Agradecemos a la Fundación de Fibrosis Quística (CFF) para la financiación de este trabajo a través de su Consorcio CFTR Estructura 3D (el número de concesión FORD08XX0). Reconocemos la enorme contribución de todos nuestros colegas en el Consorcio a este trabajo, en particular en el diseño de los genes CFTR. También reconocemos la valiosa contribución de los Dres. James Birtley (NCSR Demócrito, Grecia), Mark Young (Universidad de Cardiff, Reino Unido) y David Drew (Imperial College de Londres, Reino Unido) en las primeras etapas de la obra. Agradecemos especialmente a la Dra. Ina Urbatsch (Texas Tech. Universidad de Lubbock) para la lectura crítica del manuscrito.

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Chem. 281, 36856-36863 (2006).</li><li>Zhang, Y., Michaelis, S., Brodsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+expression+and+ER-associated+degradation+in+yeast.">CFTR expression and ER-associated degradation in yeast.</a> Methods Mol. Med. 70, 257-2565 (2002).</li><li>Drew, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-based+optimization+scheme+for+the+overexpression+and+purification+of+eukaryotic+membrane+proteins+in+Saccharomyces+cerevisiae.">GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae.</a> Nat. Protoc. 3, 784-798 (2008).</li><li>Knight, A. W., Billinton, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+wood+through+the+trees:+A+review+of+techniques+for+distinguishing+green+fluorescent+protein+from+endogenous+autofluorescence.">Seeing the wood through the trees: A review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence.</a> Anal. Biochem. 291, 175-197 (2001).</li><li>Robinson, K. M., Lemire, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+nucleotide+sequence+of+the+Saccharomyces+cerevisiae+gene+for+the+succinate+dehydrogenase+flavoprotein+subunit.">Isolation and nucleotide sequence of the Saccharomyces cerevisiae gene for the succinate dehydrogenase flavoprotein subunit.</a> J. Biol. Chem. 267, 10101-10107 (1992).</li><li>Gnann, A., Riordan, J. R., Wolf, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+transmembrane+conductance+regulator+degradation+depends+on.">Cystic fibrosis transmembrane conductance regulator degradation depends on.</a> the lectins Htm1p/EDEM and the Cdc48 protein complex in. 15, 4125-4135 (2004).</li><li>Yoo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-conventional+trafficking+of+the+cystic+fibrosis+transmembrane+conductance+regulator+through+the+early+secretory+pathway.">Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway.</a> J. Biol. Chem. 277, 11401-11409 (2002).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+cystic+fibrosis+transmembrane+conductance+regulator+protein+and+structural+changes+associated+with+phosphorylation+and+nucleotide+binding.">Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding.</a> Journal of Structural Biology. 167, 242-251 (2009).</li><li>Wellhauser, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-molecule+modulator+interacts+directly+with+deltaPhe508-CFTR+to+modify+its+ATPase+activity+and+conformational+stability.">A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability.</a> Mol. Pharmacol. 75, 1430-148 (2009).</li><li>Ramjeesingh, 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+monomer+is+the+minimum+functional+unit+required+for+channel+and+ATPase+activity+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">A monomer is the minimum functional unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator.</a> Biochemistry. 40, 10700-10706 (2001).</li><li>Huang, P., Liu, Q., Scarborough, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidylglycerol:+a+novel+effective+detergent+for+solubilizing+and+purifying+the+cystic+fibrosis+transmembrane+conductance+regulator.">Lysophosphatidylglycerol: a novel effective detergent for solubilizing and purifying the cystic fibrosis transmembrane conductance regulator.</a> Anal. Biochem. 259, 89-97 (1998).</li></ol>

No hay conflictos de interés declarado.

Este documento proporciona un método para la expresión de proteína CFTR murino en células de levadura, que debería facilitar la investigación sobre la fibrosis quística. El objetivo es vincular este trabajo con el lanzamiento de la construcción de ratón CFTR ADN, que estará disponible a través de la Fundación de Fibrosis Quística ( <a href="http://www.cff.org/research/CFFT/">http://www.cff.org/research/CFFT/</a> ). Otros orthologs debería estar disponible más tarde. La transformación de las células de levadura con el vector que contiene CFTR es sencillo, pero es importante para la detección de colonias que expresan altos niveles de CFTR. Niveles variables de expresión pueden derivarse de varios factores, pero el número de copias del plásmido por célula probablemente representa un importante grado de variación. Los pasos críticos que se describen aquí debe permitir la producción de CFTR que expresan las células de levadura y que contienen membranas microsomales CFTR. Una vez que la transformación, crecimiento, cosecha y la lisis de las células de levadura han sido dominadas, Purificación de la proteína debe ser posible, y en la Figura 3 se ha dado un ejemplo de la pureza que debe ser alcanzable en este caso como una referencia útil. No es nuestra intención en este manuscrito para proporcionar una metodología detallada para la purificación de la proteína. Sin embargo, hay algunos pasos críticos intermedios de purificación que son específicos para el sistema de expresión S.cerevisae, tales como la lisis celular y purificación microsoma, y éstos se han incluido en detalle en este manuscrito. Cabe mencionar, sin embargo, que, aparte de los dos métodos que hemos utilizado, alternativas de levadura métodos ruptura celular pueden ser empleados, tales como el uso de una célula de presión Francesa. La proteína recombinante tiene una TEV-escindible dominio C-terminal GFP que permite que la proteína a ser rastreados después de la inducción (fig. 4). La levadura tiene una proteína intrínseca 70kDa (probablemente succinato deshidrogenasa 12) que presenta fluorescencia bajo las mismas condiciones 11, y esto puede proporcionar una herramienta útil entreestándar de calibración nal para los niveles de expresión relativos de CFTR en extractos de células enteras o microsomas (fig. 3). Es evidente a partir de los datos mostrados en la Figura 4 que el momento de la recolección de células después de la inducción con galactosa es crucial. Los rendimientos de caída de CFTR precipitadamente después de aproximadamente 16 horas de inducción, de modo que apenas hay CFTR detectable en células de levadura después de 24 horas de inducción. El rendimiento de proteína purificada es de aproximadamente 1-2mg proteína CFTR por 15 litros cultura fermentador. La recuperación puede ser estimado como aproximadamente 70% del total de CFTR-proteína GFP hasta la fase de microsoma, y ​​alrededor del 25% de recuperación de la proteína purificada. Caracterización de la S. cerevisiae, expresó CFTR está en curso. Como se ve en la fig. 4, la localización de la proteína en la célula puede ser controlado por microscopía de fluorescencia. Aunque gran parte de la fluorescencia se encuentra alrededor de la periferia de la célula como se esperaba 10, parte de la proteína muestra una localización puntiforme, ya sea en ojusto dentro de la membrana plasmática, que podría ser debido a CFTR de reciclaje a través de una tarde de Golgi / endosomal vía 14 o tal vez un compartimiento posterior de la brotación de las vesículas de transporte de la sala de emergencia 4. El tratamiento con PNGasaF, una enzima que deglycosylates proteínas, mostró un cambio mínimo en la migración de la banda de proteína CFTR en SDS-PAGE, lo que implica que se no glicosilada, o tiene glicosilación mínima 15. Los experimentos en el estado de fosforilación de la proteína están en marcha. En algunos de los detergentes ensayadas hasta ahora, la proteína purificada muestra la actividad de ATPasa (que es inhibida por un inhibidor específico CFTR 16) a tasas que son similares a los publicados anteriormente 2,15. Medición de la actividad del canal CFTR requerirá la reconstitución de la proteína purificada, lo que implica una etapa de purificación final en un detergente que tiene una concentración crítica de micelas relativamente alta (CMC) 17. Levadura microsomas containing CFTR puede solublised con varios detergentes comúnmente empleados 18, incluyendo detergentes como dodecilsulfato maltósido 2, que son generalmente considerados como &quot;suave&quot;. Sin embargo la mayoría de los detergentes cmc alta han demostrado ser ineficientes para solubilsation, hasta ahora, lo que sugiere que el intercambio en estos detergentes deben ser considerados en una etapa tardía en cualquier esquema de purificación.

<table border="1"><tbody><tr><td> Nombre del reactivo / equipo </td><td> Empresa </td><td> N ° de catálogo </td></tr><tr><td> FGY217 S.cerevisiae cepa, con pep4 supresión 10 </td><td></td><td></td></tr><tr><td> La levadura base de nitrógeno sin aminoácidos </td><td> Formedium </td><td> CYN0410 </td></tr><tr><td> Completa mezcla de suplemento sin uracilo </td><td> Formedium </td><td> DCS0169 </td></tr><tr><td> Agar bacteriológico </td><td> Sigma-Aldrich </td><td> A5306 </td></tr><tr><td> D-galactosa </td><td> Pescador </td><td> BP656-500 </td></tr><tr><td> D-glucosa </td><td> Pescador </td><td> D16-500 </td></tr><tr><td> Un Pepstatina </td><td> Sigma-Aldrich </td><td> P4265 </td></tr><tr><td> Leupeptina </td><td> Merck </td><td> 108975 </td></tr><tr><td> Quimostatina </td><td> Sigma-Aldrich </td><td> C7268 </td></tr><tr><td> Phenylmethanesulfonylfluoride (PMSF) </td><td> Sigma-Aldrich </td><td> P7626 </td></tr><tr><td> Epoxysuccinyl-leucylamido-butano (E-64) </td><td> Sigma-Aldrich </td><td> E3132 </td></tr><tr><td> Aminoethylbenzenesulfonyl fluoruro (AEBSF) </td><td> Sigma-Aldrich </td><td> A8456 </td></tr><tr><td> Benzamidina clorhidrato de </td><td> Sigma-Aldrich </td><td> 434760 </td></tr><tr><td> Dimetilsulfóxido (DMSO) </td><td> Sigma-Aldrich </td><td> D8418 </td></tr><tr><td> ditiotreitol (DTT) </td><td> Sigma-Aldrich </td><td> 43815 </td></tr><tr><td> ácido etilendiaminotetraacético (EDTA) </td><td> Pescador </td><td> BP120500 </td></tr><tr><td> Tris-base </td><td> Formedium </td><td> TRIS01 </td></tr><tr><td> Tris-HCl </td><td> Pescador </td><td> P631 </td></tr&gt; <tr><td> D-sorbitol </td><td> Sigma-Aldrich </td><td> S1876 </td></tr><tr><td> Glicerol </td><td> Pescador </td><td> 065017 </td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> S6191 </td></tr><tr><td> n-dodecil-β-D maltopiranósido </td><td> Affymetrix </td><td> D310S </td></tr><tr><td> Azul de bromofenol </td><td> Sigma-Aldrich </td><td> 114391 </td></tr><tr><td> PageRuler Plus estándares preteñidos proteínas </td><td> Fermentas </td><td> SM1811 </td></tr><tr><td> NuSep tris-Gly 4-20% geles de gradiente </td><td> NuSep </td><td> NB10-420 </td></tr><tr><td> Instantánea azul de Coomassie </td><td> Novexin </td><td> ISB1L </td></tr><tr><td> Cuentas de vidrio, lavados con ácido </td><td> Sigma </td><td> G8772 </td></tr><tr><td> 50 ml estériles Tubos Falcon </td><td> Sarstedt </td><td> 62.547.254 </td></tr><tr><td> 2 mlEstériles con tapa de rosca viales </td><td> Sarstedt </td><td> 72.694.005 </td></tr><tr><td> 250 ml estéril Erlenmeyers desconcertado </td><td> BD Biosciences </td><td> 355119 </td></tr><tr><td> Erlenmeyer estéril 2l desconcertado frascos </td><td> BD Biosciences </td><td> 355131 </td></tr><tr><td> Tubos de 2 ml de microfuga </td><td> Sarstedt </td><td> 72.695 </td></tr><tr><td> 0.2μM filtro de jeringa </td><td> Sartorio </td><td> FC121 </td></tr><tr><td> tubos de ultracentrífuga </td><td> Beckman Coulter </td><td> 355618 </td></tr><tr><td> tubos de centrífuga </td><td> Beckman Coulter </td><td> 357000 </td></tr><tr><td> Potes de 1 litro de centrifugadoras </td><td> Beckman Coulter </td><td> 969329 </td></tr><tr><td> Incubadora de agitación orbital con control de temperatura </td><td> New Brunswick Scientific </td><td></td></tr><tr><td> Deltavision RT microscopio de la restauración </td><td> Visión Aplicada </td><td></td></tr><tr><td> Centrífuga de mesa </td><td> HERMLE </td><td> Z300 </td></tr><tr><td> Microcentrífuga de mesa </td><td> Pescador </td><td> 13-100-511 </td></tr><tr><td> Vortex mezclador de </td><td> Star Labs </td><td></td></tr><tr><td> El tifón escáner Trio </td><td> GE Healthcare </td><td> 63-0055-87 </td></tr><tr><td> LAS3000 sistema de imagen </td><td> Fuji </td><td></td></tr><tr><td> Recipiente fermentador 20l y unidad de control </td><td> Applikon </td><td></td></tr><tr><td> Constante de los sistemas de disruptor celular </td><td> Sistemas de constantes </td><td></td></tr><tr><td> Beadbeater disruptor celular </td><td> BioSpec </td><td> 1107900 </td></tr><tr><td> Mini-beadbeater-16 </td><td> BioSpec </td><td> 607 </td></tr><tr><td> JA-17 rotor </td><td> Beckman Coulter </td><td> 369691 </td></tr><tr><td> Optima L-100 Ultracentrífuga </td><td> Beckman Coulter </td><td> 392050 </td></tr><tr><td> Rotor de 50.2Ti </td><td> Beckman Coulter </td><td> <a href="https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=337901" target="_blank">337901</a> </td></tr></tbody></table>

expression and purification of the cystic fibrosis transmembrane conductance regulator protein in saccharomyces cerevisiae

La fibrosis quística transmembrana regulador de la conductancia (CFTR) es un canal de cloruro, que cuando está mutado, puede dar lugar a la fibrosis quística en humans.There es por lo tanto, un considerable interés en esta proteína, pero los esfuerzos para estudiar su estructura y actividad se han visto obstaculizados por la dificultad de expresión y purificación de cantidades suficientes de la proteína 1-3. Al igual que muchos de los difíciles de proteínas de membrana eucariotas, la expresión en un organismo de crecimiento rápido es deseable, pero difícil, y en la levadura S. cerevisiae, hasta ahora cantidades bajas se obtuvieron y una rápida degradación de la proteína recombinante se observó 4-9. Las proteínas involucradas en el procesamiento de CFTR en la levadura recombinante se han descrito 6.9. En este informe se describe una metodología para la expresión de CFTR en la levadura y su purificación en cantidades significativas. El protocolo describe cómo los problemas de la proteólisis anteriores se pueden superar y cómo los niveles de expresión deCFTR puede ser mejorado mediante la modificación de las condiciones de crecimiento de células y mediante el control de las condiciones de inducción, en particular, el período de tiempo antes de la recolección de células. Los reagants asociados con este protocolo (CFTR murino que expresan las células de levadura o plásmidos de levadura) se distribuyen a través de los EE.UU. Fundación de Fibrosis Quística, que ha patrocinado la investigación. Un artículo que describe el diseño y síntesis de la CFTR constructo empleado en el presente informe se publicará por separado (Urbatsch, I.;. Thibodeau, P. et al, inédito). En este artículo vamos a explicar nuestro método de inicio de la transformación de las células de levadura con la construcción de CFTR - la levadura que contiene el plásmido (Fig. 1). La construcción cuenta con una proteína verde fluorescente (GFP) fusionada a la secuencia CFTR en su extremo C-terminal y sigue el sistema desarrollado por Drew et al. (2008) 10. El GFP permite la expresión y purificación de CFTR que debe seguirse con relativa facilidad. El protoc Jove visualizaracabados ol después de la preparación de microsomas de las células de levadura, aunque incluyen algunas sugerencias para la purificación de la proteína a partir de los microsomas. Los lectores pueden añadir sus propias modificaciones en el procedimiento de purificación de microsomas, depende de los experimentos finales que se llevarán a cabo con la proteína y el equipo local a su disposición. La levadura expresa la proteína CFTR puede ser parcialmente purificada usando iones metálicos cromatografía de afinidad, usando una etiqueta polihistidina depuración intrínseca. Después de exclusión por tamaño cromatografía produce una proteína que parece ser&gt; 90% de pureza, según se juzga por SDS-PAGE y tinción con Coomassie del gel.

Watch this Scientific Journal Video about Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae at JoVE.com

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

Los intentos para expresar la transmembrana de la fibrosis quística regulador de la conductancia (CFTR) en<em&gt; Saccharomyces cerevisiae</em&gt;, Hasta ahora, dado cantidades relativamente bajas de proteína. Este protocolo y los reactivos asociados distribuidos a través de la Fundación de Fibrosis Quística debe permitir la preparación de cantidades de miligramos de esta &quot;difícil&quot; proteína de la membrana eucariota.

Expresión y purificación de la proteína transmembrana de la fibrosis quística regulador de la conductancia en la Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

expression-purification-cystic-fibrosis-transmembrane-conductance

Research

JoVE Journal

Biology

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

18.4K vistas.  University of Manchester. Los intentos para expresar la transmembrana de la fibrosis quística regulador de la conductancia (CFTR) en Saccharomyces cerevisiae, Hasta ahora, dado cantidades relativamente bajas de proteína. Este protocolo y los reactivos asociados distribuidos a través de la Fundación de Fibrosis Quística debe permitir la preparación de cantidades de miligramos de esta "difícil" proteína de la membrana eucariota.

Video: Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

La disección de Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Investigación

Biología

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 ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Phosphoproteomics cuantitativo de ácidos grasos estimulada Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

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

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

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

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

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

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

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

El análisis de microarrays para Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un ensayo de microscopía de fluorescencia para Mitophagy Vigilancia de la levadura Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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

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

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

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

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

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

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

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

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

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPR supervisado Redox La titulación de los cofactores de<em&gt; Saccharomyces cerevisiae</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...