Este trabajo fue apoyado por una subvención del NIH (5T32GM008275-22) y la American Heart beca predoctoral Asociación (a EAS), un interfaz química-biología beca del NIH (2T32GM071339-06A1) (a MED), y subvenciones de la NIH (1DP2OD002177-01 y NS067354 0110), la Ellison Medical Foundation y la Fundación Bill y Melinda Gates (a JS).

1. Expresión de Hsp104 <ol><li> El plásmido utilizado para la purificación de E. coli, pPROEX-HTB-Hsp104, contiene el Hsp104 abierto marco de lectura bajo el control inducible del promotor trc 26. El plásmido produce con su Hsp104 un N-terminal 6-etiqueta que se puede quitar por escisión de la proteasa TEV. Transformar pPROEX-HTB-Hsp104 en el codón-optimizado E. coli BL21-CodonPlus-RIL células (Stratagene, Agilent Technologies) mediante un procedimiento típico de la transformación bacteriana (por ejemplo, de acuerdo con las instrucciones del fabricante). Es importante usar un codón de E. optimizado coli cepa Hsp104 porque tiene un sesgo codón inusual. </li><li> Inocular un cultivo de 100 ml 2XYT (Receta USB.: 16 g / L de caseína peptona, 10 g / L de extracto de levadura, 5 g / l de NaCl, pH 7,0) suplementado con ampicilina y 100μg/mL 34μg/mL cloranfenicol con transformantes fresca y crecer durante la noche a 37 ° C con agitación a 200 rpm. </li><li> Inocular 10 ml de la cultura durante la noche en 6 X 1L 2XYT 100μg/mL con ampicilina y el cloranfenicol 34μg/mL en frascos de 2 litros. Agitar a 250rpm a 37 ° C hasta 600 OD = desde 0.4 hasta 0.6. Dejar de temblar y reducir la temperatura a 15 ° C. Permiten a las células para equilibrar no agitado durante 30 minutos en la incubadora hasta que llega a 15 ° C. Una vez que 15 ° C se alcanza induce la expresión de proteínas mediante la adición de IPTG a una concentración final de 1mM. Reanudar agitación a 250rpm durante la noche (16.12 horas). </li></ol> 2. Lisis de células y <ol><li> Las células por centrifugación de la cosecha (en un rotor de pre-enfriado) a 4.000 rpm durante 20 minutos a 4 ° C (se utiliza una centrífuga Sorvall RC 3BP +). Los pasos siguientes deben realizarse inmediatamente, porque Hsp104 actividad se ve disminuida cuando E. células de E. coli están congelados. Por otra parte, todos los pasos posteriores se deben realizar en hielo oa 4 º C. </li><li> Resuspender pellets de células en preenfriado (en hielo) de 10 ml buffer de lisis (40 mm de HEPES-KOH pH 7,4, KCl 500 mm, 20 mm MgCl 2, 2,5% (w / v) de glicerol, 20 mM imidazol, pepstatina 5μM A, cóctel completo inhibidor de la proteasa (1 EDTA sin tablet/50mL), y 2 mM β-mercaptoetanol). </li><li> Lisis de las células con una francesa de prensa (Emulsiflex) homogeneizador que utiliza un intercambiador de calor sumergido en agua helada. Antes de su uso, asegurar que todos los grupos de células se han solubilizado. Después de equilibrar homogeneizador con tampón de lisis, dos pasadas por el cilindro con una presión de 15,000-18,000 psi es suficiente para la lisis completa. Si la prensa francesa no está disponible, la incubación con lisozima seguido por ultrasonidos se puede utilizar (véase la discusión). Guardar una pequeña muestra de células lisadas para el análisis de SDS-PAGE (1μL) (lisado en la fig. 2). </li><li> Retirar los restos celulares por centrifugación a 16.000 rpm durante 20 minutos a 4 ° C (se utiliza un RC5C Sorvall centifuge +). Conservar el sobrenadante para el próximo paso y salvar una pequeña muestra (1μL) del sobrenadante para análisis SDS-PAGE (Carga Ni en la fig. 2). </li></ol> 3. Hsp104 Purificación <ol><li> Mezcla de sobrenadante del paso 2.4 con 12 ml (2 ml de Ni-Sepharosa cuentas por 1 litro de células) del 50% mezcla de tampón de lisis equilibrado Sepharose Ni-cuentas (GE). </li><li> Rotar la muestra lentamente durante 3 horas a 4 ° C de tal manera que Ni-Sepharose queda flotante y espuma se reduce al mínimo. Reducción de los tiempos de incubación son posibles, pero se reducirá el rendimiento. A 4 ° C en presencia de inhibidores de la proteasa, se produce muy poca degradación, y tres horas de Ni-Sepharose tiempo de incubación no disminuye la actividad del producto final. Recoger Ni-Sepharose por centrifugación durante 2 min a 4 ° C a 2.000 rpm (Eppendorf 5810R centrífuga). Guardar una pequeña muestra de sobrenadante para análisis SDS-PAGE (1μL) y desechar el resto (de flujo a través Ni (FT) en la fig. 2). </li><li> Lave el recuperado Ni-Sepharose con 25 volúmenes de columna de tampón de lavado (40 mm HEPES-KOH pH 7,4, 150 mM KCl, 20 mM MgCl 2, 2,5% (w / v) de glicerol, 20 mM imidazol, 2 mM β-mercaptoetanol), 5 volúmenes de columna de tampón de lavado con KCl 1M para eliminar los contaminantes vinculados a Hsp104 electrostáticamente, y 25 volúmenes de columna de tampón de lavado. Recoger Ni-Sepharose después de cada lavado por centrifugación durante 2 min a 4 ° C a 2.000 rpm (Eppendorf 5810R centrífuga). Después de cada ciclo de lavado y centrifugación, eliminar tampón por aspiración. </li><li> Para eluir Hsp104, mezcla de Ni-Sepharosa con tampón de elución de 1 ml (40 mm de HEPES-KOH pH 7,4, 150 mM KCl, 20 mM MgCl 2, 2,5% (w / v) de glicerol, 350mm imidazol, 2 mM β-mercaptoetanol) por 1 ml de Ni-Sepharose y girar a 4 ° C durante 20 minutos. La alta concentración de imidazol interrumpe el cordón de Ni-Su 6 interacción. Quitar Ni-Sepharose con columnas vacías 1,2 mL giro (Bio-Rad) por centrifugación durante 2 min a 4 ° C a 2.000 rpm (Eppendorf 5810R centrífuga). Guardar 1μL de eluido de análisis SDS-PAGE (eluido Ni en la fig. 2). Por cada litro de células, ~ 15 mg de Hsp104 se obtiene en este paso. </li><li> Buffer eluido cambio en Q Buffer (20 mM Tris-HCl pH 8, 0,5 mM EDTA, 5 mM MgCl2, 50 mM NaCl) con MWCO 30.000 Amicon Ultra (Millipore) 15 ml unidades concentrador centrífugo. En primer lugar, concentrado de proteína de ~ 1,5 ml a continuación, añadir 14.5mL de Buffer Q para diluir la proteína de 10 veces. Repita 3 veces para el intercambio de buffer. El aumento del pH y baja en sal asegura que Hsp104 tiene una alta carga negativa. </li><li> Purificar Hsp104 a través de cromatografía de intercambio aniónico con una memoria intermedia de Q de recursos equilibrada Q columna de 6 ml (GE). Antes de la inyección, la proteína se filtra a través de una baja en proteínas de unión Millex GP PES membrana 0.22μM filtro de jeringa (Millipore). Contamos con un caudal de 1mL/min. Lavar débilmente las proteínas de unión con el volumen de la columna 1 del 20% de búfer Q + (20 mM Tris-HCl pH 8, 0,5 mM EDTA, 5 mM de MgCl 2, 1 M NaCl). Eluir Hsp104 con gradiente lineal (20% -50% de búfer Q +) en 5 volúmenes de columna (Fig. 3). Recoger fracciones de 1 ml. Hsp104 y las variantes más típicamente eluyen a ~ 400 mm de NaCl (31mS/cm). Guardar las muestras de las fracciones de pico (1μL) para análisis SDS-PAGE (Fig. 3, recuadro). </li><li> Para quitar 6-Su etiqueta, proteína de intercambio con el concentrador Amicon centrífuga como se describe anteriormente (véase el paso 3.5) en división de amortiguación (20 mM HEPES-KOH pH 7,4, KCl 140 mm y 10 mm MgCl 2). Use proTEV proteasa (Promega) o AcTEV de la proteasa (Invitrogen) de acuerdo con las instrucciones del fabricante. Para Hsp104, hemos encontrado que el aumento de las proporciones de la proteasa TEV: Hsp104 se requieren para la escisión completa. Nosotros usamos una proporción de 1 por unidad de la proteasa 12μg Hsp104. División se debe realizar a 30 ° C durante 2-4 horas, seguido de una incubación de 16 horas a 4 ° C. La concentración final de Hsp104 debe estar entre 20-75μM monómero. Agotan los restantes Hsp104 His6 marca y la proteasa TEV con Ni-Sepharose (GE) mediante la adición de Ni-Sepharose en exceso a la cantidad de Hsp104 en la división de reacción (suponiendo que cuentas se puede unir 15 mg de proteína por ml de resina de relleno). Eliminar las perlas con columnas de centrifugado vacío (Bio-Rad) por centrifugación durante 2 minutos a 2.000 rpm a 4 ° C. Recoger muestras antes y después de la escisión (1μL) para el análisis de SDS-PAGE para asegurar la división, y la eliminación de proteínas uncleaved (Fig. 4). </li><li> Cambio en el tamaño del buffer de exclusión (20 mM HEPES-KOH pH 7,4, KCl 140 mm, 10 mm MgCl 2, 0,1 mM EDTA, 1 mM DTT) con MWCO 30.000 Amicon Ultra (Millipore) como se describe en el paso 3.5. </li><li> Además Hsp104 purificar por cromatografía de exclusión por tamaño. Uso de exclusión por tamaño Superose buffer equilibrado 6 o columna Superdex 200 (ambos de GE) (Fig. 5). Antes de la inyección, la proteína se filtra a través de una baja en proteínas de unión Millex GP PES membrana 0.22μM filtro de jeringa (Millipore). Por menos de 10 mg de proteína, el tamaño de las columnas 10/300 (24ml) se pueden utilizar y las fracciones de 0,5 ml se recogen. Para las muestras superiores a 10 mg, la columna de tamaño 12/60 (120 ml) debe ser utilizado y fracciones de 1 ml se recogen. Consulte las instrucciones del fabricante para los caudales y volúmenes de muestra para ser cargado. Después de la purificación, las fracciones Hsp104 se agrupan como se muestra en la figura. 5 y se concentró en las unidades de concentrador centrífugo Amicon (Millipore). Hsp104 se almacena como se describe a continuación. Debido a la pérdida de material en la separación de Hsp104 productos de degradación y los contaminantes del rendimiento final de purificado de larga duración Hsp104 es de ~ 1-3mg por litro a partir de la cultura. </li></ol> 4. Hsp104 Disaggregase Actividad <ol><li> Después de la purificación, es una buena práctica para evaluar Hsp104 actividad en un ensayo de disgregación. Por lo general, utilizamos un ensayo de reactivación de la luciferasa 2 (Fig. 6). En este ensayo, Hsp104 en conjunto con el sistema de chaperonas Hsp70 (Hsp70 y Hsp40) desagrega desnaturalizado urea-agregados luciferasa de luciérnaga 2. Luciferasa soluble cataliza la oxidación de la luciferina a oxyluciferin, una reacción que libera la luz. Por luminiscencia de vigilancia, la reactivación relativa, y la desagregación por lo tanto, de la luciferasa se puede determinar. Hsp104 debe ser capaz de colaborar de forma sinérgica con la co-chaperona Hsp70 sistema. La cantidad de luminiscencia recuperado depende de la exacta Hsp70: par Hsp40 utilizado. Nosotros habitualmente emplean Hsp72 y Hsp40 (diseño del ensayo). Como mínimo, los activos Hsp104 debería producir un aumento de 5 veces en la reactivación de la luciferasa en comparación con la reactivación de Hsp72: Hsp40 sola (Fig. 6). Hsp104 preparaciones que no llegan a este nivel de actividad se descartan. </li></ol> 5. Hsp104 de almacenamiento <ol><li> Para almacenamiento a corto plazo, Hsp104 pueden mantenerse a 4 ° C en tampón de exclusión por tamaño. Sin embargo, la actividad se reducirá después de 2-3 días a 4 ° C y bruscamente después de una semana a 4 ° C. Para los ensayos de disgregación se recomienda Hsp104 debe ser utilizado tan pronto como sea posible después de la purificación. Lo ideal sería que Hsp104 debe utilizarse inmediatamente para los ensayos de desagregación amiloide. </li><li> Si la proteína se debe almacenar a largo plazo, Hsp104 se intercambia en el buffer de almacenamiento (20 mM HEPES-KOH pH 7,4, KCl 140 mm, 10 mm MgCl 2, 0,1 mM EDTA, 1 mM TDT, el 10% de glicerol (w / v)). Alícuotas de 100μl se congelaron en nitrógeno líquido y almacenadas a -80 ° C. Ciclos de congelación-descongelación reducir drásticamente la actividad Hsp104y debe ser evitado. Se recomienda descongelar lentamente Hsp104 en el hielo. Amiloide disaggregase actividad disminuirá bruscamente después de un mes a -80 ° C. </li></ol> 6. Los resultados representativos y cifras: <img src="/files/ftp_upload/3190/3190fig1.jpg" alt="Figura 1"/> Figura 1. Hsp104 es un disaggregase bifuncionales. Desagregación de los agregados desordenados (que se muestra a la izquierda) se requiere la cooperación del sistema de chaperona Hsp70 (Hsp70 y Hsp40) 2. Remodelaciones Hsp104 ordenó agregados amiloides (como se muestra a la derecha) sin la ayuda de Hsp70 y Hsp40 in vitro, pero Hsp70 y Hsp40 puede mejorar Hsp104 actividad contra amiloide 26,28. Para ambos tipos de estructuras agregadas, Hsp104 parejas hidrólisis de ATP a la translocación del sustrato a través de su canal central para promover la desagregación. Tirosina-teniendo lazos de poro participar y el sustrato de transporte a través del canal central 49-52. <img src="/files/ftp_upload/3190/3190fig2.jpg" alt="Figura 2"/> Figura 2. SDS-PAGE análisis de Ni-sefarosa etapa de purificación de afinidad. Lisado, carga Ni, Ni FT y las muestras Ni eluido se fraccionaron por SDS-PAGE con un 20.4% Tris-HCl 1,0 Criterio gel (Bio-Rad) y se tiñeron con Coomassie . Tenga en cuenta que no todos los Hsp104 es capaz de unirse a la Ni-sefarosa. Amplia gama de marcadores de peso molecular (Bio-Rad) se muestran (carril de la izquierda). <img src="/files/ftp_upload/3190/3190fig3.jpg" alt="Figura 3"/> Figura 3. Recursos de purificación Q de Ni-sefarosa eluido. Rastro azul representa la absorbancia a 280 nm rastro y el verde representa buffer% Q + (máximo del 50%). El primer pico que eluye a 20% Q + contiene impurezas, productos de degradación y mal doblada Hsp104. El segundo pico y las principales contiene plegada correctamente y activa Hsp104. Caudal fue 1ml/min. Gradiente de 20 a 50% Q + es de 30 minutos o 5 volúmenes de columna. Recuadro: fracciones de los picos son resueltas por SDS-PAGE análisis utilizando un 4-20% Tris-HCl 1,0 Criterio gel (Bio-Rad) y se tiñeron con Coomassie. Amplia gama de marcadores de peso molecular (Bio-Rad) se muestran (izquierda). <img src="/files/ftp_upload/3190/3190fig4.jpg" alt="Figura 4"/> Figura 4. SDS-PAGE análisis de paso proTEV escisión de la proteasa. 6-Su Hsp104 de Recursos purificación Q fue tratada con proteasa proTEV durante 4 horas a 30 ° C y luego 16 horas a 4 ° C. Las muestras fueron fraccionadas por SDS-PAGE con un 4-20% Tris-HCl 1,0 Criterio gel (Bio-Rad) y se tiñeron con Coomassie. Tenga en cuenta que proTEV escinde Hsp104 migra más rápidamente. VET proteasa y uncleaved Hsp104 se han agotado con Ni-Sepharose como se describe en el Paso 3.7. Amplia gama de marcadores de peso molecular (Bio-Rad) se muestran (izquierda). <img src="/files/ftp_upload/3190/3190fig5.jpg" alt="Figura 5"/> Figura 5. De exclusión por tamaño de purificación de Hsp104. Escinde Hsp104 fue purificado a través de un Superose 6 columna de filtración en gel (10/300, GE). Caudal = 0.4ml/min. El pico entre las líneas de trazos representa fracciones reunidas. Recuadro: fracciones de los picos son resueltas por SDS-PAGE análisis utilizando un 4-20% Tris-HCl 1,0 Criterio gel (Bio-Rad) y se tiñeron con Coomassie. Amplia gama de marcadores de peso molecular (Bio-Rad) se muestran (izquierda). <img src="/files/ftp_upload/3190/3190fig6.jpg" alt="Figura 6"/> Figura 6. Ensayo de reactivación de la luciferasa. Desnaturalizado agregados luciferasa de luciérnaga (50nm) se incubaron con Hsp72 y Hsp40 (1μM) (ambos de los diseños de ensayo), Hsp104 (6μM monómero) o Hsp104, Hsp72 y Hsp40 de 90 minutos a 25 ° C. Luciferasa desnaturalizado sólo es plenamente reactivada en la presencia de ambos Hsp104 y Hsp40/Hsp72. Luminiscencia recuperado se mide en un lector de placas M1000 Infinito (Tecan). Los valores representan la media ± SEM (n = 3).

No hay conflictos de interés declarado.

Cronología: Para la máxima actividad Hsp104 se recomienda que el esquema de purificación se complete toda la mayor rapidez posible. Sin embargo, el número de etapas de purificación hace un exigente calendario que no siempre es práctico. Si las etapas de purificación se lleva a cabo lo más rápidamente posible, el tiempo desde el final de la expresión a través de la noche a la 02.04 horas de incubación a 30 ° C con TEV proteasa es de aproximadamente 9-11 horas. Un lugar potencial para hacer una pausa e...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> BL21-CodonPlus-RIL células competentes </td><td> Stratagene, Agilent Technologies </td><td> 230255 </td></tr><tr><td> 2XYT caldo </td><td> USB </td><td> 75864 </td></tr><tr><td> Completo, minibar, EDTA libre de tabletas de inhibidores de la proteasa </td><td> Roche </td><td> 1836170 </td></tr><tr><td> A pepstatina </td><td> Sigma </td><td> P4265 </td></tr><tr><td> Ni-Sepharose 6 Fast Flow </td><td> GE Healthcare </td><td> 17-5318-02 </td></tr><tr><td> Amicon Ultra-15 unidades centrífugas filtro (MWCO 30.000) </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> Recursos Q - columna 6 ml </td><td> GE Healthcare </td><td> 17-1179-01 </td></tr><tr><td> proTEV de la proteasa </td><td> Promega </td><td> V6052 </td></tr><tr><td> AcTEV de la proteasa </td><td> Invitrogen </td><td> 12575015 </td></tr><tr><td> Superose 6 10/300 GL </td><td> GE Healthcare </td><td> 17-5172-01 </td></tr><tr><td> Hsp40 </td><td> Diseño del ensayo </td><td> SPP-400 </td></tr><tr><td> Hsp72 </td><td> Diseño del ensayo </td><td> ADI-NSP-555 </td></tr></tbody></table>

purification of hsp104, a protein disaggregase

Hsp104 es un hexameric AAA + proteína 1 de la levadura, que se acopla la hidrólisis del ATP a la proteína de desagregación 2-10 (Fig. 1). Esta actividad se imparte dos ventajas selectivas clave. En primer lugar, la renaturalización de los agregados desordenados por Hsp104 faculta la supervivencia de hongos después de varios mal plegamiento de proteínas, destaca, como golpe de calor 3,5,11,12. En segundo lugar, la remodelación de las fibrillas de amiloide-beta cruzada por Hsp104 permite a la levadura para explotar miles de priones (amiloides infecciosas) como un reservorio de beneficios y las variaciones hereditarias fenotípica 13-22. Sorprendentemente, Hsp104 directamente remodelaciones oligómeros preamyloid y fibrillas de amiloide, incluyendo los que forman parte de las proteínas de priones de levadura Sup35 y 23-30 Ure2. Esta funcionalidad-amiloide remodelación es una faceta especializada de la levadura Hsp104. La E. orthologue coli, CLPB, no oligómeros preamyloid remodelar o fibrillas de amiloide 26,31,32. Ortólogos Hsp104 se encuentran en todos los reinos de la vida, excepto, sorprendentemente, los animales. En efecto, si las células animales poseen un sistema enzimático que desglose las parejas de proteínas para la renaturalización (en lugar de la degradación) sigue siendo desconocido 33-35. Por lo tanto, nosotros y otros han propuesto que Hsp104 podría ser desarrollado como un agente terapéutico para diversas enfermedades neurodegenerativas relacionadas con el mal plegamiento de proteínas específicas en oligómeros preamyloid tóxicos y fibrillas de amiloide 4,7,23,36-38. No existen tratamientos que afectan directamente a las especies agregados asociados a estas enfermedades. Sin embargo, se disuelve Hsp104 oligómeros tóxicos y fibrillas de amiloide compuesto de alfa-sinucleína, que se relacionan con la enfermedad de Parkinson 23, así como las formas de PrP-amiloide 39. Es importante destacar que Hsp104 reduce la agregación de proteínas y mejora la neurodegeneración en modelos de roedores de la enfermedad de Parkinson y la enfermedad de Huntington 23 38. Lo ideal sería que, para optimizar el tratamiento y minimizar los efectos secundarios, Hsp104 sería diseñado y potenciado para remodelar de forma selectiva agregados específicos en el centro de la enfermedad en cuestión 4,7. Sin embargo, la limitada comprensión estructural y mecánica de cómo Hsp104 desagrega un repertorio diverso de las estructuras de agregados y proteínas no relacionadas frustra los esfuerzos 30,40-42. Para comprender la estructura y el mecanismo de Hsp104, es esencial para el estudio de la proteína pura y reconstituir su actividad disaggregase con un mínimo de componentes. Hsp104 es una proteína con un pI 102kDa de ~ 5.3, que hexamerizes en presencia de ADP o ATP, o en las concentraciones de proteínas en ausencia de nucleótidos 43-46. Aquí se describe un protocolo optimizado para la purificación de la muy activa, estable Hsp104 de E. coli. El uso de E. coli permite simplificar en gran escala de producción y nuestro método se puede realizar de forma rápida y fiable para numerosos Hsp104 variantes. Nuestro protocolo aumenta Hsp104 pureza y simplifica 6-Su etiqueta de la eliminación en comparación con un método de purificación previa de E. coli 47. Por otra parte, el protocolo es más fácil y conveniente de dos protocolos más recientes 26,48.

Watch this Scientific Journal Video about Purification of Hsp104, a Protein Disaggregase at JoVE.com

Purification of Hsp104, a Protein Disaggregase

Aquí se describe un protocolo para la purificación de gran actividad Hsp104, un AAA hexameric + proteína de la levadura, que se acopla la hidrólisis de ATP con el desglose de proteínas. Este esquema se aprovecha de una construcción de His6-etiquetados para la purificación de afinidad de<em&gt; E. coli</em&gt; Seguido por cromatografía de intercambio aniónico, His6-tag con la eliminación de la proteasa TEV, y la cromatografía de exclusión por tamaño.

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 ...

purification-of-hsp104-a-protein-disaggregase

Research

JoVE Journal

Biology

17.3K vistas.  University of Pennsylvania. Aquí se describe un protocolo para la purificación de gran actividad Hsp104, un AAA hexameric + proteína de la levadura, que se acopla la hidrólisis de ATP con el desglose de proteínas. Este esquema se aprovecha de una construcción de His6-etiquetados para la purificación de afinidad de E. coli Seguido por cromatografía de intercambio aniónico, His6-tag con la eliminación de la proteasa TEV, y la cromatografía de exclusión por tamaño.

Video: Purification of Hsp104, a Protein Disaggregase

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

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

La purificación de la M. magneticum Tensión AMB-1 magnetosomas proteína asociada MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Expresión, la solubilización de detergente, y purificación de un transportador de membrana, la proteína de resistencia a múltiples fármacos MexB

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

La interacción hidrofóbica automatizada cromatografía en columna de selección para uso en la purificación de proteínas

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificación por cromatografía de ribosomas de levadura de gran actividad

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Purificación de proteínas ortogonales Facilitado por un marcador de afinidad biespecíficos pequeños

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identificación de los socios interacción de proteínas en tándem de purificación de afinidad

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Generación, purificación y caracterización de especies invasivas en células de proteína DISC1

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Purificación de proteínas libre de Método de Determinación afinidad de unión por termoforesis Microscale

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

La identificación de la interacción proteína-proteína en<em&gt; Drosophila</em&gt; Jefes adultas por Tandem Affinity purificación (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...