Síntesis de columna de sílica estructurada químicamente con níquel para la purificación de proteínas con cola de poli- histidina.
Cargando...
Fecha
2023
Autores
Título de la revista
ISSN de la revista
Título del volumen
Editor
Ediciones Universidad Simón Bolívar
Facultad de Ciencias Básicas y Biomédicas
Facultad de Ciencias Básicas y Biomédicas
Resumen
La purificación de proteínas actualmente demanda mucho interés en cuanto a su utilización para el diagnóstico de enfermedades y de uso terapéutico. Estos son capaces de resolver muchas de las necesidades científicas requeridas para la implementación de alternativas biotecnológicas y moleculares las cuales cumplen con las adaptaciones experimentales que promueven técnicas eficientes y de costos considerables, teniendo la facilidad de suplir con los resultados deseados. Los métodos para la purificación de proteína pueden considerarse altamente complejos de acuerdo a su utilización y sus requerimientos económicos, por lo que en el siguiente trabajo se empleó una alternativa estructurada químicamente a partir de la sílica cargada con Ni (II) con la posibilidad de retener a la proteína GFP a través de su cola de poli histidina suspendida en una matriz cromatográfica que busca obtener o sustituir otros métodos que requieran más gastos económicos y carezcan de ahorro de tiempo en cuanto a los resultados.
Para la preparación química de la sílica se utilizaron protocolos diseñados en base a información científica y a la experiencia aportada en el centro de investigación de la Universidad Simón Bolívar Adaptia. Así mismo se puso a prueba el comportamiento de la sílica basado en la experimentación de usos de reactivos y métodos necesarios para la preparación de la sílica a base de la utilización de APS (aminopropilsilano), determinado como el proceso de silanización, fundamental para iniciar con el montaje de la estructura química con la formación de grupos aminos que a su vez son necesarios para adherir el EDTA, el cual, ayudará a desarrollar y fortalecer la superficie química a la que se desea pegar el níquel. Para la unión del EDTA a la sílica se emplearon dos mecanismos con la capacidad de lograr que a la sílica silanizada se le unieran diversos EDTA, tales como; Método por reflujo y por microondas. Además, de incorporar el sulfato de níquel y el cloruro de níquel para
conseguir la adherencia de este a la matriz cromatográfica. Por otro lado, la proteína GFP requerida para probar la columna cromatográfica compuesta de sílica contaba con una cola de poli histidina, obtenida y expresada por Escherichia coli BL21, en la que solo fue necesario aplicar un protocolo de inducción con IPTG y lisis bacteriana debido a que estas ya venían transformadas con el plásmido pET28 para la obtención de GFP.
Los resultados del siguiente trabajo demostraron que para la estructuración química de la sílica los protocolos más eficientes provienen del protocolo de Adherencia del EDTA a través de microondas, lo cual, fue de gran ventaja debido a que necesita menor tiempo para elaborar el proceso y es una técnica más práctica. Así mismo, la utilización del sulfato de níquel demostró ser más preciso en cuanto a la retención de GFP en la columna. Por otro lado, en el proceso final de la obtención de GFP pura se suministraron diferentes gradientes de concentración de imidazol durante la purificación, en la cual, se logró desprender a GFP de la columna y tener la proteína purificada.
Por otra parte, se concluyó que es importante explorar diversas alternativas capaces de suplir y reemplazar métodos que demandan elevados costos y complejidad para su obtención, teniendo en cuenta que el fundamento y experimentación sean eficientes y recursivos
Protein purification currently demands much interest in terms of its use for disease diagnosis and therapeutic use capable of solving many of the scientific needs required for the implementation of biotechnological and molecular alternatives that meet the experimental adaptations that promote efficient and cost-effective techniques capable of supplying the desired results. The methods for protein purification can be considered highly complex according to their use and economic requirements, so in the following work a chemically structured alternative based on silica loaded with Ni (II) with the ability to retain the GFP protein through its polyhistidine tail suspended in a chromatographic matrix was used to replace other methods that require more economic expenses and lack of time savings in terms of results. For the chemical preparation of the silica, protocols designed on the basis of scientific information and the experience contributed by doctors and professionals of the life sciences research center of the Simón Bolívar University were used. Likewise, the behavior of the silica was tested based on the experimentation of the use of reagents and methods necessary for the preparation of the silica based on the use of APS (aminopropylsilane), determined as the silanization process, fundamental to start with the assembly of the chemical structure with the formation of amino groups that in turn are necessary to adhere the EDTA, which will help to develop and strengthen the chemical surface to which the nickel is to be bonded. For EDTA bonding to the silica, two mechanisms were used with the ability to achieve that the silanized silica is bonded to various EDTA, such as; reflux method and microwave method. In addition, nickel sulfate and nickel chloride were incorporated to achieve their adhesion to the chromatographic matrix. On the other hand, the GFP protein required to test the chromatographic column composed of silica had a polyhistidine tail, obtained and expressed by E, coli BL21, in which it was only necessary to apply an induction protocol with IPTG and bacterial lysis because these were already transformed with the plasmid pET28 to obtain GFP. The results of the following work showed that for the chemical structuring of silica the most efficient protocols come from the EDTA adhesion protocol through microwaves, which was of great advantage because it needs less time to elaborate the process and it is a more practical technique, likewise, the use of nickel sulfate proved to be more precise in terms of GFP retention in the column. For the final process of obtaining pure GFP, different concentration gradients of imidazole were supplied during the purification process to finally detach GFP from the column and have the protein in high percentages of purity. On the other hand, it was concluded that it is important to explore different alternatives capable of replacing methods that require high costs and complexity to obtain, taking into account that the foundation and experimentation must be efficient and recursive
Protein purification currently demands much interest in terms of its use for disease diagnosis and therapeutic use capable of solving many of the scientific needs required for the implementation of biotechnological and molecular alternatives that meet the experimental adaptations that promote efficient and cost-effective techniques capable of supplying the desired results. The methods for protein purification can be considered highly complex according to their use and economic requirements, so in the following work a chemically structured alternative based on silica loaded with Ni (II) with the ability to retain the GFP protein through its polyhistidine tail suspended in a chromatographic matrix was used to replace other methods that require more economic expenses and lack of time savings in terms of results. For the chemical preparation of the silica, protocols designed on the basis of scientific information and the experience contributed by doctors and professionals of the life sciences research center of the Simón Bolívar University were used. Likewise, the behavior of the silica was tested based on the experimentation of the use of reagents and methods necessary for the preparation of the silica based on the use of APS (aminopropylsilane), determined as the silanization process, fundamental to start with the assembly of the chemical structure with the formation of amino groups that in turn are necessary to adhere the EDTA, which will help to develop and strengthen the chemical surface to which the nickel is to be bonded. For EDTA bonding to the silica, two mechanisms were used with the ability to achieve that the silanized silica is bonded to various EDTA, such as; reflux method and microwave method. In addition, nickel sulfate and nickel chloride were incorporated to achieve their adhesion to the chromatographic matrix. On the other hand, the GFP protein required to test the chromatographic column composed of silica had a polyhistidine tail, obtained and expressed by E, coli BL21, in which it was only necessary to apply an induction protocol with IPTG and bacterial lysis because these were already transformed with the plasmid pET28 to obtain GFP. The results of the following work showed that for the chemical structuring of silica the most efficient protocols come from the EDTA adhesion protocol through microwaves, which was of great advantage because it needs less time to elaborate the process and it is a more practical technique, likewise, the use of nickel sulfate proved to be more precise in terms of GFP retention in the column. For the final process of obtaining pure GFP, different concentration gradients of imidazole were supplied during the purification process to finally detach GFP from the column and have the protein in high percentages of purity. On the other hand, it was concluded that it is important to explore different alternatives capable of replacing methods that require high costs and complexity to obtain, taking into account that the foundation and experimentation must be efficient and recursive
Descripción
Palabras clave
Cromatografía, Proteína, Biotecnología, E. coli, Economía, Columna, Purificación, Chromatography, Protein, Biotechnology, E. coli, Economy, Column, Purification
