Nous remercions EUROSARF de nous envoyer la levure TAP plasmides d&#39;expression. Nous sommes également reconnaissants de l&#39;aide éditoriale de Ryan Labadens. Ce travail a été soutenu par une subvention du NIH / NINDS (R01NS070962) à CW

Une. Générer SAMU-TAP-étiqueté mouches transgéniques <ol><li> Générer pUAST-TAP-étiqueté constructions d&#39;ADN. <ol><li> Décider de quel côté (N-ou C-terminale) de la protéine d&#39;appât pour la balise TAP devrait être fusionné à, sur la base de la structure / fonction de la protéine. Voir la discussion pour plus de détails. </li><li> Sous-cloner la région codante de l&#39;ADNc du gène d&#39;intérêt dans les sites de clonage multiple (MCS) des vecteurs pUAST-PAEEN ou pUAST-CTAP pour générer N-ou C-terminal-marquées transgènes UAS-TAP, respectivement. Voir la Figure 1 pour des cartes détaillées et des sites de restriction utilisables et cadres de lecture. </li></ol></li><li> Générer SAMU-TAP-étiqueté mouches transgéniques. <ol><li> Générer des mouches transgéniques suivant des protocoles standard à l&#39;aide de l&#39;élément P médiation insertion 11. Un certain nombre de services d&#39;injection sont disponibles dans le commerce. </li><li> Traverser neuronale pilote de GAL4 (c.-à-BG380-GAL4) pour chaque lignée transgénique individu et déterminer les niveaux d&#39;expression de protéinesde chaque ligne par western blot (avec les anticorps de la peroxydase anti-peroxydase) et / ou immuno-coloration (avec l&#39;anticorps anti-TAP). D&#39;une manière générale, une lignée transgénique avec un niveau d&#39;expression de protéine qui est proche de la protéine endogène est recommandé pour les procédures de la TAP. Voir la discussion pour plus de détails. </li><li> PerformGAL4/UAS-based expériences de récupération de confirmer la fonctionnalité des transgènes TAP-marqué si mutants des gènes d&#39;intérêt avec perte de fonction sont disponibles. Choisissez un transgène qui peut sensiblement sauver les phénotypes mutants pour les expériences de TAP suivants. </li></ol></li></ol> 2. Préparer les échantillons pour TAP procédure <ol><li> Générer une mouche stock qui porte à la fois un pilote neuronale GAL4 (par exemple BG380-Gal4) et le transgène TAP marqués choisi afin de faciliter l&#39;expansion des échantillons de mouches. Recueillir les descendances F1 du pilote GAL4 et la croix de la SAMU transgène dans les rares cas où la combinaison ci-dessus entraîne la survie et la croissance inconvénient. &lt;/ Li&gt; <li> Prélever de petits échantillons échelle et d&#39;optimiser l&#39;état de lyse pour solubiliser la protéine TAP-étiqueté. <ol><li> Faire une série de tampons de lyse en utilisant une combinaison des détergents non ioniques NP-40 (0,1 à 1%), NADOC (0,1-1%) et du Triton X-100 (0,05 à 0,5%). Voir le tableau 1 et la discussion pour plus d&#39;informations. </li><li> Au sommet d&#39;une garniture de CO2, utilisez # 5 pinces à disséquer 20 chefs d&#39;adultes du transgène exprimant vol TAP et les recueillir dans un tube de 1,5 ml pour tester chaque condition de tampon de lyse. </li><li> Ajouter tests tampon de lyse 100 ul dans le tube et homogénéiser les têtes en caressant de haut en bas avec un pilon en plastique, puis ajouter un autre tampon d&#39;essai de 100 ul. </li><li> Faites tourner la lysat tête à 21 500 g pendant 10 min (4 ° C), et séparer le surnageant et le culot après centrifugation. Ajouter 25 ul de tampon de charge SDS 2x au culot et le tampon de chargement 10 ul 4x SDS à 30 ul de surnageant respectivement. </li><li> Faire bouillir les deux échantillons pendant 5 min et les analyser côteCôté en utilisant SDS-PAGE et Western Blot subséquente avec l&#39;anticorps PAP. Déterminer la solubilité par le rapport entre les niveaux TAP-protéine dans le surnageant par rapport à la pastille. </li></ol></li><li> Préparer l&#39;échantillon à grande échelle <ol><li> Développer et recueillir des mouches adultes. <ol><li> Développer le stock neuronal-GAL4/UAS-TAP-transgene en bouteilles et retourner les bouteilles tous les 3 jours jusqu&#39;à cumulés 250 bouteilles sont utilisés pour la collecte. </li><li> Vieux collecter 1-3 jours mouches adultes dans 50 ml tubes coniques, mettre le tube dans de l&#39;azote liquide immédiatement au congélateur les mouches. Conserver les mouches dans un congélateur à -80 ° C. A noter que le volume de vol ne doit pas dépasser 2/3 du tube de 50 ml. </li></ol></li><li> Recueillir têtes de mouches (effectuer cette étape au-dessus de la glace sèche en poudre). <ol><li> Sortez les tamis préalablement refroidie et le mortier et le pilon du congélateur à -80 ° C et les mettre sur la glace sèche, idéalement dans un grand seau à glace. Empilez deux tamis de test standard USA avec un n ° 25 sur ee haut et n ° 40 en bas. </li><li> Prenez vol congelés sur et déposez-les dans de l&#39;azote liquide et de garder les mouches là pendant environ 10 min. Vortex ou secouer les tubes vigoureusement pour briser les têtes, les jambes et les ailes des organes. </li><li> Verser le mélange sur le tamis supérieur, puis secouer les tamis vigoureusement tout en maintenant les deux tamis ensemble. Après tamisage, les corps restent sur le tamis supérieur, voler les têtes seront retenus sur le tamis de fond, et les jambes, ailes et autres débris vont tomber sur la glace sèche. Séparer les deux tamis et soigneusement transférer les têtes de mouches au mortier froid. </li></ol></li><li> Homogénéiser les têtes de mouches <ol><li> En plus de la glace sèche, broyer les têtes avec le mortier et le pilon de particules de poudre, puis transférer la poudre à un Dounce broyeur de tissus de 15 ml en verre qui a été préalablement refroidi sur glace. </li><li> Mesurer le poids de la meuleuse avant et après l&#39;échantillon de la tête a été versé dans, puis calculer combien la samp têtele pèse. Un total de 15.6 grammes de têtes de mouches suffira pour chaque expérience TAP ajustant en conséquence les niveaux de la protéine d&#39;expression. Ajouter 15 ml de tampon d&#39;homogénéisation glacé (tampon de lyse optimisé à l&#39;étape 2.2) à la poudre, puis la course avec le grand pilon en jeu jusqu&#39;à ce qu&#39;il soit facile pour les pilon pour monter et descendre. Gardez le broyeur de verre sur la glace en tout temps. </li></ol></li><li> Préparer le surnageant pour TAP <ol><li> Transférer le broyat dans un tube de centrifugeuse à grande vitesse et un essorage pendant 20 min à ~ 50 000 x g (4 ° C). Transférer le surnageant dans un nouveau tube de centrifugation à grande vitesse et répéter la centrifugation une fois de plus. </li><li> Transférer le surnageant dans un tube d&#39;ultracentrifugation et de réaliser un 40 min ~ 250 000 xg rotation pour dégager davantage le surnageant. Le surnageant est prêt pour les opérations de purification d&#39;affinité en tandem après ultracentrifugation. </li></ol></li></ol></li></ol> 3. TAP Purification </p&gt; Les sections suivantes ont été tirées du laboratoire TAP Séraphin protocol 12 (http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/Bertran%20Seraphin%27s%20TAP%20page.pdf ) <ol><li> Effectuer IgG perle purification d&#39;affinité <ol><li> Préparer IgG sépharose perles tandis que les échantillons sont centrifugés. Laver 3x 400 ul d&#39;IgG de billes dans un tube Falcon de 15 ml avec 10 ml de tampon de lavage froid IgG. Pour chaque lavage, secouez doucement le tube pendant 2 min, puis centrifuger les perles à 1000 g pendant 2 min. A la fin du troisième lavage, éliminer le tampon et ne laisser que les billes dans le tube. </li><li> Soigneusement transférer le surnageant clarifié (~ 15 ml) dans le tube de 15 ml contenant les billes IgG. Incuber les billes et le cerveau lysat mélange à 4 ° C sur une nutator pendant 2 heures. </li><li> Mettre en place un micro colonne vide et propre avec environ 15 ml de volume total en chambre froide. Chargez le mélange de perles IgG en versant régulièrement le mélange dans la colonne; essayer de ne pas piéger toutles bulles d&#39;air à l&#39;intérieur de la colonne. Laisser les perles de s&#39;installer dans la colonne et le tampon de drainer lentement par gravité. </li><li> Laver la colonne à fond avec 10 ml de tampon de lavage froid IgG après tout du lysat de cerveau s&#39;est écoulé à travers la colonne d&#39;IgG réglée. Répétez l&#39;2x de lavage. Remarque: ne laissez jamais le cordon sécher à l&#39;air. </li></ol></li><li> Effectuer TEV clivage <ol><li> Après le troisième lavage, laver de nouveau la colonne avec 10 ml de tampon de clivage TEV. Cette étape prépare le cordon IgG que sequestrates le complexe d&#39;appât pour TEV clivage. </li><li> Juste avant la dernière goutte de tampon TEV est sur le point de s&#39;écouler, mettre un bouchon au bas de la colonne de bloquer la circulation, ajouter 1,3 ml de tampon de TEV contenant 130 unités de TEV enzyme de la colonne, et puis boucher solidement le haut de la colonne. Assurez-vous que la colonne est bien scellé aux deux extrémités. </li><li> Tournez la colonne à 18 ° C pendant 2 heures pour permettre à l&#39;enzyme TEV pour cliver le peptide sur le site de TEV et libérer la protéine WHI complexele laissant derrière la protéine A peptide de domaine lié aux perles de l&#39;IgG. </li></ol></li><li> Effectuer calmoduline perle purification d&#39;affinité <ol><li> Préparer les billes calmoduline tandis que les billes sont incubées avec des IgG de l&#39;enzyme TEV. Lavez 200 pi perles calmoduline dans un 15 ml Falcon 3x tube, chaque fois avec 10 ml de calmoduline froid de tampon de liaison. Pour chaque lavage, secouez légèrement le tube pendant 2 minutes sur un nutator, puis centrifuger les perles à 1000 g pendant 2 min. A la fin du troisième lavage, retirer toute la mémoire tampon et ne laisser que les perles dans le tube. </li><li> A la fin de l&#39;incubation TEV (étape 3.2.3), retourner la colonne IgG retour à la chambre froide et mettez-le vers le haut. Laissez les perles se contenter de 10 min. </li><li> Retirez le couvercle supérieur et le couvercle inférieur, puis recueillir le produit de clivage de 1,3 ml de TEV dans un tube Falcon de 15 ml. Laissez le tampon vider complètement. Ajouter un tampon de 200 TEV ul supplémentaire à la colonne de pousser le volume mort de la colonne, recueillir les ffaible dans le même tube. </li><li> Ajouter 4,5 ml de calmoduline et 4,5 ul de tampon de 1 M de CaCl2 de liaison au produit de clivage de 1,5 ml TEV recueillis ci-dessus. Le CaCl 2 sert à titrer l&#39;EDTA dans le tampon TEV. Transférer le mélange dans 6 ml au tube contenant les billes calmoduline et faire tourner le tube à 4 ° C sur une nutator pendant 1 heure. </li><li> Mettre en place un autre micro colonne vide et propre avec environ 10 ml de volume total en chambre froide. Chargez le mélange de perles calmoduline à la colonne et lui permettre de s&#39;écouler par gravité. </li><li> Lorsque toute la solution a traversé la colonne calmoduline constante, laver la colonne deux fois, chacune avec 10 ml de calmoduline froid de tampon de liaison. Remarque: éviter de déranger les billes calmoduline et essayer de garder la surface des perles plates que possible pendant le lavage. </li></ol></li><li> Éluer le complexe de l&#39;appât de la colonne calmoduline. Juste après le lavage, l&#39;élution de la colonne calmoduline avec cinq fractions de 200 ul Calmodu froidlin tampon d&#39;élution. Pour chaque fraction, ajouter doucement 200 ul de tampon d&#39;élution de la colonne et recueillir l&#39;éluat avec un marqué 1.5 ml tube Eppendorf. Répétez cette 4x. </li><li> Analyser le complexe de protéines par SDS-PAGE, <ol><li> Prenez une petite aliquote de chacune des cinq fractions (environ 30 pi) et ajouter un tampon de charge SDS. Faire bouillir les échantillons pendant 5 min et charger les échantillons côte-à-côte avec des marqueurs moléculaires de protéines dans un gradient (4-15%) sur gel de SDS-PAGE. </li><li> Après les échantillons ont entièrement résolu dans le gel, arrêter le électrophorèse et tacher le gel avec toutes les procédures sensibles base-G-250 colloïdales Coomassie comme «argent bleu &#39;coloration 13. Coloration à l&#39;argent est facultative, mais pas préférable car il n&#39;est pas compatible avec l&#39;analyse par spectrométrie de masse subséquente. Stocker le reste de l&#39;éluat dans un congélateur C -80 ° pour une analyse ultérieure telle que la spectrométrie de masse pour découvrir les identités moléculaires du complexe de protéine purifiée. Sediscussion de e. </li></ol></li></ol>

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Les auteurs n&#39;ont rien à révéler.

Procédé de purification par affinité en tandem (TAP) propose un protocole de double purification permettant l&#39;isolement et l&#39;enrichissement des complexes de protéines à travers deux étapes indépendantes de purification d&#39;affinité. La conception de l&#39;étiquette TAP n&#39;est pas limitée à ce qui est présenté dans ce protocole, d&#39;autres domaines et motifs de liaison de protéines sont également applicables si les conditions tampons sont ajustés en conséquence. Un bon exemple d&#39;autres...

Définir les voies moléculaires ou des réseaux qui interviennent dans un processus biologique particulier est l&#39;un des buts ultimes de la recherche biomédicale. Fly généticiens ont beaucoup compté sur la génétique à terme, en particulier modificateur écrans génétiques (deux amplificateurs et écrans suppresseurs), d&#39;identifier les facteurs qui travaillent ensemble, en parallèle avec, ou en amont ou en aval d&#39;un gène d&#39;intérêt. Cependant, les écrans de génétique à terme arrive souvent ne parviennent pas à identifier les gènes essentiels qui, lorsqu&#39;ils sont mutés, provoquent létalité à des stades précoces du développement, ou des gènes avec redondance fonctionnelle et d&#39;indemnisation dont la perte de fonction ne causer des anomalies subtiles qui sont difficiles à marquer. Une façon de surmonter cette difficulté consiste à cribler des interactions protéine-protéine directe. Pour plus d&#39;une décennie, un nombre croissant de méthodes biochimiques, y compris les deux hybrides de levure, phage display, réticulation chimique, Co-IP, Tandem Affinity Purification (TAP), etc. ont été utilisées pour étudier la protéine-les interactions protéiques. Chacune de ces approches a ses propres forces et faiblesses en ce qui concerne la sensibilité et la spécificité. Parmi eux, la méthode TAP permet la détection de l&#39;interaction physique dans des conditions quasi-physiologiques, préserve la spécificité et la cohérence 2 et comprend la possibilité d&#39;étendre à haut débit analyses 3,4. La méthode de TAP a été développé à l&#39;origine dans la levure par des collègues Rigautand 1. Dans ce procédé, une protéine d&#39;intérêt est exprimé avec une étiquette de TAP. La balise TAP abrite deux domaines d&#39;affinité de liaison indépendants: une protéine un domaine qui se lie à l&#39;IgG et un domaine liant la calmoduline. Les deux domaines sont séparés par une TEV (Tobacco Etch Virus) site de clivage. Une telle combinaison permet à deux tours indépendantes de purifications d&#39;affinité pour réduire suffisamment les liaisons non spécifiques et enrichissent fixations spécifiques 1. Pour ce cas, la méthode de TAP est une métho très puissantd pour identifier d&#39;interactions in vivo d&#39;une protéine donnée, bien que la surexpression de la protéine exogène, il peut être plus enclins à associer avec des protéines qui font normalement pas complexe avec son homologue endogène. Depuis son développement, la méthode de TAP a été appliquée dans de nombreux autres systèmes, y compris à base de culture de cellules et d&#39;autres systèmes 5,6 vivo systèmes modèles à 6-9. Nous décrivons ici l&#39;adaptation de la méthode de TAP chez la drosophile. On génère premier pUAST-NTAP et pUAST-CTAP vecteurs pour faciliter le clonage et la fusion de l&#39;étiquette TAP à l&#39;extrémité N-ou C-terminale du gène d&#39;intérêt. Le SAMU-TAP-étiqueté transgène est ensuite exprimé dans le système nerveux sous le contrôle d&#39;un chauffeur de GAL4 neuronale 10. Ensuite, un grand nombre de têtes de mouches adultes sera recueillie, qui ont une teneur élevée en cellules neurales et sont faciles à séparer des autres parties du corps après congélation en fonction des différences de taille. Les têtes d&#39;adultes sont homogénéisés et dégagé bcentrifugations successives y et le surnageant est soumis à une procédure de TAP décrit ci-dessous.

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			<td height="42" style="height:42px;width:307px;">U.S.A. standard test sieve No. 25</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-18&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">U.S.A. standard test sieve No. 40</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Kontes Dounce Tissue Grinders 15 ml</td>
			<td>Kimble Chase</td>
			<td>885300-0015</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;"></td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">IgG sepharose beads</td>
			<td>Pharmacia</td>
			<td style="width:119px;">17-0969-01</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Econo-column 0.7 cm x 20 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4721</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Econo-column 0.5 cm x 15 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4716</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Calmodulin beads</td>
			<td style="width:104px;">Stratagene</td>
			<td style="width:119px;">214303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coors Mortar and Pestle</td>
			<td style="width:104px;">CoorsTek</td>
			<td>60311</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">AcTEV Protease</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">12575-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:104px;">Roche</td>
			<td style="width:119px;">11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Mix</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction in drosophila adult heads by tandem affinity purification (tap)

Écrans génétiques effectuées à l&#39;aide Drosophila melanogaster (de la mouche des fruits) ont fait de nombreuses découvertes d&#39;étape dans le progrès des sciences biologiques. Cependant, l&#39;utilisation d&#39;écrans biochimiques visant à étendre les connaissances acquises à partir de l&#39;analyse génétique a été explorée que récemment. Ici, nous décrivons une méthode pour purifier le complexe de protéine qui s&#39;associe avec n&#39;importe quelle protéine d&#39;intérêt à partir des têtes de mouches adultes. Cette méthode tire parti du système Drosophila GAL4/UAS d&#39;exprimer une protéine appât fusionnée avec une étiquette Tandem Affinity Purification (TAP) dans les neurones de la mouche in vivo, puis met en œuvre deux cycles de purification en utilisant une procédure de TAP similaire à celui établi à l&#39;origine en une levure pour purifier le complexe protéique interagissant. A la fin de cette procédure, un mélange de plusieurs complexes protéiques dont l&#39;identité est obtenue moléculaire peut être déterminée par spectrométrie de masse. Validation des protéines candidates bénéficiera de la resource et la facilité d&#39;effectuer des études de perte de fonction chez les mouches. Des approches similaires peuvent être appliqués à d&#39;autres tissus de braguette. Nous pensons que la combinaison de manipulations génétiques et cette approche protéomique dans le système de modèle de la mouche détient un énorme potentiel pour résoudre les problèmes fondamentaux dans le domaine de la neurobiologie et au-delà.

Ici, nous démontrons notre effort pour identifier les protéines Highwire-qui interagissent dans le cerveau de la mouche. Highwire (Hiw) et ses homologues vertébrés et d&#39;invertébrés sont énormes ubiquitine ligases qui régulent le développement et la réparation du système nerveux 14. Ils partagent un certain nombre de domaines fonctionnels hautement conservées. Cependant, leurs actions moléculaires ne sont pas tout à fait clair. Les travaux effectués dans le ver, la mouche et de la souris a c...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP at JoVE.com

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophile est célèbre pour sa manipulation génétique puissant, mais pas pour son aptitude d&#39;analyse biochimique en profondeur. Ici, nous présentons une procédure basée TAP à identifier les partenaires d&#39;interaction de toute protéine d&#39;intérêt à partir du cerveau de la mouche. Cette procédure peut potentiellement conduire à de nouvelles pistes de recherche.

Identifier les interactions protéine-protéine dans<em&gt; Drosophila</em&gt; Têtes de Tandem Affinity Purification adultes (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

identifying-protein-protein-interaction-drosophila-adult-heads-tandem

Research

JoVE Journal

Biology

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

13.8K vues.  Louisiana State University Health Sciences Center.  Drosophile est célèbre pour sa manipulation génétique puissant, mais pas pour son aptitude d'analyse biochimique en profondeur. Ici, nous présentons une procédure basée TAP à identifier les partenaires d'interaction de toute protéine d'intérêt à partir du cerveau de la mouche. Cette procédure peut potentiellement conduire à de nouvelles pistes de recherche. 

Vidéo: Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Protein Purification Orthogonal Animé par une étiquette d&#39;affinité bispécifiques petits

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Recherche

Biologie

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identification des partenaires protéine interagissant Utilisation Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolement de la chromatine par purification d&#39;ARN (CHIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Un essai en phase liquide d&#39;affinité de capture utilisant des billes magnétiques pour étudier interaction protéine-protéine: Le poliovirus Exemple-Nanobodies

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identification des complexes protéiques dans<em&gt; Escherichia coli</em&gt; En utilisant séquentielle Purification par affinité peptide en combinaison avec la spectrométrie de masse en tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purification des relevés de notes et de métabolites<em&gt; Drosophila</em&gt; Têtes

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Protein Purification méthode sans la détermination de l&#39;affinité de liaison par thermophorèse Microscale

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

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) ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Identifier les sites d&#39;interaction protéine-protéine à l&#39;aide de peptides tableaux

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

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 ...