensors