Este trabajo fue apoyado por la beca NSERC 386634. Nos gustaría dar las gracias a los miembros del Laboratorio Screaton útil para los debates.

1. Preparación de los reactivos, platos, y Células <ol><li> Hacer 500 ml de tampón de lisis: 25 mM Tris pH 7,5, NaCl 150 mM, NaF 50 mM, 0,5 mM EDTA pH 8,0, 0,5% Triton X-100, 5 mM beta-glicerofosfato, 5% de glicerol. Almacenar a 4 ° C. Inmediatamente antes de usar, añadir ditiotreitol 1 mM (DTT), 1 mM de fluoruro de fenilmetilsulfonilo (PMSF), vanadato de sodio y 1 mM. Después de este paso, PMSF no se requiere en cualquier tampón de enjuague. </li><li> Hacer 20 ml de 10x tampón de quinasa: mM Tris 200 pH 7,5, 50 mM de beta-glicerofosfato (FW 216), vanadato de sodio 2 mM. Alícuota en 1 ml tubos y almacenar a -20 ° C. Inmediatamente antes de usar, añadir DTT 5 mM. </li><li> Hacer 20 ml de tampón M-ATP 10x: 300 M de adenosina trifosfato (ATP), 66 mM MgCl2, 33 mM MnCl $ 2. alícuota de 1 ml tubos y almacenar a -20 ° C. </li><li> Hacer 100 ml de sulfato de sodio dodecil 2x (SDS) tampón de lisis: 1,5 g de base Tris, 20 ml de glicerol, 30 ml de H 2 O. Disolver y ajustar el pH a 6,8 con HCl. Añadir 40ml 10% SDS, ajustar el volumen a 100 ml. Añadir 25 mg de azul de bromofenol. </li><li> Spot 4 l de 25 ng / l de plásmido de expresión de mamífero que codifican un GST-quinasa por pocillo en 14 conjuntos de placas de 96 pocillos y placas de etiquetas en consecuencia. Seal y congelación placas a -20 ° C hasta su uso. </li><li> 1-2 días antes de la transfección, las células de cultivo HEK293T en medio de Eagle modificado de Dulbecco completo (DMEM) + 10% de suero fetal bovino (FBS) y antibióticos en una incubadora a 37 ° suplementado con CO 2 (5% final). Pasaje con tripsina y no permiten que la acción se convierta en&gt; 80% de confluencia durante la expansión. Se requiere un mínimo de 6 x 10 7 células para la pantalla (aproximadamente 3 x 15 cm de platos de células HEK293T en 80% de confluencia). </li></ol> 2. La transfección Nota: Consulte la Figura 1 para un diagrama de flujo de protocolo entero. <ol><li> Si las placas que contienen plásmidos quinasa han sido congelados, descongelar a temperatura ambiente unnd centrifugar a 1900 xg durante 3 min para recoger cualquier humedad en el fondo de los pozos. </li><li> Mezclar 8,6 ml de medio de suero reducido (por ejemplo, OPTI-MEM) con 312,7 l de reactivo de transfección a base de lípidos. Deje reposar durante 5 minutos. </li><li> Añadir 10 l de medio de suero reducido a cada pocillo usando un dispensador de líquido automatizado equipado con un pequeño volumen de casete. </li><li> Añadir 10 l por pocillo de la mezcla de reactivo de medio con suero / transfección reducida desde el paso 2.2 utilizando un dispensador de líquido automatizado equipado con un pequeño volumen de casete. Deje reposar durante 20 a 45 min. </li><li> Resuspender las células 293T en 7,5 x 10 5 células / ml en 80 ml ​​de DMEM completo. Añadir 100 l de suspensión celular (es decir, 7,5 x 10 4 células) por pocillo usando un dispensador de líquido automatizado equipado con un casete de volumen estándar. </li><li> Compruebe pozos bajo el microscopio para la distribución uniforme de células y volver a la incubadora durante 24 horas. </li></ol> 3. desplegable GST-quinasa <ol><li> Haga fResh: 4 mM solución de pervanadato mediante la mezcla de 60 l de 0,2 M de vanadato de sodio con 540 l de H 2 O. En una segunda mezcla de tubo de 2,7 l de peróxido de 30% y 1,4 ml de PBS. Añadir las dos soluciones juntos y deje reposar durante 15 minutos antes de su uso. </li><li> Con una pipeta multicanal (no utilizar un dispensador de líquido automatizado, ya que crea mucha turbulencia en el pozo) prescindir de 2 l de 0,25 M CaCl2 en cada pocillo, seguido de 2,5 l de la pervanadato preparada en el paso 3.1. Incubar cada placa a 37 ° C durante 10 min y luego se coloca en hielo. </li><li> Prepare 35 ml de tampón de lisis mediante la adición de la TDT, PMSF, y vanadato de sodio, como se indica en la Sección 1 (Preparación de los reactivos). </li><li> Mantener las placas en hielo, retire medio de cada pocillo usando un aspirador de vacío. Inmediatamente añadir 50 l / pocillo de tampón de lisis de hielo frío usando un dispensador de líquido automatizado equipado con un casete estándar. Deje reposar 30 minutos en hielo para lisar (opcional: puede parar aquí si es necesario mediante plat selladocorreo y almacenamiento a -80 ° C. Para continuar, placas de deshielo en el hielo). </li><li> Placas giran a 1.900 g durante 3 min a 4 ° C. </li><li> Raspar las células de cada pocillo usando una pipeta multicanal y transferir todos los contenidos etiquetados apropiadamente a placas de 96 pocillos de fondo en V. Placas giran a 1900 xg durante 10 min a 4 ° C. </li><li> Durante el centrifugado, llenar placas revestidos con glutatión (uno para cada placa de 96 pocillos) con 100 l / pocillo de tampón de lisis de hielo frío (sin PMSF) como un enjuague. Mantenga las placas de hielo. </li><li> Retire las placas de fondo en V de la centrífuga. Uno a la vez, invertir las placas de glutatión sobre un fregadero para sacudir el tampón de lisis y mancha en una toalla de papel. Transferencia de tampón de lisis de las placas de fondo en V a las placas de glutatión por la inclinación de la placa y con una pipeta multicanal, teniendo cuidado de no perturbar el sedimento en la parte inferior. Tapar y dejar las placas en hielo durante un mínimo de 2 horas para unirse. </li><li> Cerca del final de la etapa de unión 2 hr, preparar una estación de trabajo radiactividad, asegurando tque las medidas de seguridad necesarias están en su lugar para el trabajo radiactivo. Horno de hibridación Se establece en 30 ° C. </li><li> Preparar 200 ml de tampón de lisis mediante la adición de DTT y sodio vanadato como se indica en la Sección 1. PMSF no se requiere en esta etapa. </li><li> Invertir las placas de glutatión sobre un fregadero para sacudir el tampón de lisis y secar sobre una toalla de papel. Enjuague pozos 3 veces con 100 l de tampón de lisis (sin PMSF). No deje que los pozos se sientan en seco - mantenerlos en enjuague hasta que esté listo para continuar. </li><li> Prepare 55 ml de tampón de quinasa 1x (KB) diluyendo el 10x y añadiendo la TDT, como se indica en la Sección 1. Placas de enjuague una vez con 50 l de 1x KB utilizando un dispensador de líquido automático equipado con un cassette de volumen estándar. Deja 1x KB en los pozos hasta que la solución A es listo: <ol><li> Preparar la solución A mediante la adición de 500 a 530 g del sustrato de interés, 500 g de proteína básica de la mielina (MBP), 2,65 ml de 10x KB, 13.25 l de 1 M DTT, y H 2 O hasta 15,9 ml. </li></ol></li&gt; <li> Uno a la vez, invertir las placas sobre un fregadero para eliminar enjuague 1x KB, seque sobre una toalla de papel, y de inmediato añadir 30 l de solución A mediante un distribuidor de líquido automático equipado con un pequeño casete volumen. Mantenga las placas de hielo. </li><li> Preparar la solución B en el área de trabajo radiactividad añadiendo 2,5 ml de 10x M-ATP, 32 P ATP 500 Ci gamma, y H 2 O a 10 ml. <ol><li> Añadir 20 l de solución B por pocillo usando una pipeta repetidora que ayuda en la mezcla debido a la fuerza de eyección. Cubrir e incubar en horno de hibridación 30 ° C durante 30 min. </li></ol></li><li> Después de 30 minutos, la transferencia de las placas de nuevo a hielo. Añadir 50 l de tampón de lisis 2x SDS a cada pocillo usando una pipeta multicanal. Puede proceder en este punto con el siguiente paso, o sellar las placas con papel de aluminio y guardar a -20 ° C hasta el momento conveniente. </li></ol> 4. Correr, tinción y Geles de secado Nota: Todo el trabajo debe realizarse en un área Designated la radiactividad. <ol><li> Encienda el horno de hibridación y se puso a 85 ° C. Descongelar las placas a temperatura ambiente. Una vez que el horno ha alcanzado la temperatura, placas de transferencia al horno y se incuba durante 10 minutos para desnaturalizar las muestras. </li><li> Cargar 26 pocillos geles prefabricados con 15 l de cada reacción utilizando una pipeta multicanal para llenar varios pozos a la vez. Se debe tener cuidado de que todos los consejos se alinean con las correspondientes así antes de añadir las muestras. Ejecute gel a 150 V. No deje que el tinte de seguimiento (línea azul) se escurra de la parte inferior del gel, ya que contiene la ATP no incorporado. </li><li> Desmontar los geles y cortar el ATP no incorporado (línea azul), utilizando un bisturí o borde recto ya que sobreexponer las películas. Coloca los geles en contenedores etiquetados y cubrir con tinción de Coomassie durante 15 min. </li><li> Retire la tinción de Coomassie, brevemente enjuagar los geles con agua y añadir destain solución. Destain los geles hasta que las proteínas son claramente visibles. Una banda de MBP y una banda para el sustrato debeser visible para cada muestra. </li><li> Para secar los geles: <ol><li> Cortar una hoja grande de papel de filtro y colocarlo en la secadora. </li><li> Moje una hoja de celofán en agua destilada hasta que esté suave y libre de arrugas, y colocarlo en la parte superior del papel. </li><li> Coloque los geles en la parte superior de la hoja de celofán, haciendo una nota de la orden de los geles. Moje una segunda hoja de celofán y colocar en la parte superior de los geles. </li><li> Estirar todas las burbujas (un rodillo sellado placa funciona bien para esto) para una superficie agradable uniforme. Cierre la tapa, encienda el vacío, y secar los geles durante 3 horas a 80 ° C. </li></ol></li><li> Una vez geles están secos, los exponen a una película XAR utilizando una pantalla de intensificar la señal. Envuelva la cinta con papel film o una bolsa de plástico y selle con cinta para mantener fuera a las heladas. Guarde el casete a -80 ° C durante la noche. </li></ol> 5. Desarrollar XAR Films <ol><li> Al día siguiente, eliminar el casete del congelador y dejar descongelar a temperatura ambiente. Desarrollar la película enun cuarto oscuro usando un procesador de película de acuerdo con las instrucciones del fabricante. Nota: Examine las películas para pruebas de pares quinasa-sustrato. Una segunda exposición más larga también puede ser útil para detectar eventos de fosforilación más débiles. </li></ol>

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The authors have nothing to disclose.

Desde las publicaciones originales que describen el enfoque 14,15, la biblioteca original de 180 GST-quinasas se ha ampliado a 420 miembros, o ~ 80% de la kinome proteína humana. Con la biblioteca ampliada, el protocolo como se describe toma 4-5 días y después de 1-4 días para desarrollar películas (según se considere necesario), lo que podría ser acortada mediante el uso de phosphorimaging y mejora de la señal digital. Hay varios pasos clave en las que se debe tener cuidado (Ver Figura 1</str...

Proteína modificaciones post-traduccionales (PTMs) son esenciales para la comunicación intracelular. Tal vez el mejor estudiado de todos PTM es la fosforilación, catalizada por proteínas quinasas, que regulan una gran variedad de funciones de proteínas, incluyendo su actividad bioquímica, localización subcelular, conformación, y la estabilidad. La identificación de sitios de fosforilación en las proteínas diana se puede lograr mediante mapeo de fosfopéptidos trípticos o mediante técnicas proteómicas ahora estándar usando muestras enriquecidas para los péptidos fosforilados 1,2. Aunque se espera que tres cuartas partes del proteoma expresado ser fosforilados 3 y un identificaron 200.000 sitios de fosforilación 5, con estimaciones de hasta 1 millón 6, muchos de éstos no tienen asignada la biología, la vía de señalización, o la proteína quinasa. Mientras que la identificación de sitios fosforilados es relativamente sencillo, una comparativamente mayor reto esidentificar la quinasa cognado (s) que se dirige a estos sitios, un proceso que nos referimos como mapeo de quinasa: sustrato pares. Varios enfoques para la identificación de quinasa: sustrato pares se han descrito, ya sea comenzando con una quinasa de interés y en busca de sus sustratos o comenzar con un sustrato de interés y tratando de encontrar una quinasa de modificación experimentalmente 7-11 o computacionalmente 12. Para identificar quinasas para un sustrato fosforilado conocido, la bioinformática se pueden utilizar para identificar las proteínas que contienen una secuencia corta conservada de aminoácidos que flanquean el residuo fosforilado (el sitio consenso), así como la identificación de quinasas que forman un complejo con el sustrato precipitable. Sin embargo, estos métodos requieren mucho tiempo ya menudo no cumplen con éxito. Hemos desarrollado un enfoque funcional sistemático para identificar rápidamente quinasas que pueden phosphorylate un sustrato dado 13. El ensayo de la pantalla produce una excelente específicadad, con una selección muy claro para los posibles quinasas afines. Dada la centralidad de la fosforilación de la señalización biológica, la pantalla es útil para el descubrimiento de rutas de señalización prácticamente todos los teléfonos 14-16. La pantalla implica la realización de un ensayo de quinasa a gran escala con una biblioteca de proteínas quinasas humanas. Las quinasas han sido etiquetadas con glutatión bacteriana S-proteína transferasa (GST) y se purificó a partir de extractos de células de mamíferos, lo que significa que las enzimas recombinantes - a diferencia de los preparados a partir de bacterias - se generan en presencia de las proteínas quinasas aguas arriba a menudo requeridos para la enzimas recombinantes que tienen actividad in vitro. De hecho, mientras que la actividad quinasa de serina, treonina y tirosina requiere para la activación de la quinasa aguas abajo están presentes en la levadura 10, el genoma de la levadura codifica 122 proteínas quinasas, lo que indica que el kinome de mamífero, con más de 500 genes 17, ha llegado a ser significativamente más compleja con el fin de regulate La procesos únicos para los organismos de orden superior. Por otra parte, el efecto de diferentes estímulos relevantes a la biología celular y la enfermedad humana (por ejemplo, moléculas pequeñas, factores de crecimiento, hormonas, etc.) se puede utilizar para modular la actividad de quinasa 14,15 en un contexto apropiado.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lysis buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.1</td>
		</tr>
		<tr>
			<td>10x kinase buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.2</td>
		</tr>
		<tr>
			<td>10x M-ATP</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.3</td>
		</tr>
		<tr>
			<td>Human kinase plasmids</td>
			<td>Orfeome, Invitrogen, Origene</td>
			<td></td>
			<td>GST-tagged in house</td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Fisher Scientific</td>
			<td>CS003595</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Fisher Scientific</td>
			<td>SH3002201</td>
			<td>supplement with 100U/ml penicillin, 100ug/ml streptomycin, 10% fetal calf serum.</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Sanyo</td>
			<td>MCO-17AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm cell culture dishes</td>
			<td>Fisher Scientific</td>
			<td>877224</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Invitrogen</td>
			<td>22600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated liquid dispenser</td>
			<td>Thermo Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>24073295</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>14072670</td>
			<td></td>
		</tr>
		<tr>
			<td>4mM pervanadate</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 3.1</td>
		</tr>
		<tr>
			<td>0.25 M CaCl2</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (20-200 uL)</td>
			<td>Labnet</td>
			<td>p4812-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (1-10 uL)</td>
			<td>Thermo Scientific</td>
			<td>4661040</td>
			<td></td>
		</tr>
		<tr>
			<td>V-bottom 6-well plates</td>
			<td>Evergreen Scientific</td>
			<td>290-8116-01V</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione coated 96-well plates</td>
			<td>Fisher Scientific</td>
			<td>PI-15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Biostad</td>
			<td>350355</td>
			<td></td>
		</tr>
		<tr>
			<td>GST tagged substrate</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myelin Basic Protein (MBP)</td>
			<td>Sigma</td>
			<td>M1891</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette (1 mL)</td>
			<td>Eppendorf</td>
			<td>22266209</td>
			<td></td>
		</tr>
		<tr>
			<td>32P gamma-ATP</td>
			<td>Perkin Elmer</td>
			<td>BLU502Z500UC</td>
			<td></td>
		</tr>
		<tr>
			<td>2X SDS lysis buffer (100 mL)</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.4</td>
		</tr>
		<tr>
			<td>26-well precast TGX gels</td>
			<td>BioRad</td>
			<td>567-1045</td>
			<td>gel percentage required is dependent on the molecular weight of the substrate of interest</td>
		</tr>
		<tr>
			<td>Coomassie stain</td>
			<td>Made in house</td>
			<td></td>
			<td>0.1% Coomassie R250, 10% acetic acid, 40% methanol</td>
		</tr>
		<tr>
			<td>Coomassie destain</td>
			<td>Made in house</td>
			<td></td>
			<td>10% acetic acid, 20% methanol</td>
		</tr>
		<tr>
			<td>Labeled gel containers</td>
			<td>Made in house</td>
			<td></td>
			<td>Used plastic lids from empty tip boxes, just big enough to contain one gel</td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>Fisher Scientific</td>
			<td>57144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellophane sheets (2)</td>
			<td>BioRad</td>
			<td>165-0963</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Labconco</td>
			<td>4330150</td>
			<td></td>
		</tr>
		<tr>
			<td>Double emulsion autoradiography film</td>
			<td>VWR</td>
			<td>IB1651454</td>
			<td></td>
		</tr>
		<tr>
			<td>Film cassette</td>
			<td>Fisher Scientific</td>
			<td>FBAC-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Intensifying screen</td>
			<td>Fisher Scientific</td>
			<td>FBIS-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealing rubber roller</td>
			<td>Sigma</td>
			<td>R1275</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of kinase-substrate pairs using high throughput screening

Hemos desarrollado una plataforma de cribado para identificar proteínas quinasas humanos dedicados para sustratos fosforilados que pueden utilizarse para dilucidar nuevas vías de transducción de señales. Nuestro enfoque ofrece el uso de una biblioteca de proteínas quinasas humana marcada con GST purificada y un sustrato de proteína recombinante de interés. Hemos utilizado esta tecnología para identificar MAP / microtúbulos afinidad de regulación de quinasa 2 (MARK2) como la quinasa para un sitio regulada por glucosa en CREB-regulado transcripcional Coactivador 2 (CRTC2), una proteína necesaria para la proliferación de células beta, así como la la familia de tirosina quinasas de Axl como reguladores de la metástasis de las células por la fosforilación de la proteína adaptador de ELMO. Se describe esta tecnología y discutimos cómo puede ayudar a establecer un mapa completo de cómo las células responden a estímulos ambientales.

Los resultados representativos de una pantalla se muestran en la Figura 2. 180 quinasas fueron seleccionados utilizando un sustrato peptídico marcada con GST correspondiente a los aa 268-283 de CRTC2 así como la proteína básica de la mielina sustrato clásico ensayo de quinasa (MBP). Sólo dos quinasas, MARK2 y MARK3 la quinasa altamente relacionados con el péptido CRTC2 fosforilados. MBP se incluye como un control interno en todos los ensayos, ya que contiene muchos residuos fosforilables y funcio...

Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

Identification of Kinase-substrate Pairs Using High Throughput Screening

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificación de los pares quinasa-sustrato Usando selección de alto rendimiento


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

identification-kinase-substrate-pairs-using-high-throughput

Research

JoVE Journal

Biology

8.1K vistas.  Children's Hospital of Eastern Ontario Research Institute. Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Video: Identification of Kinase-substrate Pairs Using High Throughput Screening

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Selección de alto rendimiento y biosensores fluorescentes con C. elegans Las cepas

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Investigación

Biología

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cribado de alto rendimiento de la actividad endoglucanasa por hongos en Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

De alto rendimiento mapeo físico de cromosomas utilizando automatizado<em&gt; In situ</em&gt; La hibridación

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

De alto rendimiento para los moduladores de molécula pequeña de tráfico de perfeccionamiento activo Canales rectificador de potasio

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

ARN de predicción de estructura secundaria Utilizando SHAPE de alto rendimiento

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Selección de alto rendimiento funcional utilizando un doble resplandor de ensayo de luciferasa Homemade

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Plataforma modular de selección automatizados de alto rendimiento de exopolisacáridos Junto con el análisis de carbohidratos de la huella digital de alta sensibilidad

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Multienzimático de detección sistemática mediante un sistema de cribado genético de la enzima de alto rendimiento

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...