Expressão e purificação do domínio de ligação ao receptor (RBD) da proteína Spike do vírus SARS-Cov-2 da cepa de Wuhan
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Data
2024-04-03
Autores
Vaz, Fábio Antonio Busoli [UNIFESP]
Orientadores
Tanaka, Aparecida Sadae [UNIFESP]
Tipo
Dissertação de mestrado
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Objetivos: O objetivo foi a obtenção da RBD de SARS-CoV-2 e realizar estudos de expressão, solubilização e purificação da RBD-wt recombinante a fim de encontrar uma condição ótima. Métodos: A construção plasmidial contendo a RBD-wt utilizada para obtenção da variante P1 por meio da estratégia de mutação sítio-dirigida por reações de PCR. O fragmento de DNA foi purificado e inserido no vetor pRSET-A. O DNA plasmidial da construção contendo a RBD-wt foi utilizado na transformação de células BL21(DE3) pLysS competentes, por choque térmico e, após a confirmação dos clones positivos, foram feitos os testes de expressão a fim de otimizar a produção da RBD-wt recombinante. Após os testes de expressão, foi realizado um teste de solubilidade e foi feita a detecção da RBD-wt por Western Blot. Após esses testes, foi feita a purificação da RBD-wt usando dois métodos diferentes de expressão para obter a proteína recombinante. A purificação foi realizada por cromatografia de afinidade. Resultados: Bactérias transformadas com plasmídeos contendo a sequência da RBD-wt foram confirmadas por PCR de colônia. Os clones positivos foram utilizados para a expressão da proteína recombinante que foi confirmada por SDS-PAGE apresentando uma banda proteica de aproximadamente 32 kDa. Um clone positivo foi utilizado para os testes de expressão, sob diferentes concentrações do agente indutor, tempos de incubação e temperaturas. Os resultados mostraram que as concentrações testadas do indutor não afetaram a expressão da proteína. Contudo, o tempo de incubação e a temperatura afetaram os níveis de expressão. As melhores condições observadas para a expressão de RBD-wt foram utilizando o meio de autoindução. A proteína RBD-wt recombinante foi obtida majoritariamente na forma insolúvel, dados confirmados por SDS-PAGE e Western Blotting. A proteína recombinante foi solubilizada em 8 M de ureia e purificada por cromatografia de afinidade em coluna His-Sepharose, sendo realizado o refolding dentro da coluna. Desta forma, a proteína solúvel foi obtida na eluição. A construção plasmidial contendo a sequência da RBD-P1 foi obtida com sucesso utilizando mutação sítio-dirigida e a sequência da RBD-wt como DNA-molde. A sequência correta foi confirmada por sequenciamento DNA. Conclusão: Dentre as condições testadas para a expressão da RBD-wt, não foi observado uma condição que aumentasse o rendimento da proteína recombinante solúvel quando comparada com as condições de solubilização e refolding publicadas anteriormente. Neste trabalho foi possível obter a construção plasmidial contendo a sequência de RBD-P1, que poderá ser utilizada em estudos futuros.
Objectives: The objective was to obtain the SARS-CoV-2 RBD and carry out expression, solubilization and purification studies of the recombinant RBD-wt to find an ideal condition. Methods: The plasmid construction contains the RBD-wt used to provide the P1 variant through the site-directed mutation strategy by PCR reactions. The DNA fragment was purified and inserted into the pRSET-A vector. The plasmid DNA of the construct containing the RBD-wt was used in the transformation of competent BL21(DE3) pLysS cells by heat shock and, after notification of positive clones, expression tests were carried out to optimize the production of the recombinant RBD-wt. After expression tests, a solubility test was performed and RBD-wt was detected by Western Blot. After these tests, RBD-wt was purified using two different expression methods to obtain the recombinant protein. Purification was carried out by affinity chromatography. Results: The recombinant bacteria with RBD-wt coding sequence was successfully obtained, and 4 positive clones were confirmed by colony PCR. These clones were used for the protein expression, which was observed as a 32 kDa band approximately, corresponding to the size of the RBD. One of the clones was used for the expression tests, where the inductor concentration, time and temperature of incubation varied. It was observed that the inductor concentration did not affect the protein expression. However, the time and temperature incubation resulted in different expression levels. The best conditions observed for the protein expression were using the autoinduction media. The recombinant RBD-wt protein was majority obtained insoluble, confirmed by the Western Blotting and SDS-PAGE. The protein was solubilized using 8M of urea and then purified by affinity chromatography in His-Sepharose and the on-column refolding was performed. This way, the soluble protein was then obtained with success in the elution. The plasmidial construction containing the RBD-P1 sequence was successfully obtained using site directed mutagenesis strategy and the RBD-wt sequence was used as template. The sequence was confirmed by DNA sequencing. Conclusion: Between the tested conditions for the RBD-wt expression it wasn´t observed a condition that could increase the soluble recombinant protein yield when compared with the published solubilization and refolding conditions previously. In this work, it was possible to obtain the plasmidial construction with the RBD-P1 sequence, that can be used on future studies.
Objectives: The objective was to obtain the SARS-CoV-2 RBD and carry out expression, solubilization and purification studies of the recombinant RBD-wt to find an ideal condition. Methods: The plasmid construction contains the RBD-wt used to provide the P1 variant through the site-directed mutation strategy by PCR reactions. The DNA fragment was purified and inserted into the pRSET-A vector. The plasmid DNA of the construct containing the RBD-wt was used in the transformation of competent BL21(DE3) pLysS cells by heat shock and, after notification of positive clones, expression tests were carried out to optimize the production of the recombinant RBD-wt. After expression tests, a solubility test was performed and RBD-wt was detected by Western Blot. After these tests, RBD-wt was purified using two different expression methods to obtain the recombinant protein. Purification was carried out by affinity chromatography. Results: The recombinant bacteria with RBD-wt coding sequence was successfully obtained, and 4 positive clones were confirmed by colony PCR. These clones were used for the protein expression, which was observed as a 32 kDa band approximately, corresponding to the size of the RBD. One of the clones was used for the expression tests, where the inductor concentration, time and temperature of incubation varied. It was observed that the inductor concentration did not affect the protein expression. However, the time and temperature incubation resulted in different expression levels. The best conditions observed for the protein expression were using the autoinduction media. The recombinant RBD-wt protein was majority obtained insoluble, confirmed by the Western Blotting and SDS-PAGE. The protein was solubilized using 8M of urea and then purified by affinity chromatography in His-Sepharose and the on-column refolding was performed. This way, the soluble protein was then obtained with success in the elution. The plasmidial construction containing the RBD-P1 sequence was successfully obtained using site directed mutagenesis strategy and the RBD-wt sequence was used as template. The sequence was confirmed by DNA sequencing. Conclusion: Between the tested conditions for the RBD-wt expression it wasn´t observed a condition that could increase the soluble recombinant protein yield when compared with the published solubilization and refolding conditions previously. In this work, it was possible to obtain the plasmidial construction with the RBD-P1 sequence, that can be used on future studies.
Descrição
Citação
VAZ, Fábio Antonio Busoli. Expressão e purificação do domínio de ligação ao receptor (RBD) da proteína Spike do vírus SARS-Cov-2 da cepa de Wuhan. 2024. 69 f. Dissertação (Biologia Molecular) - Escola Paulista de Medicia, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.