Les auteurs déclarent qu&#39;ils ne ont aucun intérêt financier concurrentes.

<ol>
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A. <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+recombinant+protein+derived+from+the+baculovirus+expression+system+using+glutathione+affinity+agarose.">Purification of recombinant protein derived from the baculovirus expression system using glutathione affinity agarose.</a> Methods Mol Biol. 3, 337-348 (1995).</li><li>Eckford, P. D., Li, C., Ramjeesingh, M., Bear, C. E. <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+(CFTR)+potentiator+VX-770+(ivacaftor)+opens+the+defective+channel+gate+of+mutant+CFTR+in+a+phosphorylation-dependent+but+ATP-independent+manner.">Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner.</a> J Biol Chem. 287 (44), 36639-36649 (2012).</li><li>Alkhouri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+properties+of+molecular+probes+for+the+rescue+site+on+mutant+cystic+fibrosis+transmembrane+conductance+regulator.">Synthesis and properties of molecular probes for the rescue site on mutant cystic fibrosis transmembrane conductance regulator.</a> J Med Chem. 54 (24), 8693-8701 (2011).</li><li>Eckford, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VX-809+and+Related+Corrector+Compounds+Exhibit+Secondary+Activity+Stabilizing+Active+F508del-CFTR+after+Its+Partial+Rescue+to+the+Cell+Surface.">VX-809 and Related Corrector Compounds Exhibit Secondary Activity Stabilizing Active F508del-CFTR after Its Partial Rescue to the Cell Surface.</a> Chem Biol. 21 (5), 666-678 (2014).</li><li>Kim Chiaw, P., Wellhauser, L., Huan, L. J., Ramjeesingh, M., Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+corrector+modifies+the+channel+function+of+F508del-CFTR.">A chemical corrector modifies the channel function of F508del-CFTR.</a> Mol.Pharmacol. 78 (3), 411-418 (2010).</li><li>Verkman, A. S., Galietta, L. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+alginate+secretion+across+the+bacterial+outer+membrane.">Structural basis for alginate secretion across the bacterial outer membrane.</a> Proc Natl Acad Sci U S A. 108 (32), 13083-13088 (2011).</li><li>Islam, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton-dependent+gating+and+proton+uptake+by+Wzx+support+O-antigen-subunit+antiport+across+the+bacterial+inner+membrane.">Proton-dependent gating and proton uptake by Wzx support O-antigen-subunit antiport across the bacterial inner membrane.</a> mBio. 4 (5), e00678-e00613 (2013).</li><li>Ramjeesingh, M., Conn, P. 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Les auteurs reconnaissent les conseils du Dr Mohabir Ramjeesingh au cours du développement des méthodes de purification décrites ici. Ces études ont été soutenus par des subventions de fonctionnement au CCS des Instituts de recherche en santé du Canada (IRSC) et la fibrose kystique Canada (CFC) canadiennes. PDWE a été financé en partie par des bourses de recherche par les IRSC et CFC. Les auteurs reconnaissent la fourniture de correcteur, potentialisateur et composés inhibiteurs du Dr R. Bridges (Université Rosalind de médecine et des sciences) et la distribution par les Cystic Fibrosis Foundation Therapeutics (CFFT). Les auteurs reconnaissent également un service exceptionnel par le Baylor Culture Cellulaire Facility (NIH subvention P30 CA125123) dans l&#39;expression et Poids protéine CFTR mutant en utilisant le système de baculovirus. Les inactifs VX-770 analogique, V-09 à 1188, était un don de Vertex Pharmaceuticals.

Il n&#39;y a eu qu&#39;un nombre limité de protocoles de purification de pleine longueur, l&#39;isolement de la protéine CFTR fonctionnelle, à partir d&#39;une variété de systèmes de surexpression cellulaire. Le procédé décrit ici est avantageuse car elle permet une purification rapide de WT-CFTR ou haute enrichissement de F508del- et G551D-CFTR dans des quantités modérées qui est très fonctionnel dans des dosages compris ATPase et des mesures directes de la fonction du canal, y compris les mesures de canal...

transport de chlorure à travers les membranes apicales des cellules épithéliales dans les tissus tels que poumons, intestins, le pancréas et les glandes sudoripares est principalement médiée par la fibrose kystique régulateur de la conductance transmembranaire (CFTR), un ATP et membre de phosphorylation réglementés de l&#39;ABC (ATP Binding Cassette) C sous-famille de protéines membranaires (revue dans 1). Comme d&#39;autres membres de la sous-famille SCBA, CFTR est un grand multi-enjambant protéine membranaire intégrale qui se lie l&#39;ATP à deux sites de liaison de nucleotides formée à l&#39;interface de ses domaines de nucleotides de liaison (NBD), où il possède une activité d&#39;ATPase modeste à une seule place. Cependant, contrairement à d&#39;autres membres de la sous-famille ABCC, CFTR a évolué comme un canal Cl réglementé unique, plutôt que comme un soluté transporteur actif. Des mutations dans la cause de la fibrose kystique CFTR, une maladie affectant plusieurs organes, y compris les poumons, tractus gastro-intestinal, du pancréas et des voies de reproduction, conduisant à morbidité et de la mortalité chez les jeunes adultes. Maladie pulmonaire représente généralement au début de la mortalité et de la fibrose kystique dans la plupart des cas est provoqué par la perte de fonction de la protéine CFTR à la surface de l&#39;épithélium des voies aériennes conductrices. L&#39;absence de l&#39;activité des canaux chlorure CFTR entraîne une réduction à la fois de Cl - et de circulation de l&#39;eau à travers l&#39;épithélium de surface pour modifier la couche de fluide sur la surface apicale de l&#39;épithélium cilié respiratoire. Il en résulte un liquide de surface des voies respiratoires visqueux qui altère la capacité des cellules epitheliales ciliées des voies respiratoires à des agents pathogènes efficacement claires sur les voies respiratoires. En conséquence, la plupart des patients atteints de mucoviscidose souffrent d&#39;épisodes récurrents d&#39;infection pulmonaire et une atteinte des poumons due à l&#39;inflammation. Comme prévu, les études du mécanisme d&#39;action de la protéine CFTR normale concentrent principalement sur les études électrophysiologiques détaillées de son activité voie de fenêtrage. Des études simples de canal ont montré directement que les fonctions de CFTR comme PKA-dépendante Cl- Canal qui possède une porte ATP réglementé 2. Études électrophysiologiques détaillés fournissent beaucoup d&#39;informations sur les canaux CFTR simples 1,3, mais il peut y avoir des préoccupations quant à savoir si les caractéristiques d&#39;un seul canal particulier qui a été étudié est le reflet de l&#39;ensemble de la population de canaux CFTR et donc le seul canal résultats doivent toujours être considérés ainsi que des méthodes pour étudier la population macroscopique. Dosage direct de l&#39;activité de canal de population de CFTR purifié a le potentiel de donner un aperçu du défaut moléculaire associée à des mutations pathogènes et à conduire la découverte de modulateurs chimiques qui la réparation des protéines CFTR mutant. À ce jour, il ya plus de 1900 mutations différentes dans CFTR pensé pour provoquer la fibrose kystique 4. La mutation majeure, F508del-CFTR, trouvé sur au moins un allèle dans environ 90% des patients en Amérique du Nord et en Europe conduit à un mauvais repliement des protéinesnd rétention dans le réticulum endoplasmique 5. F508del-CFTR a aussi d&#39;autres conséquences, y compris l&#39;activité du canal défectueux 6-9. L&#39;absence résultante de la protéine CFTR à la surface de cellule est associée à une maladie grave. G551D-CFTR, la mutation moins fréquente, est considérée comme étant encore correctement pliée est dysfonctionnel comme un canal chlorure à la surface de la cellule 6. Le développement de petits correcteurs de molécules et potentialisateurs a pour but de corriger de pliage et / ou le trafic de mutants tels que F508del-CFTR à la surface cellulaire, et potentialisant ou en augmentant l&#39;activité de canal de mutations telles que G551D lorsqu&#39;ils sont présents sur la surface des cellules, respectivement . Alors que les correcteurs VX-809 et VX-661 (ne sont pas encore approuvés pour une utilisation chez les patients, le potentialisateur Kalydeco (ivacaftor; VX-770) est utilisé à 150 mg toutes les 12 h chez les patients FC&gt; 6 ans avec au moins un G551D -CFTR mutation, et plus récemment pour les patients avec l&#39;un des G178R, S549N, S549R, G551S, G1244E, S1251N, S1255Pet G1349D. Kalydeco est à la fois sûr et des résultats dans l&#39;amélioration des mesures cliniques de la maladie CF 10, mais le mécanisme d&#39;action de cette petite molécule a été mal compris au moment de l&#39;approbation de la FDA pour une utilisation chez les patients. Une poignée de méthodes de purification CFTR ont été décrits précédemment 2,11-18, dont beaucoup exigent une longueur considérable de temps pour terminer. Dans une publication récente 19, un procédé de purification et reconstitution rapide unique a été décrit pour la protéine CFTR surexprimé dans le S f système d&#39;expression neuf cellulaire, et cette protéine purifiée dans les systèmes lipidiques définies a été utilisé pour développer un essai de l&#39;activité des canaux à l&#39;halogénure d&#39;CFTR pour une population de CFTR molécules. Le dosage récapitule les effets connus de phosphorylation, des nucléotides et des inhibiteurs sur la fonction de la protéine CFTR. Le système a été utilisé pour interroger les effets de la potentialisateur VX-770 / Kalydeco sur poids (-type sauvage), F508del- et G551D-CFTR et il a été montré pour la premièretemps que le médicament interagit directement avec la protéine CFTR à potentialiser son activité de canal de façon ATP-indépendant, ce qui démontre l&#39;utilité et l&#39;application de ces méthodes pour l&#39;étude de l&#39;interaction de la protéine CFTR et mutants avec des nucleotides et des petites molécules à partir d&#39;un point de vue de la population de répondre à des questions cliniquement pertinentes sur la protéine. Les procédés ont également été utilisés pour étudier d&#39;autres molécules de potentialisateur et de leurs dérivés 20, ainsi que les effets d&#39;un correcteur de petite molécule de l&#39;activité de la protéine 21. des dosages d&#39;efflux ont été utilisées dans de nombreuses études précédemment pour étudier l&#39;activité de mutants CFTR et les effets des composés CFTR-modulateurs sur son activité, y compris des dosages de cellules entières en utilisant des électrodes, des traceurs radioactifs et des fluorophores 22,23, des vésicules membranaires avec électrodes sélectives d&#39;ions 24 , et purifié CFTR reconstitué avec des traceurs radioactifs 25. Cependant thl&#39;utilisation de l&#39;e des électrodes sélectives d&#39;ions pour étudier CFTR reconstitué purifié a été rapporté récemment 19. Une adaptation de la méthode actuelle a été utilisé pour la reconstitution et la caractérisation fonctionnelle de deux protéines membranaires de Pseudomonas aeruginosa, un agent pathogène CF commun. Reconstitution de Alge purifié la protéine de la membrane externe couplé avec des mesures d&#39;efflux iodure ont été utilisés pour soutenir un modèle pour les tensioactifs anioniques alginate la sécrétion par ce transporteur 26. La reconstitution et les mesures de efflux d&#39;iodure ont été appliqués à la protéine purifiée wzx, ce qui a permis un modèle à proposer qui suggère un mécanisme de H + antiport dépendante de la translocation de lipide d&#39;oligosaccharide liée à travers la membrane bactérienne interne de 27 cette protéine. Dans les deux cas iodure a été utilisé comme un substitut pour le substrat anionique, mais à débit plus faible que l&#39;on pourrait attendre d&#39;un substrat natif. Le procédé peut être adapté pour l&#39;adaptation à d&#39;autres protéines ayant des cconduction des voies de transport ou pour des substrats anioniques ationic. Voici une procédure de purification rapide est décrit pour la protéine CFTR et sa reconstitution dans des protéoliposomes de lipide défini. La procédure de reconstitution rapide peut facilement être adapté pour une utilisation avec CFTR purifié par d&#39;autres procédés, à condition que le type de détergent utilisé dans la purification se prête à l&#39;élimination par des méthodes utilisées ici ou peut être remplacé par un détergent approprié avant la procédure de reconstitution. Procédé d&#39;efflux d&#39;iodure pour la mesure de l&#39;activité du canal CFTR de protéine purifiée et reconstituée est décrit en détail et certains résultats typiques qui peuvent être obtenus par cette méthode sont présentés.

<table border="0" cellpadding="0" cellspacing="0" width="1269">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:403px;">fos-choline 14 detergent</td>
			<td style="width:515px;">Anatrace Affymetrix (www.anatrace.affymetrix.com)</td>
			<td style="width:145px;">F312</td>
			<td style="width:207px;">Affymetrix: Anatrace Products; CAS#&#160;:77733-28-9</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete EDTA-free protease inhibitor cocktail tablets</td>
			<td>Roche (www.roche-applied-science.com)</td>
			<td>04 693 132 001</td>
			<td>EDTA-free Protease inhibitor cocktail tablets (1 in 50 ml or mini:1 in 10 mL) (Roche Diagnostics GmbH; Ref: 11873 580 001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack&#160;</td>
			<td>Roche (www.roche-applied-science.com)</td>
			<td>05 892 791 001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA Agarose</td>
			<td>Qaigen GmbH</td>
			<td align="right">1018240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fisherbrand Screening columns</td>
			<td>Fisher Healthcare</td>
			<td>11-387-50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amicon Ultra Centrifugal filters, Ultracel-100K</td>
			<td>Millipore (www.millipore.com)</td>
			<td>UFC910008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cAMP-Dependent Protein Kinase A (PKA), Catalytic Subunit</td>
			<td colspan="2">New England Biolabs (www.neb.com/products/p6000-camp-dependent-protein-kinase-pka-catalytic-subunit)</td>
			<td>Peirce PKA is also suitable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PC, Chicken Egg</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>840051C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">POPC</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PS, Porcine Brain</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>840032C</td>
			<td>(CFTR has been successfully reconstituted into Egg PC, POPC, 2:1 (w/w) Egg PC:POPC, or a mixture of PE:PS:PC:ergosterol, 5:2:2:1 (w/w))</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PE, Chicken Egg</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>841118C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ergosterol</td>
			<td>Sigma (www.sigmaaldrich.com)</td>
			<td align="right">45480</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Detergent removal spin column</td>
			<td>Thermo Scientific</td>
			<td align="right">87779</td>
			<td>1 ml capacity columns</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">valinomycin</td>
			<td>Sigma (www.sigmaaldrich.com)</td>
			<td>V-0627</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VX-770 (ivacaftor)</td>
			<td>Selleck Chemicals</td>
			<td>S1144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iodide selective microelectrode</td>
			<td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/microionn.tam)</td>
			<td>LIS-146ICM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clampex 8.1 software</td>
			<td>Axon Instruments (www.axon.com)</td>
			<td></td>
			<td>we use components of the ClampX system with a home made filter to monitor and record the response to our electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alternate Software: ArrowLabb System&#160;</td>
			<td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/ionsystems.tam)</td>
			<td>LIS-146LICM-XS</td>
			<td>Lazar Research sells a meter that can interface with a computer and software to record the probe response.&#160; This software should serve a similar function to our setup</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">small stir bars</td>
			<td>Big Science Inc (www.stirbars.com)</td>
			<td>SBM-0502-CMB</td>
			<td>choose a stir bar small enough to easily fit into a well of a 96-well plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sephadex G50, fine</td>
			<td>GE Health Care (www.gelifesciences.com)</td>
			<td>17-0042-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator</td>
			<td>Laboratory Supplies Co, Inc.</td>
			<td>G112SP1G</td>
			<td>Bath sonicators from other manufacturers should also be suitable</td>
		</tr>
	</tbody>
</table>

functional reconstitution and channel activity measurements of purified wildtype and mutant cftr protein

La fibrose kystique régulateur de la conductance transmembranaire (CFTR) est un élément de formation de canal unique de l&#39;ATP Binding Cassette (ABC) superfamille des transporteurs. L&#39;activité du canal chlorure nucléotidique et la phosphorylation dépendant de la CFTR a été étudié fréquemment dans des systèmes de cellules entières et que les canaux individuels dans patches membranaires excisées. De nombreuses mutations de fibrose kystique qui causent ont été montré de modification de cette activité. Bien qu&#39;un petit nombre de protocoles de purification ont été publiées, un procédé de reconstitution rapide qui conserve l&#39;activité de canal et un procédé approprié pour l&#39;étude de l&#39;activité du canal de la population dans un système purifié ont fait défaut. Ici méthodes rapides sont décrites pour la purification et la reconstitution fonctionnelle de la protéine CFTR pleine longueur dans des protéoliposomes de composition lipidique défini qui conserve une activité de canal à l&#39;halogénure réglementé. Cette méthode de reconstitution avec un nouvel essai à base de flux de l&#39;activité de canal est un système adapté pourétudier les propriétés du canal de la population de CFTR sauvage et les mutants pathogènes F508del- et G551D-CFTR. Plus précisément, le procédé est utile pour l&#39;étude des effets directs de la phosphorylation, les nucleotides et les petites molécules telles que potentialisateurs et des inhibiteurs sur l&#39;activité du canal CFTR. Les méthodes sont également prêtent à l&#39;étude d&#39;autres canaux / transporteurs membranaires pour les substrats anioniques.

Décrit dans cette publication écrite sont des méthodes pour purifier, reconstituer et de mesurer l&#39;activité des canaux réglementé de la protéine CFTR. Figure 1a montre le flux de travail pour la purification, la reconstitution et procédures d&#39;efflux d&#39;iodure. Méthodes pour la reconstitution et l&#39;activité du canal de flux iodure mesures sont également présentés plus en détail dans la vidéo associée. Purification et la reconstitution de la prot?...

Watch this Scientific Journal Video about Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein at JoVE.com

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Décrit ici est un procédé rapide et efficace pour la reconstitution fonctionnelle de type sauvage purifiée et une protéine de CFTR mutante qui conserve l&#39;activité de ce canal de chlorure, qui est défectueux dans la fibrose kystique. Efflux d&#39;iodure de protéoliposomes reconstituées médiées par CFTR permet des études de l&#39;activité du canal et les effets de petites molécules.

Reconstitution fonctionnelle et Activité du Chan mesures de type sauvage purifiée et Mutant protéine CFTR

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

functional-reconstitution-channel-activity-measurements-purified

Research

JoVE Journal

Biology

12.1K vues.  Hospital for Sick Children.  Décrit ici est un procédé rapide et efficace pour la reconstitution fonctionnelle de type sauvage purifiée et une protéine de CFTR mutante qui conserve l'activité de ce canal de chlorure, qui est défectueux dans la fibrose kystique. Efflux d'iodure de protéoliposomes reconstituées médiées par CFTR permet des études de l'activité du canal et les effets de petites molécules. 

Vidéo: Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

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 ...

Recherche

Biologie

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utiliser le système de fractionnement GELFREE 8100 pour les Poids Moléculaire à base de fractionnement avec récupération de la phase liquide

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

Biochemical Reconstitution of Steroid Receptor&amp;#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Reconstitution biochimique des récepteurs stéroïdiens • complexes de protéines Hsp90 et la réactivation de la liaison du ligand

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

Application de la MassSQUIRM pour des mesures quantitatives de l&#39;activité Déméthylase Lysine

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

Précipitation par affinité des actifs des Rho GEF l&#39;aide d&#39;un TPS-étiqueté Mutant Rho protéines (TPS-RhoA (G17A)) à partir de lysats cellulaires épithéliales

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

L&#39;analyse in vitro de PDZ-dépendants complexes macromoléculaires CFTR de signalisation

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Reconstitution d&#39;un canal Kv dans les membranes lipidiques des études structurales et fonctionnelles

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

Reconstitution in vitro de complexes collecteurs de lumière de plantes et d&#39;algues vertes

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

Sondage haute densité fonctionnels des puces à protéines pour détecter les interactions protéine-protéine

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Reconstitution d&#39;une protéine transmembranaire, le Ion canal de voltage-dépendants, KvAP, en géant vésicules unilamellaires pour la microscopie et Patch études Clamp

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...