Publicação
Recombinant membrane protein production using integrated strategies
| Resumo: | Membrane proteins (MP) are a crucial part for the discovery of new drugs, which requires structural and functional studies. For these studies, high amounts of high-quality (pure and active) MP are needed. Despite their importance, MP production is still challenged by their hydrophobic nature, with the process being lengthy and requiring complex downstream processing steps. Ionic liquids (ILs) can be a promising alternative for MP production, allowing for the integration of different processing steps, such as cell lysis and MP solubilization. As such, the goal of this work was to study the ability of several ILs to cause lysis of Komagataella pastoris cells, while simultaneously solubilizing the enzyme cyclooxygenase 2 (COX-2), used in this work as a model MP. The inhibition of this enzyme is therapeutically relevant due to its role in inflammatory processes. Another goal was the usage of the activity coefficient model Conductor-like screening model for realistic solvents (COSMO-RS) to study the interactions between IL and the components of the yeast cell membrane and wall, possibly creating a model to screen ILs with respect their ability to cause cell lysis. Among the investigated ILs, protic ILs based on the N,N-diethylethanolammonium cation combined with carboxylic acids, as anions, proved promising in both lysing the yeast cells and solubilizing COX-2. A sample pre-treatment method based on ultrafiltration was also developed that allows to partially remove IL interference on protein quantification methods, allowing for more accurate quantifications. Simultaneously, promising correlations were found between experimental data and COSMO-RS, with the misfit and van der Waals interactions being relevant to the cell lysis process. After the optimization of COX-2 extraction parameters, the study and development of an aqueous biphasic systems as an extraction and separation process, is proposed as future work for the initial clarification of COX-2. To sum up, it is expected that the approach herein presented will be an important step forward toward the creation of an effective and low-cost process for the production of high-quality MP. Furthermore, the potential of protic IL to simultaneously lyse yeast cells and solubilize MP is reported for the first time. |
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| Autores principais: | Fernandes, João Luís Pereira |
| Assunto: | Membrane protein Ionic liquid Bioprocess integration Cell lysis Membrane protein solubilization Komagataella pastoris COSMO-RS |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Aveiro |
| Idioma: | inglês |
| Origem: | RIA - Repositório Institucional da Universidade de Aveiro |
| Resumo: | Membrane proteins (MP) are a crucial part for the discovery of new drugs, which requires structural and functional studies. For these studies, high amounts of high-quality (pure and active) MP are needed. Despite their importance, MP production is still challenged by their hydrophobic nature, with the process being lengthy and requiring complex downstream processing steps. Ionic liquids (ILs) can be a promising alternative for MP production, allowing for the integration of different processing steps, such as cell lysis and MP solubilization. As such, the goal of this work was to study the ability of several ILs to cause lysis of Komagataella pastoris cells, while simultaneously solubilizing the enzyme cyclooxygenase 2 (COX-2), used in this work as a model MP. The inhibition of this enzyme is therapeutically relevant due to its role in inflammatory processes. Another goal was the usage of the activity coefficient model Conductor-like screening model for realistic solvents (COSMO-RS) to study the interactions between IL and the components of the yeast cell membrane and wall, possibly creating a model to screen ILs with respect their ability to cause cell lysis. Among the investigated ILs, protic ILs based on the N,N-diethylethanolammonium cation combined with carboxylic acids, as anions, proved promising in both lysing the yeast cells and solubilizing COX-2. A sample pre-treatment method based on ultrafiltration was also developed that allows to partially remove IL interference on protein quantification methods, allowing for more accurate quantifications. Simultaneously, promising correlations were found between experimental data and COSMO-RS, with the misfit and van der Waals interactions being relevant to the cell lysis process. After the optimization of COX-2 extraction parameters, the study and development of an aqueous biphasic systems as an extraction and separation process, is proposed as future work for the initial clarification of COX-2. To sum up, it is expected that the approach herein presented will be an important step forward toward the creation of an effective and low-cost process for the production of high-quality MP. Furthermore, the potential of protic IL to simultaneously lyse yeast cells and solubilize MP is reported for the first time. |
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