Purificación y caracterización de transcriptasas reversas novedosas
No hay miniatura disponible
Archivos
Fecha
2023
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
Título de la revista
ISSN de la revista
Título del volumen
Editor
Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
La enzima retrotranscriptasa (RT) es una polimerasa que forma una molécula de ADN a partir de
un templado de ARN. Se han identificado enzimas RT en virus Retroviridae y retrotransposones, además
de organismos eucariontes y procariontes. Desde su descubrimiento, se ha estudiado su estructura, función
y se le dio uso como herramienta para pruebas moleculares como RT-PCR y RT-qPCR en el diagnóstico
clínico.
Los retrotransposones son elementos genéticos móviles que generalmente codifican a enzimas con
actividad RT y pueden encontrarse en el genoma de todos los organismos. El laboratorio de virología ha
trabajado en identificar la presencia y expresión de retrotransposones en moluscos bivalvos, y hasta el
momento, se han encontrado dos de ellos: Steamer y Steamer Like Element 1 (SLE1). Ambos codifican a
una enzima RT; sin embargo, no han sido purificada ni caracterizadas. En este trabajo se buscó purificar y
evaluar la actividad RT para los dominios retrotranscriptasa de Steamer y SLE1. Las secuencias codificantes
para estos dominios fueron clonadas en vectores de expresión y transformadas en Escherichia coli, para ser
purificados por cromatografía de afinidad. Se estandarizó un protocolo para detectar actividad RT sin
radioactividad con PicoGreen y se establecieron las condiciones óptimas de expresión procarionte para las
proteínas en su estado soluble RT-Steamer y RT-SLE1. Los resultados de ese trabajo, además de contribuir
a desarrollo de nuevas herramientas, aportarán con una metodología para la purificación de proteínas RT, y
su posterior evaluación como enzimas
The enzyme retrotranscriptase (RT) is a polymerase that forms a DNA molecule from an RNA template. RT enzymes have been identified in Retroviridae viruses and retrotransposons, as well as in eukaryotic and prokaryotic organisms. Since their discovery, their structure and function have been studied and they have been used as a tool for molecular tests such as RT-PCR and RT-qPCR in clinical diagnostics. Retrotransposons are mobile genetic elements that generally encode enzymes with RT activity and can be found in the genome of all organisms. The virology laboratory has worked to identify the presence and expression of retrotransposons in bivalve mollusks, and so far, two of them have been found: Steamer and Steamer Like Element 1 (SLE1). Both encode a RT; however, no RT present in these retrotransposons has been purified or characterized. In this work we sought to purify and evaluate the RT activity for the RT domains of Steamer and SLE1. The coding sequences for these domains were cloned into expression vectors and transformed into Escherichia coli, to be purified by affinity chromatography. A protocol was standardized to detect RT activity without radioactivity with PicoGreen and optimal expression conditions were established for RT-Steamer and RT-SLE1 proteins in their soluble state. The results of this work, besides contributing to the development of new tools, will provide a methodology for the purification of RT proteins and their subsequent evaluation as enzymes.
The enzyme retrotranscriptase (RT) is a polymerase that forms a DNA molecule from an RNA template. RT enzymes have been identified in Retroviridae viruses and retrotransposons, as well as in eukaryotic and prokaryotic organisms. Since their discovery, their structure and function have been studied and they have been used as a tool for molecular tests such as RT-PCR and RT-qPCR in clinical diagnostics. Retrotransposons are mobile genetic elements that generally encode enzymes with RT activity and can be found in the genome of all organisms. The virology laboratory has worked to identify the presence and expression of retrotransposons in bivalve mollusks, and so far, two of them have been found: Steamer and Steamer Like Element 1 (SLE1). Both encode a RT; however, no RT present in these retrotransposons has been purified or characterized. In this work we sought to purify and evaluate the RT activity for the RT domains of Steamer and SLE1. The coding sequences for these domains were cloned into expression vectors and transformed into Escherichia coli, to be purified by affinity chromatography. A protocol was standardized to detect RT activity without radioactivity with PicoGreen and optimal expression conditions were established for RT-Steamer and RT-SLE1 proteins in their soluble state. The results of this work, besides contributing to the development of new tools, will provide a methodology for the purification of RT proteins and their subsequent evaluation as enzymes.
Notas
Tesis (Ingeniero en Biotecnología)
Palabras clave
Enzimas, Purificación, Reacción en Cadena de la Polimerasa de Transcriptasa Inversa, Escherichia Coli, Biotecnología