Queremos agradecer a todos los miembros del laboratorio Dinman, incluyendo Ashton Belew, Jack Karen, Musalga Sharmishtha, Sulima Sergey, Reddy Shivani, y Michael Rhodin por su ayuda y aportación en este proyecto. Este trabajo fue apoyado por becas de AM de la American Heart Association (AHA 0630163N) y JDD de los Institutos Nacionales de Salud (5R01 GM058859-12). JAL fue apoyado por una de Reinversión y Recuperación Americana de 2009, suplemento al título principal (3R01GM058859-11S1).

1. Preparación de una cisteína acusado Sulfolink resina <ol><li> Prepare Sulfolink resina (Pierce) a temperatura ambiente. </li><li> Lugar a un total de 10 ml de una mezcla del 50% de gel de acoplamiento Sulfolink suministrada por el fabricante en dos viales de 10 ml de plástico, con 5 ml distribuidos en cada vial. </li><li> En pocas palabras centrífuga viales a 850 xg y retirar con cuidado los sobrenadantes. </li><li> Lavar el gel tres veces en tampón de acoplamiento (50 mM Tris, pH 8,5, 5 mM EDTA) por resuspensión de las cuentas de 5 ml, centrifugar brevemente a 850 xg y eliminar el sobrenadante con una pipeta. </li><li> Agregue cinco ml de una solución 50 mM de L-cisteína en tampón de acoplamiento a cada tubo y mezclar la pasta durante 1 hora a 25 ° C. </li><li> Eliminar el residuo de L-cisteína por lavado como se describe en el paso 1.4. </li><li> Resuspender el gel en 10 ml de tampón de unión [20 mM HEPES, pH 7,6, 5 mM Mg (OAc) 2, 60 mM NH 4 Cl, 2 mM TDT], y equilibrar la resina por el lavado en 10 ml de tampón de unión3 veces como se describe en el paso 1.4. Resina se decanta en un 10 ml de centrífuga columna, la tapa y se almacena a 4 º C. La capacidad de la resina de unión al ARN es de ~ 20 A 260 unidades por mililitro. </li></ol> 2. Purificación por cromatografía de ribosomas con una cisteína acusado Sulfolink resina Nota: Si se trabaja con una tensión donde la actividad de la proteasa ha demostrado ser un problema, los cócteles de la proteasa inhibición puede ser utilizado en todos los topes posteriores. <ol><li> Mantener todos los componentes en el hielo y preparar todas las soluciones con RNasa libre de agua y filtración estéril. </li><li> Se cultivan las células de levadura durante la noche a mitad de la fase de registro (OD 595 = 0,6 - 1,2) en los medios de comunicación adecuados. Pellet las células por centrifugación y se lavan las células de un gramo en tampón de unión y centrifugar a 3700 xg durante 5 minutos a 4 ° C. </li><li> Resuspender las células en 1 ml de tampón de unión para un volumen total aproximado de 2 ml, e interrumpir el uso de un volumen igual de 0,5 mm perlas de vidrio se enfría a 4 ° C ucantar una Biospec Mini-batidor de bolas (Bartlesville, OK). Las muestras se pueden dividir en varios tubos según sea necesario. </li><li> Eliminar las células intactas, orgánulos, y los restos celulares por centrifugación a 30.000 xg durante 30 minutos en una ultracentrífuga Beckman-Coulter Optima Max E (Fullerton, CA). </li><li> Inmediatamente antes del uso de perdigones de la resina durante un minuto a 1.000 xg para extraer la solución de almacenamiento. Dos ml de resina se utiliza para cada gramo de las células. </li><li> Quitar sobrenadantes de células lisado de la vuelta 30 000 xg (S30), teniendo cuidado para minimizar la contaminación ya sea de la fracción lipídica en la parte superior o los restos celulares en la parte inferior de los tubos, y el lugar directamente en la suspensión Sulfolink cargos. La capa de lípidos se puede evitar, ya sea por retiro o por empujar fuera de la pipeta después de que la punta se cruzó en el sobrenadante. </li><li> Incubar la mezcla S30/Sulfolink en el hielo sin mezclar durante 15 minutos. </li><li> Coloque las columnas en 15 ml tubos cónicos y se centrifuga a 1.000 g duranter un minuto. </li><li> Coloque las fracciones de caudal contínuo de nuevo en las columnas, mezclar con la mano, y se incuba durante 15 minutos en hielo. </li><li> Coloque las columnas en 15 ml tubos cónicos y se centrifuga a 1.000 g durante un minuto. Deseche el caudal contínuo. </li><li> Columnas de la tapa y añadir 5 ml de tampón de unión. Mezcla de columnas con la mano hasta resuspendido, quite las tapas, y centrifugar la centrífuga columnas en 1000 xg durante un minuto. Repita este protocolo de lavado dos veces más. </li><li> Añadir 1,5 ml de tampón de elución (20 mM HEPES-KOH, pH 7,6, 10 mM Mg (OAc) 2, 500 mM KCl, 2 mM DTT, 0,5 mg / ml de heparina) para cada columna. Mezcla de lodos de la mano, e incubar en hielo durante 2 minutos </li><li> Columnas en lugar de nuevos tubos de 15 ml y centrifugar a 1000 xg durante 1 minuto. Recoger eluido. Repita el paso de elución una vez más, de modo que el volumen final, además de la muestra se ~ 3 ml. Resina utilizada se pueden lavar con tampón de elución y sin heparina seguida de equilibrio con buffe vinculanter, y se almacenan en volúmenes de 10 ml de tampón de unión a 4 ° C para su reutilización posterior. </li></ol> 3. Puromicina tratamiento Nota: Un método alternativo para eliminar la contaminación de especies tRNA es pasar de un nivel de glucosa rico en medios de glucosa reducido para promover la escorrentía ribosoma. Sin embargo, esto no cambia el estado metabólico de las células de levadura, lo que podría afectar la función del ribosoma y la concentración. Con el fin de despojar a los ribosomas de la endógeno peptidil-tRNA, un tratamiento con puromicina se lleva a cabo. <ol><li> Neutralizar a un pH de 7,5 una solución de 100 mM puromicina mediante la adición de KOH 1M gota a gota, a temperatura ambiente. </li><li> Añadir solución neutralizada puromicina a ~ concentración final de 1mM eluido. </li><li> Añadir 100 mM GTP hasta una concentración final de 1 mM. </li><li> Incubar durante 30 minutos a 30 ° C. </li></ol> 4. Purificación de los ribosomas por sedimentación a través de los cojines de glicerol <ol><li> Coloque un mlde tampón amortiguador (20 mM HEPES, pH 7,6, 10 mM Mg (OAc) 2, 500 mM KCl, 2 mM de DTT, 25% de glicerol) en un tubo de 4 ml de volumen ultracentrífuga policarbonato. </li><li> Suavemente la capa de puromicina tratados fracciones de elución en la parte superior de la almohadilla, y las muestras de centrifugar a 100.000 xg durante la noche a 4 ° C. </li><li> Retire los tubos de los rotores de centrifugación, y aspirar sobrenadante. </li><li> Lávese las pastillas que contienen ribosomas purificados dos veces con 1 ml de tampón de amortiguación. </li><li> Resuspender los ribosomas en 100 l de tampón amortiguador por la interrupción suave usando una varilla de vidrio. </li><li> Después de la interrupción, cubrir los tubos con parafilm y agitar a una velocidad moderada en un vórtice cuarto frío durante una hora. </li><li> Transferir el contenido de un tubo de microcentrífuga, centrifugar durante 5 minutos a velocidad máxima a 4 ° C, y eliminar los sobrenadantes a tubos nuevos. </li><li> Repita los pasos 4,1 a 4,7 con 2 ml de tampón de amortiguación y, salvo que l 100 de buffer de almacenamiento (50 mM HEPES-KOH pH 7,6, 50 mM NH 4 Cl, 5 mM Mg(OAc) 2, 1 mM DTT, 25% de glicerol) se utiliza en los pasos 4.4 y 4.5 en lugar de buffer amortiguador. </li><li> Cuantificar los ribosomas purificados por espectrofotometría (1 A 260 = 20 pmoles de los ribosomas de levadura), y almacenar a -80 ° C. Los resultados típicos de 1 gramo de levadura son 300-400 pmoles de los ribosomas. </li></ol> 5. Los resultados representativos: Un ejemplo de las especies de ARN extraído de cada uno de los tres pasos principales del protocolo se muestra en la Figura 2. Mientras rRNAs son las principales especies presentes en lisados ​​celulares totales (T), estos también contienen un gran número de otras especies de ARN. Ribosomas purificados de la columna Sulfolink (SL) también contienen una gran cantidad de tRNAs debido a la alta afinidad del lecho de la columna para estas especies también. El tratamiento de esta fracción con los resultados de puromicina en la hidrólisis de los péptidos de la peptidil-ARNt, y promueve la disociación de estas especies de los ribosomas. La puromicina muestras tratadas (Pm) la falta de co-purifying especies de tRNA, y por lo tanto representan los ribosomas completamente puro. Como se informó anteriormente 8, estos ribosomas son altamente intactos y activos bioquímicamente, lo que los soportes ideales para detallado análisis funcional y estructural. <img src="/files/ftp_upload/3214/3214fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de flujo del método. Purificación cromatográfica de los ribosomas con la cisteína vinculada sulfolink resina se representa. <img src="/files/ftp_upload/3214/3214fig2.jpg" alt="Figura 2"/> Figura 2. Análisis representativo de los preparativos del ribosoma. Especies de ARN total se extrajeron a partir de lisados ​​celulares totales (T), Sulfolink ribosomas purificados (SL) y ribosomas Sulfolink purificado posteriormente fueron tratadas con puromicina y sedimentado a través de un colchón de glicerol (Pm). Carril M representa el tamaño de marcadores de ARN. Las bandas que representan rRNAs 25S y 18S, así como los ARNt y otras especies de ARN se indican. Todoslos carriles fueron cargados con 2 g de ARN.

<ol>
	<li>Palade, G. E., Siekevitz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+microsomes;+an+integrated+morphological+and+biochemical+study.">Liver microsomes; an integrated morphological and biochemical study.</a> J. Biophys. Biochem. Cytol. 2, 171-200 (1956).</li><li>Fabry, M., Kalvoda, L., Rychlik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interactions+of+Escherichia+coli+ribosomes.">Hydrophobic interactions of Escherichia coli ribosomes.</a> Biochim. Biophys. Acta. 652, 139-150 (1981).</li><li>Jelenc, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+highly+active+ribosomes+from+Escherichia+coli.">Rapid purification of highly active ribosomes from Escherichia coli.</a> Anal. Biochem. 105, 369-374 (1980).</li><li>Le goffic, F., Moreau, N., Chevelot, L., Langrene, S., Siegrist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+bacterial+ribosomes+using+chloramphenicol+and+erythromycin+columns.">Purification of bacterial ribosomes using chloramphenicol and erythromycin columns.</a> Biochimie. 62, 69-77 (1980).</li><li>Meskauskas, A., Petrov, A. N., Dinman, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+functionally+important+amino+acids+of+ribosomal+protein+L3+by+saturation+mutagenesis.">Identification of functionally important amino acids of ribosomal protein L3 by saturation mutagenesis.</a> Mol. Cell Biol. 25, 10863-10874 (2005).</li><li>Algire, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+reconstituted+yeast+translation+initiation+system.">Development and characterization of a reconstituted yeast translation initiation system.</a> RNA. 8, 382-397 (2002).</li><li>Maguire, B. A., Wondrack, L. M., Contillo, L. G., Xu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chromatography+system+to+isolate+active+ribosomes+from+pathogenic+bacteria.">A novel chromatography system to isolate active ribosomes from pathogenic bacteria.</a> RNA. 14, 188-195 (2008).</li><li>Leshin, J. A., Rakauskaite, R., Dinman, J. D., Meskauskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purity,+activity+and+structural+integrity+of+yeast+ribosomes+purified+using+a+general+chromatographic+method.">Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method.</a> RNA. Biol. 7, 354-360 (2010).</li><li>Triana, F., Nierhaus, K. H., Chakraburtty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+RNA+binding+to+80S+ribosomes+from+yeast:+evidence+for+three+sites.">Transfer RNA binding to 80S ribosomes from yeast: evidence for three sites.</a> Biochem. Mol. Biol. Int. 33, 909-915 (1994).</li><li>Azzam, M. E., Algranati, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+puromycin+action:+fate+of+ribosomes+after+release+of+nascent+protein+chains+from+polysomes.">Mechanism of puromycin action: fate of ribosomes after release of nascent protein chains from polysomes.</a> Proc. Natl. Acad. Sci. U. S. A. 70, 3866-3869 (1973).</li><li>Nathans, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Puromuycin+inhibition+of+protein+synthesis:+incorporation+of+puromycin+into+peptide+chains.">Puromuycin inhibition of protein synthesis: incorporation of puromycin into peptide chains.</a> Proc. Natl. Acad. Sci. U. S. A. 51, 585-592 (1964).</li><li>Rabinovitz, M., Fisher, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dissociative+effect+of+puromycin+on+the+pathway+of+protein+synthesis+by+Ehrlich+ascites+tumor+cells.">A dissociative effect of puromycin on the pathway of protein synthesis by Ehrlich ascites tumor cells.</a> J. Biol. Chem. 237, 477-481 (1962).</li><li>Allen, D. W., Zamecnik, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+puromycin+on+rabbit+reticulocyte+ribosomes.">The effect of puromycin on rabbit reticulocyte ribosomes.</a> Biochim. Biophys. Acta. 55, 865-874 (1962).</li><li>Yarmolinsky, M. B., Haba, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+by+puromycin+of+amino+acid+incorporation+into+protein.">Inhibition by puromycin of amino acid incorporation into protein.</a> Proc. Natl. Acad. Sci. U. S. A. 45, 1721-1729 (1959).</li><li>Becker-Ursic, D., Davies, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+phosphorylation+of+ribosomal+proteins+by+protein+kinases+from+Saccharomyces+cerevisiae.">In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.</a> Biochemistry. 15, 2289-2296 (1976).</li></ol>

No hay conflictos de interés declarado.

Protocolos de purificación de ribosomas en esencia, de las células de lisis, cosecha una fracción citosólica de un giro a baja velocidad, y luego granulación ribosomas por centrifugación de alta velocidad 1. Mientras que un nuevos métodos se han utilizado algunos para la purificación de los ribosomas bacterianos, la misma no había sido cierto para los eucariotas 2-4. A pesar de las medidas adicionales han sido añadidos a lo largo de los años, por ejemplo, se lava la sal, y los cojines de...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Sulfolink acoplamiento de resina </td><td> Atravesar </td><td> 20402 </td></tr><tr><td> Mini-batidor de cuentas 16 </td><td> Biospec </td><td> 607 </td></tr><tr><td> Optima Max E ultracentrífuga </td><td> Beckman Coulter </td><td></td></tr><tr><td> Leyenda RT </td><td> Sorvall </td><td></td></tr><tr><td> 0.5 Cuentas de vidrio mm </td><td> Biospec </td><td> 11079105 </td></tr><tr><td> Tris </td><td> Sigma-Aldrich </td><td> 252859 </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E9884 </td></tr><tr><td> TDT </td><td> Sigma-Aldrich </td><td> 43815 </td></tr><tr><td> HEPES </td><td> Sigma-Aldrich </td><td> 54459 </td></tr><tr><td> NH 4 Cl </td><td> Sigma-Aldrich </td><td> A9434 </td></tr><tr><td> KCl </td><td> Sigma-Aldrich </td><td> 60128 </td></tr><tr><td> Mg (OAc) 2 </td><td> Sigma-Aldrich </td><td> M5661 </td></tr><tr><td> KOH </td><td> Sigma-Aldrich </td><td> 221473 </td></tr><tr><td> Glicerol </td><td> Sigma-Aldrich </td><td> G5516 </td></tr><tr><td> Puromicina </td><td> Sigma-Aldrich </td><td> P7255 </td></tr><tr><td> GTP </td><td> Fermentas </td><td> R0461 </td></tr></tbody></table>

chromatographic purification of highly active yeast ribosomes

Ribosomas eucariotas son mucho más lábiles en comparación con sus contrapartes eubacterial y archael, lo que plantea un importante reto para los investigadores. Particularmente preocupante es el hecho de que la lisis de las células libera una gran cantidad de proteasas y nucleasas que pueden degradar los ribosomas. Por lo tanto, es importante separar los ribosomas de estas enzimas lo más rápido posible. Desafortunadamente, los métodos convencionales de ultracentrifugación diferencial ribosomas hojas expuestas a estas enzimas por períodos excesivamente largos de tiempo, afectando su integridad estructural y funcionalidad. Para solucionar este problema, utilizamos un método de cromatografía de cisteína con un cargo Sulfolink resina. Esta aplicación sencilla y rápida reduce significativamente co-purificación de las actividades proteolíticas y nucleolytic, produciendo altos rendimientos de los ribosomas intactos, levadura muy bioquímicamente activas. Sugerimos que este método debería ser aplicable también a los ribosomas de mamíferos. La simplicidad deel método, y la pureza mejorada y la actividad de los ribosomas cromatográficas representa un avance técnico importante para el estudio de los ribosomas eucarióticos.

Watch this Scientific Journal Video about Chromatographic Purification of Highly Active Yeast Ribosomes at JoVE.com

Chromatographic Purification of Highly Active Yeast Ribosomes

La contaminación de las preparaciones de los ribosomas eucarióticos purificado por los métodos tradicionales de purificación de nucleasas y proteasas co-incide negativamente en posteriores análisis bioquímicos y estructurales. Un método de cromatografía de purificación rápida y sencilla se utiliza para resolver este problema con los ribosomas levadura como un sistema modelo.

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.  ...

chromatographic-purification-of-highly-active-yeast-ribosomes

Research

JoVE Journal

Biology

16.7K vistas.  University of Maryland. La contaminación de las preparaciones de los ribosomas eucarióticos purificado por los métodos tradicionales de purificación de nucleasas y proteasas co-incide negativamente en posteriores análisis bioquímicos y estructurales. Un método de cromatografía de purificación rápida y sencilla se utiliza para resolver este problema con los ribosomas levadura como un sistema modelo.

Video: Chromatographic Purification of Highly Active Yeast Ribosomes

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 Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purificación de las mitocondrias de las células de levadura

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Preparación de corazón de rata altamente acoplado mitocondrias

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

El aislamiento de los ribosomas traducir contienen peptidil-ARNt para el Análisis Funcional y Estructural

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Colonia de levaduras Método de anidamiento

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

En la reconstitución in vitro de los activos T. castaneum telomerasa

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

La levadura de alto rendimiento de la pantalla sobreexpresión plásmido

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Pantallas competitiva Genómica de Bibliotecas de levadura con código de barras

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Florecimiento extracción de proteínas de levadura y purificación para el Estudio de la función, las interacciones y modificaciones post-traduccionales

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

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

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

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

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

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

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

Methods Article

Purificación por cromatografía de ribosomas de levadura de gran actividad