Nous remercions les National Institutes of Health (1R21CA138373 et Stand Up to Cancer (SU2C) pour les supports financiers de ce travail. HY est reconnaissante pour le Prix 2009 Elion de l&#39;American Association of Cancer Research, une Scholar Award de la Fondation Kimmel Sidney Kimmel pour Recherche sur le Cancer (SKF-08-101), et la National Science Foundation Faculté de carrière Prix précoce (NSF0954819).

1. Considérations clonage <ol><li> Oligonucléotides préparés commercialement représentant le TMD d&#39;intérêt flanqué Nhel et des sites de restriction BamHI et 5&#39;-phosphorylés peuvent être ligaturés dans pTox7 (modifié dans notre laboratoire par insertion d&#39;une paire de base directement après le site de restriction BamHI 14) (figure 3) digéré séquentiellement avec BamHI et Nhel. Un oligonucléotide exemple est illustré ci-dessous: 5&#39;ctagcTMDSEQUENCEg3 &#39; 3 &#39;gTMDSEQUENCEcctag5&#39; </li></ol> La séquence TMD devrait être de 12 à 24 résidus (courtes séquences seront probablement allongée par des résidus hydrophobes vecteur codé). Afin d&#39;étudier l&#39;interface, quatre variantes de la conception TMD doivent être créés là où les insertions de résidus séquentiels et concomitants de résidus suppressions entraîner la rotation de la relative au TMD ToxR. 15,16 Enfin, la concentration d&#39;arabinose doit être variée entre 0,001 et 0,01% (p / v) d&#39;identifier les différences de concentration où maximale β-galactosidase signaux entre les différentes séquences de TMD sont observées; tests différents niveaux d&#39;expression est recommandé d&#39;identifier les conditions dans lesquelles des affinités différentes peuvent être distinguées meilleurs. En plus de l&#39;arabinose et des antibiotiques, 0,4 mM d&#39;IPTG peut être utilisé pour améliorer les différences d&#39;affinités entre les différents TMD. La mesure doit être effectuée ToxR au moins en quatre exemplaires. Toute la procédure doit être répétée au moins trois fois avec différentes transformations plasmide. 2. Croissance de cultures bactériennes <ol><li> Doucement le dégel FHK12 cellules compétentes (200 pi) sur la glace et le transfert dans un tube de culture de 15 ml. Ajouter l&#39;ADN plasmidique (200 ng) et incuber les cellules sur la glace pendant 30 min. </li><li> Choc thermique des cellules par incubation pendant 90 s à 42 ° C, suivie d&#39;une incubation sur la glace pendant 2 min. </li><li> Ajouter SOC médias (800 pi) et incuber les échantillons à 37 ° C avec agitation (300 rpm) pendant une heure </li><li> Inoculer 5 ml avec du milieu LB chloramphénicol (30 pg / ml) et de l&#39;arabinose (0,0025% p / v) avec 50 ul du mélange de transformation dans des tubes de 15 ml de culture en trois exemplaires. Incuber les échantillons à 37 ° C avec agitation (300 rpm) pendant 20 h. (Alternativement 5 ul de la culture peut être utilisée pour inoculer 100 pi de milieu dans une plaque de 96 puits. Cette méthode est utile lorsqu&#39;il s&#39;agit de grands nombres d&#39;échantillons, bien que les erreurs seront légèrement plus élevés. Afin d&#39;éviter l&#39;évaporation, ce qui entraînerait à l&#39;erreur, remplir les puits ultrapériphériques avec les médias, mais ne pas les utiliser pour des échantillons. Enfin, double-envelopper le joint entre le couvercle et la plaque avec du parafilm). </li></ol> 3. Mesure de la β-galactosidase <ol><li> Préchauffer le lecteur de plaques à 28 ° C. </li><li> Transférer le Z-buffer dans un réservoir avec une grande pointe de la pipette, en veillant à ne prendre jusqu&#39;à la partie supérieure (aqueuse) couche. Transférer 100 ul d&#39;Z-buffer/chloroform fraîchement préparés dans les puits d&#39;une plaque de 96 puits. Transférer 5 ul de chaque culture dans les puits de la plaque en quatre exemplaires. Omettre la culture à partir de quatre puits qui servira de vide. </li><li> Mesurer la DO 595 de la plaque afin de déterminer la densité cellulaire. </li><li> Ajouter 50 ul d&#39;Z-buffer/SDS à tous les puits de la plaque. Agiter la plaque dans le lecteur de plaques pendant 10 minutes pour lyser les cellules. Assurez-vous que les suspensions cellulaires sont claires après lyse et répétez l&#39;étape secouer si nécessaire. Lyse incomplète suggère la Z-buffer/chloroform n&#39;a pas été fraîchement préparés. </li><li> Ajouter 50 ul fraîchement préparés Z-buffer/ONPG à tous les puits et retourner la plaque pour le lecteur de plaque et de mesurer OD 405 toutes les 30 s pendant 20 min. </li><li> Calculer β-galactosidase en utilisant l&#39;équation suivante (n&#39;oubliez pas de soustraire le vide). Le ratio de 405 OD / min devrait être calculé en utilisant tous les points de données dans la DO 405 entre 0.0 et 1.0 en utilisant un ajustement du modèle linéaire. <img src="files/ftp_upload/2721/2721eq1.jpg" alt="L&#39;équation 1" /> Miller unités diffèrent parfois quand enregistrées sur plusieurs jours. Par conséquent, une construction de référence comme AMP devrait être mesuré dans chaque essai. Ses valeurs peuvent être utilisées pour la normalisation des valeurs ToxR. </li></ol> 4. Contrôle pour l&#39;expression protéique <ol><li> Effectuer Western blot pour vérifier l&#39;expression des protéines, même entre les constructions. Mélanger 50 pl de la cultures en triple et centrifuger (2000 rpm, 4 min) dans une microcentrifugeuse. Eliminer le surnageant par pipetage et resuspendre le culot dans 2 résiduelle de tampon de chargement x échantillon. </li><li> Charge 7,5 ul sur un gel de la norme de 8% et de réaliser les électrophorèses à 125 V pendant 1 heure 5 min. Après le transfert, incuber avec des anti-MBP HRP-anticorps conjugué et de visualiser, la protéine chimère est observée à environ 70 kDa avec certains produits de dégradation parfois vu environ 48 kDa. MBP endogène est également observée à 45 kDa (voir Figure 5). </li></ol>5. Contrôle d&#39;insertion membranaire correcte Une lignée cellulaire déficiente en protéine de liaison du maltose est utilisé pour évaluer insertion membranaire correcte de la construction chimérique TMD. Lorsqu&#39;il est cultivé sur un milieu minimal avec des maltose comme seule source de carbone, que les cellules exprimant une membrane intégrale produit d&#39;expression avec le maltose binding protein correctement localisé dans le périplasme sont capables de croître. <ol><li> Transformez PD28 cellules (comme décrit pour FHK12 cellules) et inoculer 2 ml de milieu LB. Cultiver les cellules à 37 ° C avec agitation (300 rpm) pendant la nuit. </li><li> Pellet les cellules par centrifugation à 3500 rpm, 10 min, 4 ° C et laver à la remise en suspension dans du PBS (2 ml) par pipetage doux avec un gros pourboire ou vortex doux. Pellet les cellules (comme ci-dessus), laver avec du PBS pour une deuxième fois, granulés et finalement remettre en suspension dans du PBS (1 ml). </li><li> Utilisez 25 ul de cellules en suspension pour inoculer 5 ml de milieu minimum en trois exemplaires et incuber à 37 ° C avec agitation (300 rpm). Prenez OD 595 lectures entre 15-25 h, environ toutes les 2 heures en transférant 200 ul de chaque échantillon dans une plaque de 96 puits et de la lecture en utilisant le lecteur de plaques. </li></ol> 6. Les résultats représentatifs: Un exemple de l&#39;utilisation du dosage de journaliste transcriptionnelle ToxR d&#39;analyser la propension oligomérisation des domaines transmembranaires dans la figure 4. Auparavant, nous avons étudié l&#39;oligomérisation des domaines transmembranaires de la membrane multispanning intégrante de membrane latente protéines protein-1 (LMP-1) par diverses techniques, y compris ToxR 14 domaine transmembranaire cinq (TM5) a été montré à exposer une forte propension à oligomériser.; Cela est démontré par les unités de Miller élevés, comparables à du contrôle positif, GPA, une séquence bien établie dimérisation. Une mutation délétère dans TM5, D150A, réduit la capacité de la séquence à oligomériser. LMP-1 TM1 ne modifie pas significativement oligomériser et présente un signal d&#39;unité très faible Miller, juste au-dessus du signal pour le blanc, non-transformé FHK12 cellules. <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="Figure 1" /> Cartoon Figure 1. Illustrant le dosage journaliste ToxR. Domaine transmembranaire (TMD) entraîné des résultats d&#39;oligomérisation dans la dimérisation de ToxR et l&#39;activation de la transcription de LacZ. Le produit du gène de LacZ, β-galactosidase peut être quantifiée comme une mesure de la propension d&#39;un TMD pour oligomériser. <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="Figure 2" /> Figure 2. Le clivage hydrolytique d&#39;ONPG par la β-galactosidase résultats dans la production de l&#39;espèce absorbant la lumière o-nitrophénolate (ONP). <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="Figure 3" /> Figure 3. Carte du plasmide pToxR7. <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="Figure 4" /> Figure 4. Représentant ToxR test journaliste de la transcription d&#39;analyser la propension oligomérisation de la membrane protein-1 latente domaines transmembranaires. 5 domaine transmembranaire (TM5) oligomérise fortement, alors que 1 domaine transmembranaire (TM1) ne présente qu&#39;une faible interaction. D150A mutation dans TM5 réduit considérablement sa capacité à oligomériser. GPA est inclus comme une séquence de contrôle positif pour la dimérisation forte. Blank représente non transformées FHK12 cellules. <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="Figure 5" /> Figure 5. Western blot pour l&#39;expression des protéines. <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="Figure 6" /> Figure 6. PD28 test complémentation pour contrôler insertion membranaire correcte pour le périplasme. Contrôle négatif représente une construction déficiente en protéine de liaison du maltose.

<ol>
	<li>Parker, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+the+heptahelical+G+protein+coupling+receptors:+a+case+for+association+using+transmembrane+helices.">Oligomerization of the heptahelical G protein coupling receptors: a case for association using transmembrane helices.</a> Mini Rev Med Chem. 9, 329-339 (2009).</li><li>Mo, W., Zhang, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+human+ATP-binding+cassette+transporters+and+its+potential+significance+in+human+disease.">Oligomerization of human ATP-binding cassette transporters and its potential significance in human disease.</a> Expert Opin Drug Metab Toxicol. 5, 1049-1063 (2009).</li><li>von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-protein+topology.">Membrane-protein topology.</a> Nat Rev Mol Cell Biol. 7, 909-918 (2006).</li><li>Van Horn, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+nuclear+magnetic+resonance+structure+of+membrane-integral+diacylglycerol+kinase.">Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase.</a> Science. 324, 1726-1729 (2009).</li><li>Hattori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mg(2+)-dependent+gating+of+bacterial+MgtE+channel+underlies+Mg(2+)+homeostasis.">Mg(2+)-dependent gating of bacterial MgtE channel underlies Mg(2+) homeostasis.</a> Embo J. 28, 3602-3612 (2009).</li><li>Ulmschneider, M. B., Sansom, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+distributions+in+integral+membrane+protein+structures.">Amino acid distributions in integral membrane protein structures.</a> Biochim Biophys Acta. 1512, 1-14 (2001).</li><li>Deber, C. M., Goto, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+proteins+into+membranes.">Folding proteins into membranes.</a> Nat Struct Biol. 3, 815-818 (1996).</li><li>Gerber, D., Shai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+hetero-association+of+glycophorin-A+and+its+mutants+within+the+membrane.">In vivo detection of hetero-association of glycophorin-A and its mutants within the membrane.</a> J Biol Chem. 276, 31229-31232 (2001).</li><li>Langosch, D., Brosig, B., Kolmar, H. p., Fritz, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerisation+of+the+glycophorin+A+transmembrane+segment+in+membranes+probed+with+the+ToxR+transcription+activator.">Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator.</a> J Mol Biol. 263, 525-530 (1996).</li><li>Russ, W. P., Engelman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TOXCAT:+a+measure+of+transmembrane+helix+association+in+a+biological+membrane.">TOXCAT: a measure of transmembrane helix association in a biological membrane.</a> Proc Natl Acad Sci U S A. 96, 863-868 (1999).</li><li>Berger, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+motif+for+integrin+transmembrane+helix+association.">Consensus motif for integrin transmembrane helix association.</a> Proc Natl Acad Sci U S A. 107, 703-708 (2010).</li><li>Oates, J., King, G., Dixon, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+oligomerization+behavior+of+PDGFbeta+receptor+transmembrane+domain+and+its+regulation+by+the+juxtamembrane+regions.">Strong oligomerization behavior of PDGFbeta receptor transmembrane domain and its regulation by the juxtamembrane regions.</a> Biochim Biophys Acta. 1798, 605-615 (2010).</li><li>Sal-Man, N., Gerber, D., Shai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hetero-assembly+between+all-L-+and+all-D-amino+acid+transmembrane+domains:+forces+involved+and+implication+for+inactivation+of+membrane+proteins.">Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: forces involved and implication for inactivation of membrane proteins.</a> J Mol Biol. 344, 855-864 (2004).</li><li>Sammond, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+peptides+used+to+investigate+the+homo-oligomeric+interface+and+binding+hot-spot+of+the+oncogene,+latent+membrane+protein+1.">Transmembrane peptides used to investigate the homo-oligomeric interface and binding hot-spot of the oncogene, latent membrane protein 1.</a> , (2010).</li><li>Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization+of+the+transmembrane+domain+of+Integrin+alphaIIb+subunit+in+cell+membranes.">Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes.</a> J Biol Chem. 279, 26666-26673 (2004).</li><li>Ruan, W., Becker, V., Klingmuller, U., Langosch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interface+between+self-assembling+erythropoietin+receptor+transmembrane+segments+corresponds+to+a+membrane-spanning+leucine+zipper.">The interface between self-assembling erythropoietin receptor transmembrane segments corresponds to a membrane-spanning leucine zipper.</a> J Biol Chem. 279, 3273-3279 (2004).</li><li>Finger, C., Escher, C., Schneider, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single+transmembrane+domains+of+human+receptor+tyrosine+kinases+encode+self-interactions.">The single transmembrane domains of human receptor tyrosine kinases encode self-interactions.</a> Sci Signal. 2, ra56-ra56 (2009).</li></ol>

Le dosage de ToxR journaliste de la transcription est un moyen aisé d&#39;identifier les séquences transmembranaires ayant le potentiel de oligomériser. Depuis les interactions se produisent dans la membrane bactérienne interne, ce test contourne les problèmes liés à la validité des systèmes d&#39;étudier dans la membrane-mimétiques environnements. Étant donné que le clonage des PTM multiples en un seul plasmide peut facilement être fait en parallèle et le dosage complet peut être réalisée en format 96...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Enzyme de restriction BamHI </td><td> Invitrogen </td><td> 15201023 </td><td> Invitrogen enzymes ont été jugées plus efficaces que les fournisseurs alternatifs </td></tr><tr><td> Enzyme de restriction Nhel </td><td> Invitrogen </td><td> 15444011 </td><td> Invitrogen enzymes ont été jugées plus efficaces que les fournisseurs alternatifs </td></tr><tr><td> 15 tubes de culture ml </td><td> Fisher Scientific </td><td> 14 à 956-1J </td><td></td></tr><tr><td> SOC des médias </td><td> Teknova </td><td> S0225 </td><td> Constitué au volume approprié et stérilisé par autoclavage. </td></tr><tr><td> Milieu LB </td><td> Sigma-Aldrich </td><td> L7275 </td><td> Constitué au volume approprié et stérilisé par autoclavage. </td></tr><tr><td> Le chloramphénicol </td><td> Sigma-Aldrich </td><td> CO 3 78 </td><td> Solution mère de 30 mg / ml dans l&#39;éthanol stocké dans un congélateur </td></tr><tr><td> Arabinose </td><td> Fluka </td><td> 10839 </td><td> Solution mère de 2,5% (p / v) dans de l&#39;eau stockée dans un congélateur </td></tr><tr><td> Na 2 HPO 4 </td><td> Sigma-Aldrich </td><td> S9390 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> Sigma-Aldrich </td><td> S9638 </td><td></td></tr><tr><td> KCl </td><td> Produits chimiques Mallinckrodt </td><td> 6858-06 </td><td></td></tr><tr><td> MgSO 4 .7 H 2 O </td><td> Sigma-Aldrich </td><td> 63138 </td><td></td></tr><tr><td> Dodécylsulfate de sodium (SDS) </td><td> Sigma-Aldrich </td><td> L6026 </td><td></td></tr><tr><td> 2-nitrophényl β-D-galactopyranoside (ONPG) </td><td> Sigma-Aldrich </td><td> 73660 </td><td></td></tr><tr><td> Z-buffer </td><td></td><td></td><td> 16,1 g de Na 2 HPO 4 5.5g de NaH 2 PO 4 0,75 g de KCl 0.246g MgSO 4 Compléter à 1 l, pH 7,0 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 ml β-mercaptoéthanol, 2 ml de chloroforme, faire jusqu&#39;à 20 mL avec du Z-buffer. Vortex pendant 1 min, centrifuger 1 min à 800 tpm. Faire frais pour chaque plaque. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 mg de SDS dissout dans 10 ml de Z-buffer </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40 mg ONPG dans 10 ml de Z-buffer. Faire frais pour chaque plaque </td></tr><tr><td> β-mercaptoéthanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Anti-MBP anticorps monoclonal (HRP conjugué) </td><td> ONE </td><td> E8038S </td><td></td></tr><tr><td> Milieu minimal avec le maltose </td><td></td><td></td><td> 1 x sels M9, le maltose de 0,4%, à 1 mg / ml de thiamine, de 2 mM MgSO 4 </td></tr><tr><td> Plaque de 96 puits à fond plat </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> Plate-lecteur </td><td> Beckman Coulter </td><td> DTX880 détecteur multimode </td><td></td></tr><tr><td> Bain-marie </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> Incubateur agitateur </td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

La conception simpliste des domaines transmembranaires des protéines comme de simples points d&#39;ancrage dans les bicouches phospholipidiques a depuis longtemps été réfuté. Dans de nombreux cas transmembranaire des protéines ont développé des mécanismes sophistiqués de l&#39;action. 1-3 Une façon dont les protéines membranaires peuvent moduler leurs structures et leurs fonctions se fait par contact direct et spécifique des hélices hydrophobes, formant des oligomères transmembranaire structuré. 4,5 récente grande travaux ont porté sur la répartition des acides aminés de préférence dans l&#39;environnement de membrane en comparaison à la solution aqueuse et les forces intermoléculaires différentes que l&#39;association de protéines disque 6,7. Néanmoins, les études de reconnaissance moléculaire à domaine transmembranaire des protéines est toujours en retard ceux de solubles dans l&#39;eau des régions. Un obstacle majeur demeure: malgré la spécificité remarquable et d&#39;affinité qui oligomérisation transmembranaire peut atteindre, 8 mesure directe de leur association est un défi. Méthodologies traditionnelles appliquées à l&#39;étude de la fonction des protéines membranaires intégrale peut être entravée par l&#39;insolubilité des séquences inhérentes à l&#39;étude. Aperçus tirés de l&#39;étude biophysique peptides synthétiques représentant domaines transmembranaires peuvent fournir un aperçu utile de structure. Toutefois, la pertinence biologique de la micellaire détergent ou systèmes liposomes utilisés dans ces études pour imiter les membranes cellulaires est souvent contestée, ne peptides adoptent une structure native-like dans ces conditions et leur comportement ne reflètent véritablement fonctionnelle du mode d&#39;action au sein d&#39;une membrane native ? Afin d&#39;étudier les interactions des séquences transmembranaires dans les bicouches de phospholipides naturels, le laboratoire Langosch au point des dosages ToxR journaliste de la transcription. 9 Le domaine transmembranaire d&#39;intérêt est exprimé comme une protéine chimérique avec protéine de liaison du maltose pour la localisation dans le périplasme et ToxR de fournir un rapport du niveau d&#39;oligomérisation (figure 1). Dans la dernière décennie, plusieurs autres groupes (par exemple Engelman, DeGrado, Shai) encore optimisé et appliqué ce test journaliste ToxR. 10-13 Les dosages différents ToxR sont devenus un standard en or pour tester les interactions entre protéines dans les membranes cellulaires. Nous présentes démontrent une opération typique expérimentales menées dans notre laboratoire que suit principalement les protocoles développés par Langosch. Cette méthode est généralement applicable utile pour l&#39;analyse de domaine transmembranaire auto-association dans E. coli, où β-galactosidase de production est utilisée pour évaluer la propension oligomérisation TMD. Sur TMD induite dimérisation, ToxR se lie au promoteur ctx provoquant une régulation positive du gène LacZ par β-galactosidase. Une lecture colorimétrique est obtenue par addition d&#39;ONPG à cellules lysées. Clivage hydrolytique d&#39;ONPG par la β-galactosidase résultats dans la production de l&#39;espèce absorbant la lumière o-nitrophénolate (ONP) (figure 2).

Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Une procédure efficace pour évaluer la propension d&#39;oligomérisation des domaines transmembranaires un seul passage (TMDS) est décrite. Des protéines chimères comprenant le TMD fusionnée à ToxR sont exprimés dans une souche E. coli journaliste. TMD induite oligomérisation provoque la dimérisation du ToxR, activation de la transcription et la production de la protéine rapporteur,-galactosidase.

Propension oligomérisation domaine transmembranaire déterminée par dosage ToxR

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

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15.1K vues.  University of Colorado at Boulder. Une procédure efficace pour évaluer la propension d'oligomérisation des domaines transmembranaires un seul passage (TMDS) est décrite. Des protéines chimères comprenant le TMD fusionnée à ToxR sont exprimés dans une souche E. coli journaliste. TMD induite oligomérisation provoque la dimérisation du ToxR, activation de la transcription et la production de la protéine rapporteur,-galactosidase.

Vidéo: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

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

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

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

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

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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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 ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

Caractérisation des récepteurs de protéines G couplées par une mobilisation de calcium dosage à base de fluorescence

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

L&#39;échelle du génome interaction protéine-protéine Le dépistage par Protein-fragment complémentation Assay (PCA) dans les cellules vivantes

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

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 ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Cartographie optique du commerce intra-réticulum sarcoplasmique Ca<sup&gt; 2+</sup&gt; Et potentiel transmembranaire dans le Lapin Coeur Langendorff perfusé

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal glucocorticoïdes Analyse: Surveillance surrénale non invasive en Equidés

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Approches génétiques et biochimiques pour<em&gt; In Vivo</em&gt; et<em&gt; In vitro</em&gt; Évaluation de Protein oligomérisation: Étude de cas Ryanodine Receptor

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Etude de réticulum endoplasmique et mitochondries Interactions par<em&gt; In Situ</em&gt; Proximité Ligation Assay dans des cellules fixes

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...