The authors would like to acknowledge Natalie Doude and David Westerway (University of Alberta) for providing us Sho cDNA, Christophe Chevalier (INRA) for plasmid construction, Michel Br&#233;mont (INRA) for providing us BL21Sol bacteria; Christine Longin (INRA) for TEM assistance and Edith Pajot-Augy (INRA) for comments and proofreading.

1. Generación de plásmido y Shadoo Expresión Nota: La proteína murina Shadoo gen que codifica (Shadoo 25 a 122), se subclonó en el vector de expresión pET-28 11. Este clon se transformó en E. SoluBL21 coli cepa bacteriana, para expresar la proteína Shadoo con una etiqueta de hexahistidina N-terminal, (HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG) como se explicó previamente 11. El plásmido se puede solicitar a los autores. El peso molecular de Shadoo calculado a partir de su secuencia de aminoácidos es 12,243.2 Da. Nota: Realice todas las manipulaciones con bacterias bajo una campana de flujo laminal o cerca de Bunsen llama. <ol><li> Transformar bacterias SoluBL21 competentes con el 28-Su plásmido pET -Shadoo 6, utilizando un protocolo de choque térmico estándar. Para esta mezcla 1 a 5 l de la plásmido (típicamente 10 a 100 pgng) en 50 l de las bacterias en un tubo. Realice el choque térmico a 42 ° C durante 45 segundos. </li><li> Cultura bacterias transformadas a 37 ° C hasta 600 A de 0,5 a 0,7 en un medio Luria-Bertani suplementado con 40 mg / ml de kanamicina (Figura 1B). </li><li> Inducir Shadoo expresión O / N mediante la adición de 240 mg / ml de isopropil-β-D-tiogalactopiranósido, ITPG, a la cultura. Cultura de las bacterias bajo agitación a 150 rpm a 37 ° C. Nota: Por lo general, la cultura bacteriana alcanza un 600 de 3 O / N. Nota: Transformado bacterias pueden ser almacenadas a -80 ° C para la futura producción de proteínas. Para ello, agregue el 10% -50% (v / v) de glicerol estéril en alícuotas de cultivo bacteriano y congelar a -80 ° C. </li></ol><img alt="Figura 1" src="/files/ftp_upload/53432/53432fig1.jpg" /> Figura 1. Esquema de Shadoo construcción de expresión, las bacterias transformación y la inducción de la expresión Shadoo (como se describe en el paso 1). </strong&gt; (A) El constructo de expresión para Shadoo codifica una etiqueta de hexahistidina fusionado al extremo N-terminal de la proteína, Nt. El esquema muestra que la proteína Shadoo contiene repeticiones básicos arginina / glicina (cilindro verde), un dominio hidrófobo (HD), y un único sitio de N-glicosilación (CHO), seguido de un terminal C (Ct). (B) pET-28-Su 6 -Shadoo plásmido se transforma en bacterias SoluBL21 por choque térmico de 45 segundos a 42 ° C. Bacterias transformadas se sembraron en agar selectivo que contenía 40 mg / ml de kanamicina. Una sola colonia de la placa se utiliza para inocular un cultivo en un matraz de agitador de 1.000-3.000 ml. La expresión de la proteína recombinante se indujo con IPTG 1 mM. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig1highres.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a> 2. Purificación de Shadoo proteínas por baja presión cromatografía líquida (LPLC) <ol><li> La lisis celular e Inconclusión preparación cuerpos <ol><li> Recoger la suspensión bacteriana y la transfiere en tubos de centrifugación (Figura 2). </li><li> Giran a 2500 xg, durante 20 min a 4 ° C. Eliminar el sobrenadante por decantación y pellets de resuspender en 15 ml de tampón (Tris 50 mM, EDTA 10 mM, pH 8 con cóctel anti-proteasa) para cada pellet derivado de 500 ml de cultivo celular. Nota: Las bolitas resuspendidas pueden ser congelados y almacenados a -20 ° C durante varios días. La congelación facilita la lisis de células bacterianas. </li><li> Compruebe la sobre-expresión de la proteína recombinante utilizando bacterias crudo gránulos mediante análisis de SDS-PAGE y tinción de Coomassie. <ol><li> Para ello, de centrífuga de 1 ml de cultivo bacteriano para sedimentar las células. Añadir 100 l solución de Laemmli de desnaturalización (mM Tris-HCl 277,8, pH 6,8, 4,4% de SDS, 44,4% w / v de glicerol, 0,02% azul de bromofenol) para sedimentar y se calienta a 100 ° C durante 5 min. </li><li> Cargar 10 l de la muestra a un gel de acrilamida al 12%, ejecute SDS-PAGE y visualize separa proteínas por tinción de Coomassie. </li></ol></li><li> Añadir Triton X-100 hasta una concentración final 0,5% al ​​sedimento resuspendido completamente a partir de 2.1.2). </li><li> Se incuba la suspensión en baño de agua a 37 ° C durante 30 min. </li><li> Sonicar la suspensión durante 5 min a 6-12 micras amplitud pico a pico en agua enfriada con hielo para lisar células bacterianas. </li><li> Transferencia sonicó la suspensión en 50 ml tubos de centrifugación limpio. </li><li> Tubos centrífuga a 15.000 xg durante 15 min a 4 ° C. </li><li> Eliminar el sobrenadante por decantación y añadir 5 ml de tampón de unión (20 mM de tampón Tris-HCl, pH 7,4, NaCl 0,5 M, 8 M urea, 5 mM imidazol) por tubo. </li><li> Colocar los tubos en la rotación de la rueda O / N para volver a suspender por completo las proteínas a partir de cuerpos de inclusión. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53432/53432fig2.jpg" /> Figura 2. Esquema de purificación de inclusióncuerpos (como se describe en el paso 2.1). transformadas bacterias que expresan Shadoo se recogen por centrifugación y se resuspendieron en el tampón de lisis. La suspensión se incubó a 37 ° C durante 30 min, y luego se sometió a ultrasonidos para romper mecánicamente las células bacterianas. La sonicación se lleva a cabo en hielo para evitar el calentamiento de la muestra. Células rotas se recogieron por centrifugación y se resuspendieron en un tampón que contiene urea 8 M para solubilizar proteínas a partir de cuerpos de inclusión. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig2highres.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a> </li><li> Cromatografía de Ni 2+ -affinity Nota: La cromatografía se realiza en un sistema de FPLC usando Ni2 + -charged, columna quelante de 5 ml de Sepharose (Figura 3A). Todas las soluciones se cargan en la columna con un caudal de 1 ml / min. <ol><li> Inyectar 10 ml de solución 4 agua 0,2 M NiSO _ en la columna de Sepharose con el fin de cargarlacon iones de Ni 2+. </li><li> Equilibrar la columna cargada con Ni 2+ iones mediante la inyección de un 30-50 ml de tampón de unión que contiene la una baja concentración de imidazol (20 mM de tampón Tris-HCl, pH 7,4, NaCl 0,5 M, 8 M urea, 5 mM imidazol). </li><li> Cargar las proteínas de urea-solubilizado con un bucle de inyección de 50 ml. </li><li> Recuperar la fracción de flujo a través lavando a fondo la columna con tampón de 10 ml bajo imidazol de unión (20 mM de tampón Tris-HCl, pH 7,4, NaCl 0,5 M, 8 M urea, 5 mM imidazol) con el fin de eliminar un máximo de proteínas no unidas de la columna. </li><li> Lavar la columna con 50-100 ml de tampón de lavado (20 mM Tris-HCl, pH 7,4, NaCl 0,5 M, 8 M urea, 80 mM imidazol) para eluir las proteínas unidas de forma no específica a la columna. </li><li> Aplicar un gradiente lineal 15 min de 80 a 800 mM imidazol en el tampón que contiene 20 mM Tris-HCl, pH 7,4, NaCl 0,5 M, 8 M urea. Recoger fracciones de 1 ml durante la aplicación del gradiente. Nota: Las proteínas etiquetadas-Su se eluyen ingeniogradiente ha de tampón que contiene imidazol (usado como competidor). </li><li> Tomar una alícuota de 8 l de cada fracción y añadir 4 l solución de desnaturalización 4x Laemmli (ver 2.1.3.1). Soluciones de calor a 100 ° C durante 5 min. </li><li> Cargar 10 l de tamaño proteína marcadora y 10 l de cada muestra en el gel de acrilamida al 12%, correr SDS-PAGE y visualizar proteínas separadas por tinción de Coomassie. Nota: El peso molecular calculado de Shadoo formar su secuencia de aminoácidos es 12,243.2 Da. </li><li> Piscina todas las fracciones que contienen Shadoo. </li></ol><img alt="figura 3" src="/files/ftp_upload/53432/53432fig3.jpg" /> Figura 3. procedimiento de purificación (tal como se describe en los pasos 2.2-2.4). (A) Ni 2+ iones pueden ser coordinados por los residuos de histidina, lo que permite la purificación selectiva de proteínas polihistidina-etiquetado. El imidazol se utiliza para eluir la proteína, a través desu capacidad de coordinar con el 2+ -ion Ni y desplazar la proteína unida. (B) Las proteínas de diferente radio hidrodinámico que fueron retenidos en el Ni2 + -columna están separados en una columna de exclusión de tamaño. Las proteínas se eluyen de la más grande a los más pequeños. (C) Antes de su uso, las fracciones que contenían Shadoo se reúnen y se desalaron para eliminar la urea, imidazol y sales. Ensayos de control de calidad incluyen SDS-PAGE / tinción Coomassie, Western Blot. Purificada Shadoo puede ser liofilizado. Liofilizado Shadoo es estable durante meses cuando se almacena a -20 ° C. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig3highres.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a> </li><li> Cromatografía de exclusión por tamaño Nota: Una segunda etapa de purificación consiste en la cromatografía de exclusión por tamaño que separa la proteína Shadoo de las otras proteínas co-eluidos durante la primera etapa de purificación(Figura 3B). <ol><li> Equilibrar la columna Superdex de 120 ml de volumen total del lecho mediante la inyección de 250 ml de 20 mM de tampón Tris-HCl, pH 7,4, NaCl 0,5 M, 8 M urea en la velocidad de flujo de 1,5 ml / min. </li><li> Cargar fracciones reunidas que contienen Shadoo de 2.2.9 en la columna equilibrada. Recoger fracciones de 1 ml después de la volumen vacío de la columna de 40 ml sobre el flujo constante del tampón anterior. </li><li> Tomar una alícuota de 10 l de cada fracción y le realizará un análisis de SDS-PAGE y tinción con Coomassie como se describió anteriormente para saber qué contienen fracciones Shadoo. </li><li> Fracciones que contienen Shadoo piscina. </li></ol></li><li> Desalación Nota: procedimiento de desalación elimina sales, imidazol y urea para obtener la proteína recombinante Shadoo en un tampón adecuado para posteriores estudios biofísicos y bioquímicos (Figura 3C). El mismo resultado se puede obtener muestras de diálisis contra el tampón. <ol><li> Equilibrar una columna de desalación de 53 ml con 150 mlde 10 mM de tampón de acetato de amonio, pH 5, a un caudal de 5 ml / min. </li><li> Cargar agruparon fracciones Shadoo en la columna. Tras la inyección del tampón de acetato de amonio recoger manualmente fracciones que muestran un aumento de la señal a 280 nm en la salida del detector de UV. </li><li> Liofilizar la Shadoo desalada y almacenarla a -20 ° C. </li><li> Disolver Shadoo liofilizado en un tampón adecuado, mM tampón tal como acetato de sodio 10, pH 5, antes de su uso. Nota: La etiqueta de polihistidina se puede eliminar con enteroquinasa. </li></ol></li></ol> 3. En Vitro Montaje de Shadoo en amiloide fibrillas en Physiological pH Nota: Los agregados de proteínas Shadoo y espontáneamente forman fibrillas a pH ≥ 7 11. <img alt="Figura 4" src="/files/ftp_upload/53432/53432fig4.jpg" /> Figura 4. Esquema de Shadoo replegamiento a las fibrillas de amiloide (como se descricama en el paso 3). purificada Shadoo convierte espontáneamente a las fibrillas de amiloide cuando se disuelve en tampón de pH ≥ 7. En una solución ácida, Shadoo convierte a las fibrillas de amiloide tras la incubación con M Gdn-HCl 1 mientras se agitaba. Ensayos de control de calidad incluyen la microscopía electrónica y tinción ThT. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig4highres.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a> <ol><li> Diluir Shadoo a una concentración final de 2 mM en un tampón mM Tris-HCl 20, pH 7,4. Medir la concentración midiendo la densidad óptica de la solución a 280 nm y usando el coeficiente de extinción deducida a partir de la composición de aminoácidos Shadoo de 20.970. </li><li> Incubar 500 l de la solución en un tubo de plástico cónico a 4 ° C durante un par de semanas, para permitir la conversión espontánea de proteínas a las estructuras amiloides (Figura 4). </li><li> Agregar recién preparada tioflavina T (ThT) a un conce definitivantration de 10 micras a una alícuota de 100 l de la solución Shadoo. Incubar la solución durante 5 min a RT. Medida de emisión de fluorescencia 460-520 nm usando una longitud de onda de excitación de 435 nm para verificar la presencia de estructuras amiloides 17. </li></ol> 4. En Vitro Montaje de Shadoo de amiloide fibrillas en ácida pH <ol><li> Diluir Shadoo en 1 M GdnHCl, 20 mM de tampón Na-acetato, pH 5 a una concentración final de 2 mM. </li><li> Incubar 500 l de esta solución en un tubo cónico con agitación continua a 37 ° C, durante 3-7 días. </li><li> Compruebe la presencia de fibrillas amiloides añadiendo ThT a una alícuota de la solución como en 3.3. </li><li> Dializar suspensión obtenida contra 10 mM de tampón Na-acetato, pH 5,0, para purificar las fibrillas de amiloide Shadoo. </li><li> Guarde fibrillas purificados a 4 ° C. </li></ol><img alt="Figura 5" src="/files/ftp_upload/53432/53432fig5.jpg"/&gt; Figura 5. Los resultados representativos. Las fracciones proteicas eluidas de Ni 2+ -charged columna quelante de (A) y la columna de exclusión por tamaño (B) se analizaron mediante SDS-PAGE y tinción de Coomassie. Identificación de Shadoo purificado se evaluó a través de anticuerpo anti-Shadoo SPRN (C). <a href="https://www.jove.com/files/ftp_upload/53432/53432fig5large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

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The authors have nothing to disclose.

Se presenta un protocolo fácil y eficiente tanto para la expresión y purificación de una gran cantidad de proteína Shadoo recombinante de ratón. El método descrito permite la solubilización y purificación de (Su 6) proteína recombinante Shadoo-etiquetados éxito. Es importante, como se ha señalado en el título, que cuando se expresa en una bacteria procariota E. coli Shadoo se acumula en cuerpos de inclusión. Antes de la purificación Shadoo, las bacterias tienen que ser lisadas por sonic...

Enfermedades neurodegenerativas causadas por priones, que incluyen la encefalopatía espongiforme bovina en el ganado, la tembladera en las ovejas y la enfermedad de Creutzfeldt-Jakob en humanos, son fatales e incurable. Las enfermedades priónicas se caracterizan por cambios conformacionales de la proteína priónica celular compuestos principalmente de alfa-hélices, en el β-hoja de cruz enriquecido amiloide confórmero 1,2. Cruz-ß-estructuras contienen densas y ß-hojas-cristalinas como altamente ordenadas estabilizados con enlaces de hidrógeno 3,4. Amiloides priónicas puede auto-replicarse debido a los beta-capítulos en el borde de crecimiento que proporcionan una plantilla para el reclutamiento y la conversión de una unidad de proteína monomérica. De acuerdo con la hipótesis de &quot;única proteína&quot;, el confórmero de amiloide de la proteína prión es el único agente infeccioso. Sin embargo, se han propuesto algunas otras biomoléculas ser esencial para los trastornos por priones. Por ejemplo, las membranas celulares se cree que ser un lugar donde conversien su lugar y algunos toma lípidos cargados negativamente se ha demostrado para mejorar el proceso de conversión de 5,6. Además, algunas proteínas también pueden estar implicados en patologías priónicas. En concreto, hay otros dos miembros de la familia de proteínas de priones: Doppel y Shadoo 7,8. Doppel tiene una estructura relativamente rígida estabilizado con puentes di-sulfuro y no autopolimerizarse y el agregado de fibrillas amiloides 9. En contraste, similar a la proteína prión, Shadoo puede adoptar una estructura enriquecida en β-lámina y autoasociarse en estructuras de fibrillas amiloides. Se demostró que Shadoo puede fibrilar en condiciones nativas o tras la unión membranas cargadas negativamente 10,11. La conversión de Shadoo en fibras de tipo amiloide puede estar asociada con patologías. De hecho, los mecanismos por los cuales las estructuras de tipo amiloide causan la enfermedad son poco conocidos. Shadoo tiene un dominio hidrófobo (HD) y una serie de tándem Arg / Gly repite t similareso la parte N-terminal de la proteína prión (Figura 1A). A medida que la parte N-terminal de la proteína prión, Shadoo está altamente cargado positivamente y parece ser una proteína nativa no estructurada 11,12. Shadoo parece ser funcionalmente relacionadas con los trastornos por priones, ya que puede unirse directamente la proteína del prión. Además, su expresión es regulada hacia abajo durante la patología prión 13,14. Sin embargo, el papel de Shadoo en la enfermedad priónica todavía no se ha establecido. Hemos desarrollado un plásmido que porta la secuencia de codificación del gen Shadoo ratón. El plásmido se utilizó para transformar E. coli para la producción de N-terminal Su proteína Shadoo 6 de fusión. Este sistema de expresión está bien establecida en nuestro laboratorio y se utiliza comúnmente en nuestros proyectos en curso 6,15,16. Una cuestión importante para la expresión de Shadoo es la elección de la E. coli cepa. Mientras cepa bacteriana BL21 competente se utiliza comúnmente para la expresión de pro recombinanteproteínas Shadoo se expresó y se purifica sólo de la cepa bacteriana transformada SoluBL21 éxito. SoluBL21 E. competente coli es un mutante mejorada de cepa huésped BL21 desarrollado para la producción de proteínas cuya expresión en la BL21 padre no dio producto soluble detectable. Una expresión de alto nivel de Shadoo en SoluBL21 E. coli conduce a la acumulación de la proteína en cuerpos de inclusión. Como característica general, cuando una proteína no nativa es altamente expresado en E. coli, esta proteína tiende a acumularse en los cuerpos de inclusión insolubles. Shadoo probablemente agregados a través de interacciones hidrófobas no covalentes o iónicos (o una combinación de ambos) para estructuras dinámicas altamente enriquecido formadas por la proteína plegada en diversos grados. En consecuencia, la purificación comprende al menos dos etapas: (i) una separación selectiva de la proteína a partir de otras biomoléculas en un medio de desnaturalización, y (ii) una renaturalización de la proteína purificada utilizando en el plegamiento in vitrotécnicas. La separación selectiva de Shadoo se logró en una solución tampón que contiene urea 8 M (o, alternativamente, guanidina-HCl 6 M). La eliminación de urea y de la renaturalización de la proteína se puede hacer mediante la aplicación de diversos protocolos de: (i) la renaturalización en una solución de pH ácido para obtener una proteína Shadoo monomérica no estructurada, o (ii) la renaturalización en una solución de pH ≥ 7 para obtener Shadoo polimerizado en fibras amiloides estables con motivos característicos cruz β-hoja.

<table>
	<tbody>
		<tr>
			<td>AKTA FPLC&#160;</td>
			<td>GE, Amersham</td>
			<td># 1363</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/60 Superdex</td>
			<td>GE Healthcare</td>
			<td>17-1069-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap IMAC</td>
			<td>GE Healthcare</td>
			<td>GE17-0920-05</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep26/10 Desalting column</td>
			<td>GE Healthcare</td>
			<td>GE17-5087-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SoluBL21 Competent E. coli</td>
			<td>Genlantis</td>
			<td>C700200</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-28b+</td>
			<td>Novogen</td>
			<td>69865-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Broth medium</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Biophotometar</td>
			<td>Eppendorf</td>
			<td>550507804</td>
			<td></td>
		</tr>
		<tr>
			<td>Trito X-100</td>
			<td>Life Technologies</td>
			<td>85111</td>
			<td></td>
		</tr>
		<tr>
			<td>NiSO4</td>
			<td>Sigma-Aldrich</td>
			<td>656895</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Gdn-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr>
			<td>protein size marker&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>26620</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli

El sistema de expresión de Escherichia coli es una poderosa herramienta para la producción de proteínas recombinantes eucariotas. Lo utilizamos para producir Shadoo, una proteína perteneciente a la familia de priones. Un método cromatográfico para la purificación de (His) 6-etiquetados Shadoo recombinantes expresadas como cuerpos de inclusión se describe. Los cuerpos de inclusión se solubilizan en 8 M urea y unidos a una columna de Ni 2+ -charged para llevar a cabo la cromatografía de afinidad de iones. Las proteínas unidas se eluyen mediante un gradiente de imidazol. Las fracciones que contienen la proteína Shadoo son sometidas a cromatografía de exclusión por tamaño para obtener una proteína altamente purificada. En la etapa final de Shadoo purificada se desala para eliminar las sales, urea e imidazol. Shadoo proteína recombinante es un reactivo importante para los estudios biofísicos y bioquímicos de los trastornos de la conformación de proteínas que ocurren en las enfermedades priónicas. Muchos informes han demostrado que las enfermedades neurodegenerativas causadas por priones originan a partir de la deposición de puñaladale ordenó fibrillas amiloides. / Se presentan valores de pH básicos protocolos de ejemplo que describen cómo fibrilar Shadoo en fibrillas amiloides en ácido y neutro. Los métodos sobre cómo producir y fibrilar Shadoo puede facilitar la investigación en laboratorios que trabajan sobre las enfermedades priónicas, ya que permite la producción de grandes cantidades de proteína en una manera rápida y de bajo coste.

Acerca de 5-10 mg de proteína purificada Shadoo se obtiene por litro de cultivo bacteriano. Se necesita una purificación de dos pasos para obtener Shadoo recombinante de alta pureza. El primer paso se lleva a cabo mediante cromatografía de afinidad con la columna -charged una de Ni 2+ que conserva sus-proteínas etiquetadas. Las fracciones eluidas se sometieron a SDS-PAGE y se tiñeron con azul brillante de Coomassie (Figura 5A). Las fracciones que contenían la proteína Shadoo se reúnen...

Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Purificación y replegamiento de amiloide fibrillas de (Su)<sub&gt; 6</sub&gt;-etiquetados proteína recombinante Shadoo Expresado como cuerpos de inclusión en<em&gt; E. coli</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

purification-refolding-to-amyloid-fibrils-his6-tagged-recombinant

Research

JoVE Journal

Biology

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

14.1K vistas.  INRA. A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures. 

Video: Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Investigación

Biología

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagénesis y funcional protocolos de selección de la evolución dirigida de proteínas en E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

La purificación de Hsp104, una proteína Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificación de los complejos de proteínas en<em&gt; Escherichia coli</em&gt; La purificación mediante afinidad del péptido secuencial en combinación con espectrometría de masas tándem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Los ensayos acoplados para Monitoreo replegamiento de proteínas en<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

La purificación de la fibrosis quística regulador de conductancia transmembrana de proteínas expresadas en<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening Expresión cuantitativa y Purificación Aplicada a Recombinante Disulfide ricos en proteínas del veneno Producido en<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

La purificación no cromatográfico de polipéptidos recombinantes Elastina-como y sus fusiones con Péptidos y Proteínas de<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.