Síntesis de resina de Cromatografía de Afinidad de Iones Metálicos Inmovilizados (IMAC) para la purificación de proteínas recombinantes con etiquetas de Histidina
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Fecha
2024
Autores
Avila Carbono, Adan De Jesus
Lara Vargas , Guillermo Enrique
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Ediciones Universidad Simón Bolívar
Facultad de Ciencias Básicas
Facultad de Ciencias Básicas
Resumen
La purificación de proteínas es un proceso crucial en la investigación biomédica y
biotecnológica, y la cromatografía de afinidad de metales inmovilizados (IMAC) ha
demostrado ser una herramienta poderosa y altamente eficaz para este fin. Esta
técnica se basa en la interacción entre los grupos donantes de electrones de los
residuos de histidina o cisteína presentes en la proteína de interés y los iones
metálicos inmovilizados en una matriz cromatográfica. La proteína se une
selectivamente a la columna IMAC, mientras que las impurezas pasan sin unirse.
La proteína purificada se elude posteriormente mediante un cambio en las
condiciones del buffer, rompiendo la interacción entre la proteína y el ion metálico.
IMAC se utiliza ampliamente para purificar proteínas recombinantes que llevan una
etiqueta de polihistidina. Esta etiqueta se fusiona genéticamente a la proteína diana
durante la expresión y proporciona una alta afinidad por los iones metálicos
divalentes, lo que permite una purificación eficiente y específica. La técnica es
versátil y se puede utilizar para purificar una amplia gama de proteínas
recombinantes de diferentes fuentes, incluyendo bacterias, levaduras, células de
mamíferos y plantas. Para lograr resultados eficaces con IMAC, es importante
considerar varios aspectos, como la naturaleza de la matriz cromatográfica, la
selección del ligando adecuado, la concentración de iones metálicos y las
condiciones del buffer. La optimización de estas variables es crucial para obtener
un alto rendimiento y pureza de la proteína purificada.
El proyecto de investigación descrito en el texto tiene como objetivo desarrollar un
protocolo de estandarización para la síntesis de columnas de cromatografía IMAC
para la purificación "in-house" de proteínas recombinantes marcadas con etiquetas
de histidina. El proyecto evaluó la eficiencia de columnas previamente sintetizadas
y propone un modelo a mayor escala para optimizar las variables del proceso. La
estandarización de la técnica IMAC permitirá obtener columnas de alta calidad y
eficiencia para la purificación de proteínas recombinantes en investigación y
producción
This study describes the research carried out to develop a standardisation protocol for the synthesis of IMAC columns for the in-house purification of recombinant histidine-tagged proteins. Previously synthesised columns were evaluated at the Centro de Investigación e Innovación en Cambio Climático y Biodiversidad (ADAPTIA) of the Universidad Simón Bolívar in Barranquilla, Colombia. A proposal was formalised to optimise the process variables, such as the power and amidation time of the APTS by microwave method, the pH of the EDTA solution, the concentration of residual Ni, the concentration of the elution solution and the presence of the GFP protein. The results confirmed that IMAC chromatography is a highly efficient method for the purification of GFP protein from E. coli BL21 transformed with plasmid pET28a+GFP. The nickel-loaded silica column was able to capture GFP specifically, taking advantage of the affinity between nickel ions and the polyhistidine tag. Conditions for GFP binding were optimised using a pH of 2.2 during EDTA binding on the column. Polyacrylamide gels and fluorescence under UV light confirmed the high purity of GFP obtained after the purification process. It was observed that the higher the molar concentration of the imidazole buffer in the elution step, the higher the effectiveness of the process. The efficiency of IMAC purification may vary depending on the specific protein and experimental conditions. Pilot testing is recommended to optimise the process for each specific application. The IMAC column can also be used as a matrix to immobilise enzymes containing a polyhistidine tag in their structure. The high concentrations of imidazole required for protein elution are not found naturally in the environment, which makes this technique especially useful in industrial or bioremediation processes, where immobilised enzymes can convert a substrate into a desired product. Immobilised metal affinity chromatography is presented as a robust, efficient and versatile tool for the purification of recombinant proteins, with great potential for diverse applications, including biomedical research, pharmaceuticals and bioremediation.
This study describes the research carried out to develop a standardisation protocol for the synthesis of IMAC columns for the in-house purification of recombinant histidine-tagged proteins. Previously synthesised columns were evaluated at the Centro de Investigación e Innovación en Cambio Climático y Biodiversidad (ADAPTIA) of the Universidad Simón Bolívar in Barranquilla, Colombia. A proposal was formalised to optimise the process variables, such as the power and amidation time of the APTS by microwave method, the pH of the EDTA solution, the concentration of residual Ni, the concentration of the elution solution and the presence of the GFP protein. The results confirmed that IMAC chromatography is a highly efficient method for the purification of GFP protein from E. coli BL21 transformed with plasmid pET28a+GFP. The nickel-loaded silica column was able to capture GFP specifically, taking advantage of the affinity between nickel ions and the polyhistidine tag. Conditions for GFP binding were optimised using a pH of 2.2 during EDTA binding on the column. Polyacrylamide gels and fluorescence under UV light confirmed the high purity of GFP obtained after the purification process. It was observed that the higher the molar concentration of the imidazole buffer in the elution step, the higher the effectiveness of the process. The efficiency of IMAC purification may vary depending on the specific protein and experimental conditions. Pilot testing is recommended to optimise the process for each specific application. The IMAC column can also be used as a matrix to immobilise enzymes containing a polyhistidine tag in their structure. The high concentrations of imidazole required for protein elution are not found naturally in the environment, which makes this technique especially useful in industrial or bioremediation processes, where immobilised enzymes can convert a substrate into a desired product. Immobilised metal affinity chromatography is presented as a robust, efficient and versatile tool for the purification of recombinant proteins, with great potential for diverse applications, including biomedical research, pharmaceuticals and bioremediation.
Descripción
Palabras clave
Cromatografia, IMAC, Resina, Purificación, Proteína
