Ce travail a été soutenu par l&#39;Institut canadien de recherche en santé. SCC a été financé par une bourse de recherche postdoctorale en sciences naturelles et en génie du Canada. FD est une chaire de recherche du Canada.

<html> Processus de reconstitution globale Le processus de reconstitution commence par mélanger la protéine d&#39;échafaudage membranaire (MSP) avec le complexe purifié MalFGK 2 en présence d&#39;un détergent-solubilisées phospholipides. L&#39;étape est suivie de l&#39;élimination lente de la lessive par un matériau adsorbant en polystyrène appelé Bio-Beads ou Amberlite (figure 1). Le processus d&#39;auto-assemblage se produit probablement en raison des interactions entre les phospholipides apolaires hydrophobes, le 2 MalFGK complexes et la surface de la protéine MSP amphipathique. Le produit final est une particule discoïde composé de deux molécules d&#39;emballage MSP à travers le MalFGK complexe 2. Les particules sont séparées des produits d&#39;addition et d&#39;agrégats par ultra-centrifugation analytique et chromatographie d&#39;exclusion stérique. Les particules sont caractérisées par une électrophorèse sur gel natif et dynamique spectroscopie de diffusion de lumière. titre &quot;&gt; 1. Préparation de la membrane des protéines d&#39;échafaudage, MSP <ol><li> Le son MSP-marqué (version MSP1D1 1) est produit à partir du plasmide dans E. pMSP1D1 coli BL21 (DE3) induite à DO 600 ~ 0,5 avec 0,5 mM d&#39;isopropyl β-D-1-thiogalactopyranoside pendant 3 heures à 37 ° C. </li><li> Les cellules sont récoltées par centrifugation à 5000 xg pendant 10 min à 4 ° C et remises en suspension dans TSG10 tampon contenant 100 uM fluorure de phénylméthanesulfonyle. </li><li> Les cellules sont lysées avec une presse française (3x) à 8.000 livres par pouce carré et l&#39;insoluble est éliminé par centrifugation à 5000 xg pendant 10 min à 4 ° C. La fraction soluble contenant la MSP est isolé par ultracentrifugation à 125.000 g pendant 45 min. </li><li> Le MSP est purifié par chromatographie de chélation nickel en utilisant ~ 1,5 ml Ni Sepharose HP TSG10 tampon. Contaminants sont éliminés par lavage avec TSG10 tampon contenant 50 mM d&#39;imidazole. Le MSP est éluée avec un tampon contenant TSG10 600 mMimidazole. La protéine purifiée est dialysée dans un tampon TSG10 et stocké dans -70 ° C à une concentration en protéine de ~ 10-15 mg / ml. </li></ol> 2. Préparation de la MalFGK 2 Complexe <ol><li> Le 2 MalFGK complexe, sa-marquées à l&#39;extrémité C-terminale de MalK, est exprimée à partir du plasmide pBAD22-FGK dans E. coli BL21 (DE3) et induit à DO 600 ~ 0,5 avec 0,2% de L-arabinose pendant 3 heures à 37 ° C. </li><li> Suite à la lyse des cellules et centrifugation comme dans l&#39;étape 1,3, le culot est remis en suspension dans la membrane TSG20 tampon à une concentration finale de 5 mg / ml. Le matériau est solubilisé à 1% p / v de n-dodécyl-β-maltopyranoside (DDM) pendant 3 heures à 4 ° C sous agitation douce. </li><li> L&#39;insoluble est éliminé par ultra-centrifugation comme à l&#39;étape 1.3 et le surnageant contenant le solubilisée 2 MalFGK complexe est recueilli et purifié comme à l&#39;étape 1,4, mais sans dialyse. </li><li> Further purification est réalisée par chromatographie par filtration sur gel dans un tampon TSGD sur une colonne Superdex 200 HR 10/300 de colonne à un débit de 0,5 ml / min. </li></ol> 3. Préparation des phospholipides <ol><li> Un E. coli lipidique totale extraire dissous dans du chloroforme est séparée en 1.000 nmol aliquotes dans des tubes à centrifuger de vis d&#39;assemblage. Le solvant est évaporé sous un léger courant d&#39;azote et séché encore pendant une nuit dans un dessiccateur sous vide. </li><li> Le film lipidique est dissoute dans un tampon TS à une concentration de 5 nM final et vortexé vigoureusement et ultrasons. Les lipides dissous apparaîtra légèrement opaque en raison de leur insolubilité générale dans le tampon TS aqueuse, mais ils doivent rester en suspension. DDM est ajouté à une concentration finale de 0,5% (~ 10 mM), à laquelle la solution devient claire. </li><li> Le mélange de lipides est placé dans un bain d&#39;eau et d&#39;impulsions ultrasons pendant 5 heures à ~ 5 sec. Le mélange de lipides est stocké à 4 ° C pendant un maximum de 1 week. </li></ol> 4. Préparation de Bio-Beads <ol><li> Environ 10-15 ml (volume sec) Bio-Beads est placé dans un tube de 50 ml. </li><li> Les perles sont lavées successivement avec 50 ml de méthanol à 100%, éthanol à 95%, milliQ H 2 O, et enfin tampon TS (deux fois). </li><li> Les billes lavées sont conservés à 4 ° C dans 10 ml de tampon ~ TS. </li></ol> 5. Reconstitution Nanodisc <ol><li> Le MSP est dilué dans du tampon TSGD à la concentration finale d&#39;environ 7 mg / ml (~ 0,3 mM). </li><li> ~ 2 nmol de 2 purifié MalFGK complexe sont mélangés à une protéine: PVC: lipide rapport 1:3:60 ou dans un tampon TSGD 1:3:400. La concentration finale est de 6 uM MalFGK 2, 18 uM MSP et les lipides pM 360 (1:3:60) ou 2,4 mM de lipides (1:3:400). Le volume final est de 300 ul. La concentration finale est DDM 0,08% (~ 1,6 mM). La concentration finale en glycérol dépend de la quantité de lipide ajouté mais reste autour de 5-10% v / v </li&gt; <li> ~ 50 pi de Bio-perle suspension est ajouté dans le tube et le mélange est incubé pendant une nuit sur une table à bascule à 4 ° C. </li><li> Les billes sont sédimentées par gravité et la solution est introduit à la pipette à travers une pointe étroite à éviter autant Bio-Beads que possible. </li><li> Gros précipités sont éliminés par ultra-centrifugation à 100.000 xg pendant 20 min. Les disques sont purifiées par chromatographie par filtration sur gel comme à l&#39;étape de 2,4 TSG10 tampon. Les fractions contenant les disques sont mis en commun et une partie aliquote doit être réinjecté dans la colonne de filtration sur gel même de vérifier la stabilité de la préparation. </li><li> Les disques purifiés peuvent stocké à -70 ° C pendant une période de temps prolongée. Après le dégel de la glace, les disques doivent être soumis à l&#39;ultra-centrifugation comme à l&#39;étape 5.5 à retirer potentiel précipités. Une aliquote doit être analysé par chromatographie de filtration sur gel afin de s&#39;assurer que l&#39;agrégation ou une précipitation importante n&#39;a pas eu lieu lors de la décongélationprocessus. </li></ol> 6. Électrophorèse sur gel natif <ol><li> 1 uM de NANODISQUES (~ 0,2 mg / ml) sont analysés par PAGE native 6, 7 (Tris-HCl pH 8,8, 4-12%) pour évaluer la qualité de la reconstitution (figure 3A). </li><li> La liaison de l&#39;Homme (1 uM) à la 2-complexe MalFGK reconstitué dans NANODISQUES est également évalué en natif-PAGE (figure 3A). </li><li> Après électrophorèse, le gel est coloré au bleu de Coomassie pendant 10 min, et décolorés pendant ~ 1 heure. </li></ol> 7. Diffusion de la lumière dynamique (DLS) <ol><li> NANODISQUES sont purifiées par chromatographie par filtration sur gel comme dans l&#39;étape 2,4 à l&#39;aide d&#39;un tampon TSG10 Superdex 200 HR 10/300 de colonne à un débit de 0,1 ml / min. Les fractions contenant NANODISQUES sont réunies et concentrées à environ 10 mg / ml avec un filtre Amicon centrifuge. </li><li> L&#39;échantillon est filtré deux fois (0,22 um filtre) avant l&#39;analyse par DLS utilisantun DynaPro nanostar instrument (Wyatt Technology) dans un volume intérieur 1 pl cuvette en quartz. Les données s&#39;effectue à l&#39;aide du logiciel DYNAMIQUE (Wyatt Technology) pour estimer le diamètre et le poids moléculaire des particules. </li></ol> 8. Mesures ATPase <ol><li> L&#39;activité ATPase de MalFGK 2-reconstituées NANODISQUES est déterminée en utilisant un dosage colorimétrique 8. </li><li> 1 uM de disques purifiés et 1 mM d&#39;ATP sont mélangés avec des quantités croissantes de MalE et incubées à 37 ° C pendant 20 minutes. La libération de phosphate inorganique est mesurée à 660 nm. </li><li> La quantité de phosphate libéré est comparée à une courbe d&#39;étalonnage générée avec une solution standard de phosphore. </li></ol> 9. Les résultats représentatifs Les NANODISQUES sont purifiés par chromatographie de filtration sur gel (Figure 2A, à gauche). Le chromatogramme montre que la majorité de la reconstitution dCSI (trace noire) éluer forme d&#39;un pic unique, tandis que les disques à base de lipides en excès (rouge trace) éluent dans le volume de vide et comme une série de pics larges. La qualité des NANODISQUES est ensuite analysée par électrophorèse sur gel natif et dynamique spectroscopie diffusion de la lumière (DLS). Disques correctement reconstitué migrer comme une bande nette sur le gel alors que ceux reconstitué en présence d&#39;un excès de lipides comme un test de migration (figure 2A, à droite). Analyse par DLS montre que la population est homogène disque avec un diamètre moyen de 11,4 nm (Figure 2B). Les disques reconstituées ont un poids moléculaire apparent de 215 kDa sur la base de l&#39;approximation DLS. Les échantillons reconstitués en présence d&#39;excès de lipides affichage largement distribué rayons autour de 100 nm, ce qui est typique pour les échantillons non homogènes. La qualité de la MalFGK complexe 2 est évaluée par électrophorèse sur gel natif et son activité ATPase parmesures (figure 3). Le mâle protéine liant le maltose se lie avec une haute affinité pour le transporteur 2 MalFGK 9, 10. Utilisation non électrophorèse sur gel dénaturant, il est possible de détecter un complexe entre l&#39;homme et MalFGK 2 (figure 3A). La stimulation de l&#39;activité ATPase MalK 2 par mâle est montré sur la figure 3B. <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="Figure 1"/> Figure 1. Organigramme type pour le protocole de reconstitution. <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="Figure 2"/> Figure 2. Contrôle de la qualité de la préparation nanodisc. A. Analyse par filtration sur gel (Superdex 200 HR 10/300 colonnes) des NANODISQUES reconstitué à lipide faible (03/01/60; trace noire) ou le taux de lipides élevé (1/3/400; trace rouge). Électrophorèse sur gel natif de la préparation même disque. Molmarqueurs de poids ecular à kDa sont indiquées. B. Analyse dynamique diffusion de la lumière de la préparation même disque. <a href="http://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="Figure 3"/> Figure 3. L&#39;analyse de la MalFGK 2-nanodisc particules. A. L&#39;analyse de retard sur gel de MalE incubés avec des quantités croissantes de MalFGK 2-nanodisc particules. B. ATPase de la MalFGK 2-nanodisc particules en fonction de la concentration de sexe masculin.

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Aucun conflit d&#39;intérêt déclaré.

Nous décrivons une procédure simple pour la reconstitution du transporteur maltose en NANODISQUES. Le transporteur est ATPase active et l&#39;interaction avec le mâle partenaire de liaison soluble peut être recréé (figure 3). La reconstitution réussie du transporteur en NANODISQUES ouvrir la voie à l&#39;analyse biophysique et biochimique supplémentaire. D&#39;un intérêt particulier sera l&#39;analyse systématique de l&#39;ATPase MalK et de l&#39;activité de transport du maltose dans du dé...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Amicon Ultra-4 50K filtre centrifuge </td><td> Millipore </td><td> UFC805008 </td><td> Suivre le protocole du fabricant pour une utilisation correcte </td></tr><tr><td> Bio-Beads SM-2 Adsorbant </td><td> Bio-Rad </td><td> 152-3920 </td><td></td></tr><tr><td> E. coli lipides totaux </td><td> Avanti Polar Lipids </td><td> 100500C </td><td> Dissous dans du chloroforme, gérer comme il convient pour un solvant organique </td></tr><tr><td> Ni Sepharose HP résine </td><td> GE Healthcare </td><td> 17-5268-01 </td><td></td></tr><tr><td> Solution étalon de phosphore </td><td>Sigma-Aldrich </td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20061 </td><td></td></tr><tr><td> Superdex 200 HR 10/300 </td><td> GE Healthcare </td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tableau I. réactifs spécifiques. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Nom </td><td> Composition </td><td> Commentaires </td></tr><tr><td> DDM actions </td><td> 10% v / v DDM </td><td> Remettre en suspension dans l&#39;eau milliQ et conserver à -20 ° C </td></tr><tr><td> MalFGK 2 stock </td><td> 1-2 mg / ml 50 mM Tris-HCl, pH 7,9 NaCl 100 mM 10% v / v de glycérol 0,03% p / v DDM </td><td> Conserver à -70 ° C après purification </td></tr><tr><td> MSP actions </td><td> 10-15 mg / ml 50 mM Tris-HCl, pH 7,9 NaCl 100 mM 10% v / v de glycérol </td><td> Conserver à -70 ° C après purification en aliquotes de 1 ml et &lt;éviter de trop cycles gel / dégel </td></tr><tr><td> Actions phospholipides </td><td> 5 nM E. coli en lipides totaux 0,5% p / v (10 mM) DDM 50 mM Tris-HCl, pH 7,9 50 mM de NaCl </td><td> Conserver à 4 ° C pendant 1 semaine </td></tr><tr><td> TS tampon </td><td> 50 mM Tris-HCl, pH 7,9 50 mM de NaCl </td><td> Stocker à 4 ° C </td></tr><tr><td> TSG10 tampon </td><td> 50 mM Tris-HCl, pH 7,9 NaCl 100 mM 10% v / v de glycérol </td><td> Stocker à 4 ° C </td></tr><tr><td> TSG20 tampon </td><td> 50 mM Tris-HCl, pH8 NaCl 100 mM 20% v / v de glycérol </td><td> Stocker à 4 ° C </td></tr><tr><td> Tampon TSGD </td><td> 50 mM Tris-HCl, pH 7,9 NaCl 100 mM 10% v / v de glycérol 0,03% p / v DDM </td><td> Conserver à 4 ° C et ajouter DDM juste avant l&#39;utilisation </td></tr><tr><td colspan="3"> Tableau II. Solution recettes. </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

Le nanodisc est une particule en forme de disque (~ 10-12 nm grande) que les protéines membranaires piège dans une petite parcelle de bicouche phospholipidique. Le nanodisc est une option particulièrement intéressante pour l&#39;étude des protéines membranaires, en particulier dans le contexte de interactions ligand-récepteur. Le procédé mis au point par Sligar et ses collaborateurs est basée sur les propriétés amphipathiques d&#39;une ingénierie très une hélice protéine extraite de l&#39;échafaudage apolipoprotéines A1. Les faces hydrophobes de la protéine d&#39;échafaudage interagir avec les acyles gras des chaînes latérales de la bicouche lipidique, alors que les régions polaires face à l&#39;environnement aqueux. L&#39;analyse des protéines membranaires ont NANODISQUES avantages significatifs par rapport liposome, car les particules sont petites, homogène et soluble dans l&#39;eau. En outre, les méthodes biochimiques et biophysiques normalement réservés à des protéines solubles peuvent être appliqués, et de part et d&#39;autre de la membrane. Dans ce protocole visuelle, nous présentons une reconstitution étape par étape, d&#39;un caractère bientérisée transporteur ABC bactérien, le mâle-MalFGK complexe 2. La formation du disque est un processus d&#39;auto-assemblage qui dépend des interactions hydrophobes qui ont lieu au cours de l&#39;élimination progressive du détergent. Nous décrivons les étapes essentielles et nous soulignons l&#39;importance de choisir un bon protéine-lipide rapport afin de limiter la formation d&#39;agrégats et les grands polydisperses liposomes comme des particules. Contrôle qualité simples telles que la chromatographie de filtration sur gel, l&#39;électrophorèse sur gel natif et dynamique spectroscopie de diffusion de lumière en sorte que les disques ont été correctement reconstitué.

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A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

NANODISQUES sont de petites particules discoïdes qui incorporent des protéines membranaires dans un petit lopin de bicouche phospholipidique. Nous fournir un protocole visuelle montre que l&#39;incorporation étape par étape du transporteur MalFGK2 dans un disque.

Une méthode étape par étape pour la reconstitution d&#39;un transporteur ABC dans des particules lipidiques Nanodisc

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

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Biology

25.3K vues.  University of British Columbia. NANODISQUES sont de petites particules discoïdes qui incorporent des protéines membranaires dans un petit lopin de bicouche phospholipidique. Nous fournir un protocole visuelle montre que l'incorporation étape par étape du transporteur MalFGK2 dans un disque.

Vidéo: A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Expression, Solubilisation détergent, et purification d&#39;un transporteur membranaire, la protéine MexB multirésistance

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Recherche

Biologie

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Chromatographie d&#39;affinité lipidique des vésicules médiée utilisant magnétique tri cellulaire activé (LIMACS): une nouvelle méthode pour analyser les interactions protéines-lipides

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Une méthode simple pour l&#39;imagerie Arabidopsis Feuilles utilisant perfluorodécaline comme un moyen d&#39;imagerie infiltratives

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

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

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

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

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

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

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 ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Procédure pour évaluer l&#39;efficacité de floculants pour l&#39;élimination des particules dispersées dans des extraits de plantes

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

Une méthode de Transposition efficace In Vitro par un système de beauté dormir transcriptionnellement réglementé empaquetée dans un vecteur Lentiviral défectueux de l’intégration

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear : Un tissu méthode de compensation pour l’imagerie en trois dimensions du tissu adipeux

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

Un modèle épithélial bronchique d’interface air-liquide pour l’exposition réaliste et répétée par inhalation aux particules aéroportées pour l’essai de toxicité

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...