Nous remercions le Cystic Fibrosis Foundation (CFF) pour le financement de ce travail par le biais de son Consortium Structure CFTR 3D (numéro de subvention FORD08XX0). Nous reconnaissons l&#39;énorme contribution de tous nos collègues au sein du Consortium à ce travail, en particulier dans la conception des gènes CFTR. Nous reconnaissons également la contribution inestimable des Drs. James Birtley (CNRS Demokritos, Grèce), Mark Young (Université de Cardiff, Royaume-Uni) et David Drew (Imperial College, Londres, Royaume-Uni) dans les premiers stades du travail. Nous remercions tout particulièrement le Dr Ina Urbatsch (Texas Tech. University, Lubbock) pour la lecture critique du manuscrit.

<ol>
	<li>Ford, R. C., Birtley, J., Rosenberg, M. F., 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=CFTR+three-dimensional+structure.">CFTR three-dimensional structure.</a> Methods Mol. Biol. 741, 329-346 (2011).</li><li>Rosenberg, M. F., Kamis, A. B., Aleksandrov, L. A., Ford, R. C., Riordan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+crystallization+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">Purification and crystallization of the cystic fibrosis transmembrane conductance regulator.</a> CFTR). J. Biol. Chem. 279, 39051-39051 (2004).</li><li>Zhang, L., Aleksandrov, L. A., Riordan, J. R., Ford, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+location+within+the+cystic+fibrosis+transmembrane+conductance+regulator+protein+investigated+by+electron+microscopy+and+gold+labelling.">Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling.</a> Biochim. Biophys. Acta. 1808, 399-404 (2011).</li><li>Kiser, 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=Expression+and+degradation+of+the+cystic+fibrosis+transmembrane+conductance+regulator+in+Saccharomyces+cerevisiae.">Expression and degradation of the cystic fibrosis transmembrane conductance regulator in Saccharomyces cerevisiae.</a> Arch. Biochem. Biophys. 390, 195-205 (2001).</li><li>Huang, P., Stroffekova, K., Cuppoletti, J., Mahanty, S. K., 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=Functional+expression+of+the+cystic+fibrosis+transmembrane+conductance+regulator+in+yeast.">Functional expression of the cystic fibrosis transmembrane conductance regulator in yeast.</a> Biochim. Biophys. Acta. 1281, 80-90 (1996).</li><li>Ahner, A., Nakatsukasa, K., Zhang, H., Frizzell, R. A., 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=Small+heat-shock+proteins+select+deltaF508-CFTR+for+endoplasmic+reticulum-associated+degradation.">Small heat-shock proteins select deltaF508-CFTR for endoplasmic reticulum-associated degradation.</a> Mol. Biol. Cell. 18, 806-814 (2007).</li><li>Fu, L., Sztul, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER-associated+complexes+(ERACs)+containing+aggregated+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR)+are+degraded+by+autophagy.">ER-associated complexes (ERACs) containing aggregated cystic fibrosis transmembrane conductance regulator (CFTR) are degraded by autophagy.</a> Eur. J. Cell. Biol. 88, 215-226 (2009).</li><li>Sun, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derlin-1+promotes+the+efficient+degradation+of+the+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR)+and+CFTR+folding+mutants.">Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants.</a> J. Biol. 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>

Pas de conflits d&#39;intérêt déclarés.

Ce document fournit une méthode pour l&#39;expression de la protéine CFTR murin des cellules de levure, ce qui devrait faciliter la recherche sur la fibrose kystique. L&#39;objectif est de relier le présent document avec la sortie de la souris CFTR construction d&#39;ADN, qui sera disponible à travers le Cystic Fibrosis Foundation ( <a href="http://www.cff.org/research/CFFT/">http://www.cff.org/research/CFFT/</a> ). Autre orthologues devrait être disponible plus tard. Transformation des cellules de levure avec le vecteur contenant la protéine CFTR est simple, mais il est important à l&#39;écran pour les colonies exprimant des niveaux élevés de la protéine CFTR. Les niveaux d&#39;expression variables peuvent provenir de plusieurs facteurs, mais le nombre de copies de la cellule plasmide par représente probablement un degré significatif de la variation. Les étapes critiques décrites ici devrait permettre la production de la protéine CFTR des cellules exprimant la protéine CFTR et de levure contenant des membranes microsomales. Une fois la transformation, la croissance, la récolte et la lyse des cellules de levure ont été maîtrisées, Purification de la protéine devrait être possible, et à la figure 3, nous avons donné un exemple de la pureté qui devrait être réalisable dans ce cas comme une référence utile. Ce n&#39;est pas notre intention dans ce manuscrit de fournir une méthodologie détaillée pour la purification de la protéine. Cependant, il ya certaines étapes critiques de purification en aval qui sont spécifiques au système d&#39;expression S.cerevisae, tels que la lyse cellulaire et de purification des microsomes, et ceux-ci ont été inclus en détail dans ce manuscrit. Il convient de mentionner, cependant, qu&#39;en dehors des deux méthodes que nous avons utilisés, des méthodes alternatives de cellules de levure de perturbation peuvent être employées, telles que l&#39;utilisation d&#39;une cellule de pression française. La protéine recombinante a un domaine VET-clivable GFP C-terminal qui permet à la protéine à être suivis après induction (fig. 4). Levure ont une protéine intrinsèque 70kDa (probablement succinate déshydrogénase 12) qui entre en fluorescence dans les mêmes conditions 11, ce qui peut fournir une inter utileétalon interne pour les niveaux d&#39;expression relatifs des CFTR dans des extraits cellulaires ou des microsomes entiers (fig. 3). Il est clair à partir des données présentées dans la figure 4 que le moment de la récolte des cellules après induction par le galactose est crucial. Les rendements de baisse CFTR précipitamment après environ 16 heures d&#39;induction, de sorte que il n&#39;y a guère CFTR détectable dans les cellules de levure après 24 heures d&#39;induction. Le rendement de la protéine purifiée est d&#39;environ 1-2mg protéine CFTR par 15 litres culture du fermenteur. La récupération peut être estimée à environ 70% du total CFTR-protéine GFP jusqu&#39;au stade microsomes, et la récupération d&#39;environ 25% de protéine purifiée. Caractérisation de la S. cerevisiae a exprimé la protéine CFTR est en cours. Comme on le voit dans la figure. 4, emplacement de la protéine dans la cellule peut être contrôlée par microscopie de fluorescence. Bien que une grande partie de la fluorescence se trouve autour de la périphérie de la cellule en tant 10 attendue, une partie de la protéine présente une localisation ponctuée, soit dans, oujuste à l&#39;intérieur de la membrane plasmique, qui pourrait être due à CFTR de recyclage grâce à une fin de Golgi / endosome voie 14 ou peut-être un peu en aval du compartiment de le bourgeonnement des vésicules de transport de l&#39;ER 4. Le traitement par PNGaseF, une enzyme qui deglycosylates protéines, ont montré des changements minimes dans la migration de la bande protéine CFTR sur SDS-PAGE, ce qui implique qu&#39;il est non glycosylé, ou a glycosylation minime 15. Expériences sur l&#39;état de phosphorylation de la protéine sont en cours. Dans quelques-unes des détergents testés jusqu&#39;à présent, la protéine purifiée présente une activité ATPase (qui est inhibée par un inhibiteur de la protéine CFTR spécifique 16) à des taux qui sont semblables à ceux publiés antérieurement 2,15. Mesure de l&#39;activité du canal CFTR, il faudra la reconstitution de la protéine purifiée, ce qui impliquerait une étape de purification finale dans un détergent qui a un niveau relativement élevé concentration micellaire critique (cmc) 17. Levure microsomes containing CFTR peut être solublised avec plusieurs détergents couramment utilisés, y compris 18 des détergents tels que le dodécyl maltoside 2, qui sont généralement considérées comme «douces». Cependant, la plupart des détergents haute cmc se sont avérés inefficaces pour solubilsation, jusqu&#39;à présent, ce qui suggère que l&#39;échange dans ces détergents devraient être examinées à un stade tardif de tout régime de purification.

<table border="1"><tbody><tr><td> Nom de réactif / équipements </td><td> Entreprise </td><td> Catalogue Pas </td></tr><tr><td> FGY217 souche S. cerevisiae, avec suppression pep4 10 </td><td></td><td></td></tr><tr><td> Base azotée de levure sans acides aminés </td><td> Formedium </td><td> CYN0410 </td></tr><tr><td> Remplissez mélange supplément sans uracile </td><td> Formedium </td><td> DCS0169 </td></tr><tr><td> Agar agar bactériologique </td><td> Sigma-Aldrich </td><td> A5306 </td></tr><tr><td> D-galactose </td><td> Pêcheur </td><td> BP656-500 </td></tr><tr><td> D-glucose </td><td> Pêcheur </td><td> D16-500 </td></tr><tr><td> Pepstatine A </td><td> Sigma-Aldrich </td><td> P4265 </td></tr><tr><td> Leupeptine </td><td> Merck </td><td> 108975 </td></tr><tr><td> Chymostatine </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-butane (E-64) </td><td> Sigma-Aldrich </td><td> E3132 </td></tr><tr><td> Fluorure Aminoethylbenzenesulfonyl (AEBSF) </td><td> Sigma-Aldrich </td><td> A8456 </td></tr><tr><td> Chlorhydrate de benzamidine </td><td> Sigma-Aldrich </td><td> 434760 </td></tr><tr><td> Le diméthylsulfoxyde (DMSO) </td><td> Sigma-Aldrich </td><td> D8418 </td></tr><tr><td> dithiothréitol (DTT) </td><td> Sigma-Aldrich </td><td> 43815 </td></tr><tr><td> acide éthylènediaminetétraacétique (EDTA) </td><td> Pêcheur </td><td> BP120500 </td></tr><tr><td> Tris-base </td><td> Formedium </td><td> TRIS01 </td></tr><tr><td> Tris-HCl </td><td> Pêcheur </td><td> P631 </td></tr&gt; <tr><td> D-sorbitol </td><td> Sigma-Aldrich </td><td> S1876 </td></tr><tr><td> Glycérol </td><td> Pêcheur </td><td> 065017 </td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> S6191 </td></tr><tr><td> n-dodécyl-β-D-maltopyranoside </td><td> Affymetrix </td><td> D310S </td></tr><tr><td> Bleu de bromophénol </td><td> Sigma-Aldrich </td><td> 114391 </td></tr><tr><td> PageRuler plus les normes de protéines précolorés </td><td> Fermentas </td><td> SM1811 </td></tr><tr><td> NuSep tris-gly gels de gradient 4-20% </td><td> NuSep </td><td> NB10-420 </td></tr><tr><td> Instantanée Bleu de Coomassie Stain </td><td> Novexin </td><td> ISB1L </td></tr><tr><td> Perles de verre, lavé à l&#39;acide </td><td> Sigma </td><td> G8772 </td></tr><tr><td> 50ml stériles Tubes Falcon </td><td> Sarstedt </td><td> 62.547.254 </td></tr><tr><td> 2mlStériles vis-top flacons </td><td> Sarstedt </td><td> 72.694.005 </td></tr><tr><td> 250ml Erlenmeyer stérile dérouté </td><td> BD Biosciences </td><td> 355119 </td></tr><tr><td> 2l Erlenmeyer stérile dérouté flacons </td><td> BD Biosciences </td><td> 355131 </td></tr><tr><td> Microtubes 2ml </td><td> Sarstedt </td><td> 72.695 </td></tr><tr><td> 0,2 um filtre à seringue </td><td> Sartorius </td><td> FC121 </td></tr><tr><td> tubes ultracentrifugeuse </td><td> Beckman Coulter </td><td> 355618 </td></tr><tr><td> tubes à centrifuger </td><td> Beckman Coulter </td><td> 357000 </td></tr><tr><td> Des pots à centrifuger 1l </td><td> Beckman Coulter </td><td> 969329 </td></tr><tr><td> Orbital incubateur agitateur avec contrôle de température </td><td> New Brunswick Scientific </td><td></td></tr><tr><td> DeltaVision RMicroscope de restauration T </td><td> Applied Vision </td><td></td></tr><tr><td> Centrifugeuse de paillasse </td><td> HERMLE </td><td> Z300 </td></tr><tr><td> Paillasse microcentrifugeuse </td><td> Pêcheur </td><td> 13-100-511 </td></tr><tr><td> Vortex </td><td> Labs étoile </td><td></td></tr><tr><td> Scanner Trio Typhoon </td><td> GE Healthcare </td><td> 63-0055-87 </td></tr><tr><td> Système d&#39;imagerie LAS3000 </td><td> Fuji </td><td></td></tr><tr><td> La cuve de fermentation 20l et l&#39;unité de contrôle </td><td> Applikon </td><td></td></tr><tr><td> Constant perturbateur des systèmes à pile </td><td> Systèmes constants </td><td></td></tr><tr><td> Perturbateur cellule Beadbeater </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 Ultracentrifugeuse </td><td> Beckman Coulter </td><td> 392050 </td></tr><tr><td> Rotor 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

Le régulateur de la conductance transmembranaire de la mucoviscidose (CFTR) est un canal chlorure, que lorsqu&#39;il est muté, peut donner lieu à la fibrose kystique dans humans.There est donc un intérêt considérable dans cette protéine, mais les efforts pour étudier sa structure et l&#39;activité ont été entravés par la difficulté d&#39;exprimer et de purification de quantités suffisantes de la protéine 1-3. Comme beaucoup de &#39;difficiles&#39; protéines membranaires eucaryotes, l&#39;expression dans un organisme en pleine croissance est souhaitable, mais difficile, et dans la levure S. cerevisiae, dans la mesure de faibles quantités ont été obtenues et de la dégradation rapide de la protéine recombinante a été observée 4-9. Les protéines impliquées dans le traitement de la protéine CFTR chez la levure recombinante ont été décrites 6-9. Dans ce rapport, nous décrivons une méthodologie pour l&#39;expression de la protéine CFTR chez la levure et sa purification en quantités importantes. Le protocole décrit comment les problèmes de protéolyse antérieures peuvent être surmontés et les niveaux d&#39;expression de la façon dontCFTR peut être grandement améliorée en modifiant les conditions de croissance des cellules et en contrôlant les conditions d&#39;induction, en particulier la période de temps avant la récolte des cellules. Les reagants associés à ce protocole (CFTR murin exprimant des cellules de levure ou des plasmides de levure) seront distribués par l&#39;intermédiaire de la Fondation de la fibrose kystique aux États-Unis, qui a parrainé la recherche. Un article décrivant la conception et la synthèse de la protéine CFTR Construct employé dans le présent rapport sera publié séparément (Urbatsch, I.;. Thibodeau, P. et al, non publié). Dans cet article nous allons expliquer notre méthode de début de la transformation des cellules de levure avec le CFTR construire - la levure contenant le plasmide (Fig. 1). Le produit de construction a une protéine fluorescente verte (GFP) fusionnée à la séquence CFTR à son extrémité C-terminale et suit le système développé par Drew et al. (2008) 10. La GFP permet l&#39;expression et la purification de la protéine CFTR à suivre relativement facilement. Le protocole JoVE visualiséesfinitions ol après la préparation de microsomes des cellules de levure, bien que nous incluons quelques suggestions pour la purification de la protéine à partir des microsomes. Les lecteurs peuvent ajouter leurs propres modifications à la procédure de purification microsome, en fonction des derniers essais à effectuer avec la protéine et l&#39;équipement local à leur disposition. La levure a exprimé la protéine CFTR peut être partiellement purifié à l&#39;aide d&#39;ions métalliques chromatographie d&#39;affinité, en utilisant un marqueur polyhistidine intrinsèque de purification. Après chromatographie d&#39;exclusion stérique donne une protéine qui semble être&gt; 90% de pureté, à en juger par SDS-PAGE et Coomassie-coloration du 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

Les tentatives pour exprimer le régulateur de la conductance transmembranaire de la mucoviscidose (CFTR) dans<em&gt; Saccharomyces cerevisiae</em&gt; Ont, jusqu&#39;à présent, abouti à des quantités relativement faibles de protéines. Ce protocole et les réactifs associés distribués par la Cystic Fibrosis Foundation devrait permettre la préparation de quelques milligrammes de cette «difficile» protéine de la membrane eucaryote.

Expression et purification de la protéine transmembranaire de la fibrose kystique conductance Regulator dans Saccharomyces cerevisiae

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

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

18.4K vues.  University of Manchester. Les tentatives pour exprimer le régulateur de la conductance transmembranaire de la mucoviscidose (CFTR) dans Saccharomyces cerevisiae Ont, jusqu'à présent, abouti à des quantités relativement faibles de protéines. Ce protocole et les réactifs associés distribués par la Cystic Fibrosis Foundation devrait permettre la préparation de quelques milligrammes de cette «difficile» protéine de la membrane eucaryote.

Vidéo: 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.

Dissection de Saccharomyces cerevisiae Asques

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

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Biologie

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.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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.

Phosphoprotéomique quantitatives en acide gras Stimulé 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.

Purification de la M. magneticum Strain AMB-1 Protein magnétosome Associated 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.

Analyse de Microarray pour 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 test microscopie de fluorescence pour Mitophagy surveillance dans la levure<em&gt; 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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<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.

Les analyses couplées pour la surveillance protéines repliement<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;

Purification de la fibrose kystique régulateur de la conductance transmembranaire protéine exprimée dans<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 surveillé Redox titrage des cofacteurs 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 ...