Sobreexpresión heteróloga y purificación de TAQ ADN polimerasa en Escherichia coli Rosetta (DE3)
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Fecha
2025
Autores
Jimenez Bilbao, Gabriela
Sanjuan Nieto, Saymy Geysay
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Ediciones Universidad Simón Bolívar
Facultad de Ciencias Básicas y Biomédicas
Facultad de Ciencias Básicas y Biomédicas
Resumen
La creciente necesidad de desarrollo científico e innovación tecnológica trae consigo una alta demanda de insumos para su ejecución, entre ellos, en la investigación e industria biotecnológica los reactivos y materiales para técnicas de laboratorio y ensayos como la reacción en cadena de la polimerasa (PCR). En épocas de crisis como las emergencias sanitarias provocadas por patógenos emergentes, la PCR se convierte en una herramienta ampliamente utilizada para el diagnóstico de infecciones y/o enfermedades, por ello, se aumenta la demanda de los reactivos necesarios para su realización como primers, ADN polimerasas, nucleótidos o kits de diagnóstico, los cuales son priorizados para los países productores. En Colombia, la producción de insumos biológicos como las vacunas y productos orgánicos era alta, sin embargo, con el cierre de la planta de producción biológica del Instituto Nacional de Salud en la década de 1990, la producción de insumos biológicos y biotecnológicos decayó, por lo cual la dependencia biotecnológica a la importación de reactivos e insumos se convierte en una problemática, principalmente en tiempos de crisis. A pesar de esto, el auge de tecnologías de ADN recombinante como la expresión de proteínas recombinantes surge como una estrategia ideal para mitigar esta problemática, por esta razón la presente investigación busca producir Taq ADN polimerasa mediante un sistema de sobreexpresión heteróloga de proteínas en Escherichia coli Rosetta. Para lograr la producción de Taq polimerasa termoestable, se realizó una revisión de la literatura para identificar las características genéticas y fenotípicas de la cepa seleccionada que favorecen una mayor productividad y eficiencia en la expresión de proteínas. Con base en esta información, se estandarizaron de métodos y protocolos de cultivo y expresión para la producción de Taq ADN polimerasa, partiendo de células de E. coli biotecnológica Rosetta (DE3) transformadas con el vector de expresión pOpen_taq. El proceso experimental contempló la evaluación de distintas condiciones experimentales con el objetivo de optimizar la concentración de IPTG (isopropilo-β-D-1-tiogalactopiranósido) como molécula inductora, el tiempo de incubación y la temperatura del cultivo.
The growing need for scientific development and technological innovation brings with it a high demand for supplies for its execution, including reagents and materials for laboratory techniques and tests such as the polymerase chain reaction (PCR) in research and the biotechnology industry. In times of crisis, such as health emergencies caused by emerging pathogens, PCR becomes a widely used tool for diagnosing infections and/or diseases. Therefore, the demand for the reagents necessary for its implementation, such as primers, DNA polymerases, nucleotides, and diagnostic kits, increases, and these are prioritized for producing countries. In Colombia, the production of biological supplies such as vaccines and organic products was high; however, with the closure of the National Institute of Health's biological production plant in the 1990s, the production of biological and biotechnological supplies declined, making biotechnology's dependence on imported reagents and supplies a problem, especially in times of crisis. Despite this, the rise of recombinant DNA technologies such as recombinant protein expression emerges as an ideal strategy to mitigate this problem. The present research seeks to produce Taq DNA polymerase through a heterologous protein overexpression system in Escherichia coli Rosetta (DE3). To achieve the production of thermostable Taq polymerase, a literature review was conducted to identify the genetic and phenotypic characteristics of the selected strain that favor greater productivity and efficiency in protein expression. Based on this information, culture and expression methods and protocols were standardized for the production of Taq DNA polymerase, starting from the biotechnological strain E. coli Rosetta (DE3) cells transformed with the pOpen_taq expression vector. The experimental process contemplated the evaluation of different experimental conditions with the aim of optimizing the concentration of IPTG (isopropyl-βD-1-thiogalactopyranoside) as an inducing molecule, the incubation time and the culture temperature.
The growing need for scientific development and technological innovation brings with it a high demand for supplies for its execution, including reagents and materials for laboratory techniques and tests such as the polymerase chain reaction (PCR) in research and the biotechnology industry. In times of crisis, such as health emergencies caused by emerging pathogens, PCR becomes a widely used tool for diagnosing infections and/or diseases. Therefore, the demand for the reagents necessary for its implementation, such as primers, DNA polymerases, nucleotides, and diagnostic kits, increases, and these are prioritized for producing countries. In Colombia, the production of biological supplies such as vaccines and organic products was high; however, with the closure of the National Institute of Health's biological production plant in the 1990s, the production of biological and biotechnological supplies declined, making biotechnology's dependence on imported reagents and supplies a problem, especially in times of crisis. Despite this, the rise of recombinant DNA technologies such as recombinant protein expression emerges as an ideal strategy to mitigate this problem. The present research seeks to produce Taq DNA polymerase through a heterologous protein overexpression system in Escherichia coli Rosetta (DE3). To achieve the production of thermostable Taq polymerase, a literature review was conducted to identify the genetic and phenotypic characteristics of the selected strain that favor greater productivity and efficiency in protein expression. Based on this information, culture and expression methods and protocols were standardized for the production of Taq DNA polymerase, starting from the biotechnological strain E. coli Rosetta (DE3) cells transformed with the pOpen_taq expression vector. The experimental process contemplated the evaluation of different experimental conditions with the aim of optimizing the concentration of IPTG (isopropyl-βD-1-thiogalactopyranoside) as an inducing molecule, the incubation time and the culture temperature.
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Palabras clave
ADN polimerasa, Sobreexpresión de proteínas, PCR
