Diese Arbeit wurde von der Canadian Institute of Health Research unterstützt. CSC wurde von einem Postdoc-Stipendium von der Natural Sciences and Engineering Research Council of Canada finanziert. FD ist ein Tier-II Canada Research Chair.

<html> Insgesamt Rekonstitutionsverfahren Die Rekonstitution Prozess beginnt durch Mischen der Membran Gerüstprotein (MSP) mit dem gereinigten MalFGK 2-Komplex in Gegenwart von Detergens-solubilisierten Phospholipide. Der Schritt wird durch die langsame Entfernen des Detergens durch ein Adsorptionsmittel Polystyrolmaterial genannte Bio-Beads oder Amberlite (Abbildung 1) verfolgt. Selbststartendes Montageprozess tritt am wahrscheinlichsten aufgrund der Wechselwirkungen zwischen den apolaren hydrophoben Phospholipide, die 2 MalFGK Komplexes und der Oberfläche der MSP amphipathisches Protein. Das Endprodukt ist eine scheibenförmige Teilchen von zwei Molekülen MSP Umschlingung der MalFGK 2-Komplex hergestellt. Die Partikel werden von den Addukten und Aggregate durch Ultrazentrifugation und analytische Größenausschluss-Chromatographie getrennt. Die Partikel werden durch native-Gelelektrophorese und dynamische Lichtstreuung Spektroskopie charakterisiert. Titel &quot;&gt; 1. Vorbereitung der Membrane Scaffold Protein, MSP <ol><li> Die his-markierte MSP (Version MSP1D1 1) wird von dem Plasmid in E. hergestellt pMSP1D1 coli BL21 (DE3) bei OD 600 ~ 0,5 mit 0,5 mM Isopropyl-β-D-1-thiogalactopyranosid für 3 h induziert bei 37 ° C. </li><li> Die Zellen werden durch Zentrifugation bei 5.000 xg für 10 min bei 4 ° C geerntet und in TSG10 Puffer mit 100 uM Phenylmethansulfonylfluorid. </li><li> Die Zellen werden mit einem Französisch Presse (3x) bei 8000 psi lysiert und das unlösliche Material wird durch Zentrifugation bei 5000 × g für 10 min entfernt wird bei 4 ° C. Die lösliche Fraktion, die das MSP durch Ultrazentrifugation bei 125.000 × g für 45 min isoliert. </li><li> Der MSP wird durch Nickel-Chelat-Chromatographie mit ~ 1,5 ml Ni Sepharose HP in TSG10 Puffer gereinigt. Verunreinigungen werden mit auswärts TSG10 Puffer, der 50 mM Imidazol gewaschen. Die MSP ist mit TSG10 Puffer mit 600 mM eluiertImidazol. Das gereinigte Protein wird in TSG10 Puffer dialysiert und in -70 ° C bei einer Proteinkonzentration von ~ 10-15 mg / ml. </li></ol> 2. Vorbereitung der MalFGK 2 Complex <ol><li> Die MalFGK 2-Komplex, an dem C-Terminus von MalK his-markierte, wird aus dem Plasmid-pBAD22 FGK exprimiert in E. coli BL21 (DE3) und induziert bei OD 600 ~ 0,5 mit 0,2% L-Arabinose für 3 Stunden bei 37 ° C. </li><li> Nach Zellaufschluß und Zentrifugation wie in Schritt 1.3 wird die Membran Pellet in TSG20 Puffer in einer Endkonzentration von 5 mg / ml resuspendiert. Das Material wird mit 1% w / v n-Dodecyl-β-maltopyranosid (DDM) während 3 Stunden bei 4 ° C unter vorsichtigem Schütteln gelöst. </li><li> Das unlösliche Material wird durch Ultrazentrifugation nach Stufe 1.3 entfernt und der Überstand, der das solubilisierte MalFGK 2-Komplex wird gesammelt und gereinigt, wie in Schritt 1.4, aber ohne Dialyse. </li><li> Further Reinigung erfolgt durch Gelfiltrationschromatographie in TSGD Puffer auf einer Superdex 200 HR 10/300 Säule erreicht bei einer Fließgeschwindigkeit von 0,5 ml / min. </li></ol> 3. Vorbereitung der Phospholipide <ol><li> Ein E. coli insgesamt Lipidextrakt in Chloroform gelöst in 1.000 nmol Aliquots in Schraubverschluss Mikrozentrifugenröhrchen getrennt. Das Lösungsmittel wird unter einem leichten Stickstoffstrom eingedampft und getrocknet weitere Nacht in einem Vakuumexsikkator. </li><li> Der Lipidfilm wird in TS-Puffer bei 5 nM Endkonzentration gelöst und gevortext kräftig und beschallt. Die gelösten Lipide erscheint leicht opak aufgrund ihrer allgemeinen Unlöslichkeit in wässrigen TS-Puffer, aber sie sollten in der Schwebe bleiben. DDM wird bis zu einer Endkonzentration von 0,5% (~ 10 mM), bei dem die Lösung klar wird zugegeben. </li><li> Das Lipidgemisch wird in ein Wasserbad gestellt und Puls beschallt 5 mal für ~ 5 sek. Das Lipidgemisch wird bei 4 ° C für maximal 1 wee gespeichertenk. </li></ol> 4. Vorbereitung der Bio-Beads <ol><li> Etwa 10-15 ml (Volumen im trockenen Zustand) Bio-Beads wird in einem 50 ml Röhrchen gegeben. </li><li> Die Perlen werden nacheinander mit 50 ml 100% Methanol, 95% Ethanol, milliQ H 2 O und schließlich TS-Puffer (zweimal) gewaschen. </li><li> Die gewaschenen Kügelchen werden bei 4 ° C in 10 ml ~ TS-Puffer. </li></ol> 5. Nanodisc Rekonstitution <ol><li> Der MSP in TSGD Puffer bei einer Endkonzentration von ~ 7 mg / ml (~ 0,3 mM) verdünnt. </li><li> MSP: Lipid-Verhältnis von 1:3:60 oder 1:3:400 in TSGD Puffer ~ 2 nmol von gereinigtem MalFGK 2-Komplex an ein Protein gemischt. Die Endkonzentration beträgt 6 uM MalFGK 2, 18 und 360 pM MSP uM Lipide (1:3:60) oder 2,4 mM Lipide (1:3:400). Das Endvolumen beträgt 300 ul. Die endgültige Konzentration DDM beträgt 0,08% (~ 1,6 mm). Die Endkonzentration von Glycerin ist abhängig von der Menge an Lipid zugesetzt bleibt aber etwa 5-10% v / v; </li&gt; <li> ~ 50 ul Bio-Bead Suspension wird in das Röhrchen gegeben und die Mischung wird über Nacht auf einem Schüttler Tabelle bei 4 ° C inkubiert </li><li> Die Perlen werden durch Schwerkraft sedimentiert und die Lösung wird durch eine schmale Spitze pipettiert, so viel Bio-Beads wie möglich zu vermeiden. </li><li> Große Präzipitate werden durch Ultrazentrifugation bei 100.000 × g für 20 min entfernt. Die Scheiben werden durch Gelfiltrationschromatographie, wie in Schritt 2.4 in TSG10 Puffer gereinigt. Fraktionen, welche die Scheiben werden gepoolt und ein Aliquot sollte auf demselben Gelfiltrationssäule, um die Stabilität der Zubereitung testen erneut injiziert werden. </li><li> Die gereinigten Scheiben können bei -70 ° C über einen längeren Zeitraum gelagert. Nach dem Auftauen auf Eis, sollten die Scheiben Ultrazentrifugation wie in Schritt 5,5 unterzogen werden, um zu entfernen potenziellen ausfällt. Ein Aliquot sollte durch Gelfiltrationschromatographie analysiert werden, um sicherzustellen, dass eine nennenswerte Aggregation oder Ausfällung nicht während des Auftauens auftretenProzess. </li></ol> 6. Nativer Gelelektrophorese <ol><li> 1 uM Nanoscheiben (~ 0,2 mg / ml) werden durch native PAGE 6, 7 (Tris-HCl pH 8,8, 4-12%), um die Qualität der Rekonstitution (3A) zu beurteilen analysiert. </li><li> Die Bindung der männlichen (1 uM) zur MalFGK 2-Komplex rekonstituiert in Nanoscheiben wird auch durch native-PAGE (3A) beurteilt. </li><li> Nach der Elektrophorese wird das Gel mit Coomassieblau für 10 min gefärbt und entfärbt für ~ 1 Stunde. </li></ol> 7. Dynamic Light Scattering (DLS) <ol><li> Nanoscheiben werden durch Gelfiltrationschromatographie, wie in Schritt 2.4 in TSG10 Puffer unter Verwendung einer Superdex 200 HR 10/300 Säule bei einer Fließgeschwindigkeit von 0,1 ml / min gereinigt. Enthaltenden Fraktionen werden vereinigt und Nanoscheiben konzentriert, um ~ 10 mg / ml mit einer Amicon Filterzentrifuge. </li><li> Die Probe wird zweimal (0,22 um Filter) gefiltert vor der Analyse unter Verwendung von DLSein DynaPro NANOSTAR Instrument (Wyatt Technology) in einem 1 ul Innenvolumen Quarzküvette. Daten werden unter Verwendung des eingepasst DYNAMICS Software (Wyatt Technology), um den Durchmesser und das Molekulargewicht der Teilchen zu schätzen. </li></ol> 8. ATPase Messungen <ol><li> Die ATPase-Aktivität von 2-MalFGK rekonstituierten Nanoscheiben wird unter Verwendung eines kolorimetrischen Assay 8. </li><li> 1 uM des gereinigten Scheiben und 1 mM ATP zusammen mit steigenden Mengen von männlichen und bei 37 ° C für 20 min gemischt. Die Freisetzung von anorganischem Phosphat wird bei 660 nm gemessen. </li><li> Die Menge an Phosphat-Freigabe mit einer Standardkurve, die mit einer Standardlösung erzeugte Phosphor verglichen. </li></ol> 9. Repräsentative Ergebnisse Die Nanoscheiben werden durch Gelfiltrationschromatographie (2A, links) gereinigt. Das Chromatogramm zeigt, dass die Mehrheit der rekonstituierten dISCs (schwarze Kurve) eluieren als einzelner Peak, während Discs mit überschüssigem Lipide (rote Linie) eluieren im Leervolumen und als eine Reihe von breiten Peaks hergestellt. Die Qualität der Nanoscheiben wird weiter durch native Gelelektrophorese und dynamische Lichtstreuung Spektroskopie (DLS) analysiert. Richtig rekonstituierten Discs als scharfe Bande auf dem Gel wandern während diejenigen rekonstituierten in Gegenwart eines Überschusses von Lipiden als eine Schmierschicht (2A, rechts) zu migrieren. Analyse durch DLS zeigt, dass die Scheibe Population homogen mit einem durchschnittlichen Durchmesser von 11,4 nm (2B) ist. Die rekonstituierten Scheiben haben ein scheinbares Molekulargewicht von 215 kDa auf dem DLS-Näherung berechnet. Proben rekonstituiert in Gegenwart eines Überschusses an Lipiden Display weithin Radien um 100 nm verteilt sind, was typisch ist für inhomogene Proben. Die Qualität der MalFGK 2-Komplex wird durch native-Gelelektrophorese und seine Aktivität durch ATPase beurteiltMessungen (Abbildung 3). Die Maltosebindeprotein MalE bindet mit hoher Affinität an den Transporter MalFGK 2 9, 10. Verwendung nicht-denaturierender Gelelektrophorese, ist es möglich, einen Komplex zwischen männlichen und MalFGK 2 (Abbildung 3A) zu detektieren. Die Stimulation des MalK 2 ATPase-Aktivität von männlichen in 3B gezeigt. <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Typische Ablaufplan für die Wiederherstellung Protokoll. <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Qualitätskontrolle der nanodisc Vorbereitung. A. Gelfiltrationsanalyse (Superdex 200 HR 10/300 Spalte) der Nanoscheiben rekonstituiert bei niedrigen Lipid-Verhältnis (1/3/60; schwarze Kurve) oder hoher Lipid-Verhältnis (1/3/400, rote Kurve). Nativer Gelelektrophorese derselben Disk Zubereitung. Molecular Gewicht Marker in kDa angegeben. B. dynamische Lichtstreuung Analyse der gleichen Disc Vorbereitung. <a href="http://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen</a> . <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Analyse des MalFGK 2-nanodisc Teilchen. A. Gel-Shift-Analyse MalE mit steigenden Mengen MalFGK 2-nanodisc Partikeln inkubiert. B. ATPase Aktivität des MalFGK 2-nanodisc Teilchen als Funktion der männlichen Konzentration.

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Wir beschreiben ein einfaches Verfahren für die Wiederherstellung der Maltose Transporter in Nanoscheiben. Der Transporter ist ATPase aktiv und die Interaktion mit dem löslichen Bindungspartner MalE können wiederhergestellt (Abbildung 3). Die erfolgreiche Rekonstitution des Transporters in Nanoscheiben öffnen den Weg für weitere biophysikalische und biochemische Analyse. Von besonderem Interesse ist dabei die systematische Analyse werden die MalK ATPase und Maltose Transportaktivität in Wasch-, Li...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Amicon Ultra-4 50K Zentrifugalfilter </td><td> Millipore </td><td> UFC805008 </td><td> Folgen Sie dem Protokoll des Herstellers für die ordnungsgemäße Verwendung </td></tr><tr><td> Bio-Beads SM-2 Adsorbent </td><td> Bio-Rad </td><td> 152-3920 </td><td></td></tr><tr><td> E. coli Gesamtlipide </td><td> Avanti Polar Lipids </td><td> 100500C </td><td> Gelöst in Chloroform, zu behandeln, wie es sich für ein organisches Lösungsmittel </td></tr><tr><td> Ni-Sepharose HP Harz </td><td> GE Healthcare </td><td> 17-5268-01 </td><td></td></tr><tr><td> Phosphor Standardlösung </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> Tabelle I. Spezifische Reagenzien. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Name </td><td> Zusammensetzung </td><td> Kommentare </td></tr><tr><td> DDM Lager </td><td> 10% w / v DDM </td><td> Resuspendieren in milliQ Wasser und bei -20 ° C </td></tr><tr><td> MalFGK 2 stock </td><td> 1-2 mg / ml 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v Glycerin 0,03% w / v DDM </td><td> Lagerung bei -70 ° C nach der Reinigung </td></tr><tr><td> MSP Lager </td><td> 10-15 mg / ml 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v Glycerin </td><td> Lagerung bei -70 ° C nach der Reinigung in &lt;1 ml Aliquots und vermeiden Sie übermäßige Einfrieren / Auftauen </td></tr><tr><td> Phospholipid Lager </td><td> 5 nM E. coli Gesamtlipide 0,5% w / v (10mM) DDM 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Lagerung bei 4 ° C für 1 Woche </td></tr><tr><td> TS-Puffer </td><td> 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Lagerung bei 4 ° C </td></tr><tr><td> TSG10 Puffer </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v Glycerin </td><td> Lagerung bei 4 ° C </td></tr><tr><td> TSG20 Puffer </td><td> 50 mM Tris-HCl, pH8 100 mM NaCl 20% v / v Glycerin </td><td> Lagerung bei 4 ° C </td></tr><tr><td> TSGD Puffer </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v Glycerin 0,03% w / v DDM </td><td> Lagerung bei 4 ° C und fügen DDM kurz vor Gebrauch </td></tr><tr><td colspan="3"> Tabelle II. Lösung Rezepte. </td></tr></tbody></table></td></tr></tbody></table>

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

Die nanodisc ist eine scheibenförmige Teilchen (~ 10-12 nm groß), dass trap Membranproteine ​​in einem kleinen Fleckchen Phospholipiddoppelschicht. Die nanodisc ist eine besonders attraktive Option zur Untersuchung Membranproteine, insbesondere im Kontext von Ligand-Rezeptor-Wechselwirkungen. Die Methode erstmals von Sligar und Kollegen auf den amphipathischen Eigenschaften eines technisch hoch ein-helikalen Gerüstprotein aus dem Apolipoprotein A1 abgeleitet. Die hydrophoben Flächen des Gerüstprotein interagieren mit den Fettacyl Seitenketten der Lipiddoppelschicht während die polaren Regionen der wässrigen Umgebung zugewandt. Analysen von Membranproteinen in Nanoscheiben deutliche Vorteile gegenüber Liposom weil die Teilchen kleine, homogene und wasserlösliche sind. Zusätzlich können biochemische und biophysikalische Methoden normalerweise löslichen Proteinen vorbehalten angewandt werden, und von beiden Seiten der Membran. In dieser visuellen Protokoll, präsentieren wir eine Schritt-für-Schritt Rekonstitution eines gut charakterisiertgekenn zeichnet bakteriellen ABC-Transporter, die männlich-MalFGK 2-Komplex. Die Bildung der Platte eine Selbstorganisation, die auf hydrophoben Wechselwirkungen stattfinden während der progressiven Entfernung des Detergens abhängig ist. Wir beschreiben die wesentlichen Schritte und wir die Bedeutung der Wahl eines korrekten Protein-zu-Lipid-Verhältnis, um die Bildung von Aggregaten und größer polydispersen Liposomen-ähnliche Partikel zu begrenzen. Einfache Qualitätskontrollen wie Gelfiltrationschromatographie, native Gelelektrophorese und dynamische Lichtstreuung Spektroskopie sicherzustellen, dass die Scheiben wurden ordnungsgemäß wiederhergestellt.

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

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

Nanoscheiben sind kleine scheibenförmige Partikel, die Membranproteine ​​in einem kleinen Fleckchen Phospholipiddoppelschicht integrieren. Wir stellen eine visuelle Protokoll, das die Schritt-für-Schritt Einarbeitung des MalFGK2 Transporters zu einer Scheibe zeigt.

Eine Schritt-für-Schritt-Verfahren zur Rekonstitution eines ABC-Transporter in Nanodisc Lipid Particles

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 ...

a-step-step-method-for-reconstitution-an-abc-transporter-into

Research

JoVE Journal

Biology

25.3K Aufrufe.  University of British Columbia. Nanoscheiben sind kleine scheibenförmige Partikel, die Membranproteine ​​in einem kleinen Fleckchen Phospholipiddoppelschicht integrieren. Wir stellen eine visuelle Protokoll, das die Schritt-für-Schritt Einarbeitung des MalFGK2 Transporters zu einer Scheibe zeigt.

Serie: 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, Waschmittel Solubilisierung und Reinigung von einer Membran Transporter, der 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 ...

Forschung

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.

Lipidvesikel-vermittelte Affinitätschromatographie unter Verwendung von Magnetic Activated Cell Sorting (LIMACS): Eine neue Methode zur Protein-Lipid-Wechselwirkungen Analyze

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.

In vitro Rekonstitution der 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 ...

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.

Eine einfache Methode zur Bildgebung Arabidopsis Leaves Mit Perfluordecalin als 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 ...

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.

Rekonstitution eines Kv-Kanal in Lipidmembranen für Strukturelle und Funktionelle Studien

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.

Rekonstitution der ein Transmembranprotein, das spannungsgesteuerten Ionenkanal, KvAP, in Riesen unilamellare Vesikel für Mikroskopie und Patch-Clamp-Studien

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.

Vorgehensweise Um die Effizienz von Flockungsmittel für die Beseitigung von Dispersed Partikel aus Pflanzenextrakten zur Bewertung

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.

Eine effiziente In-vitro- Umsetzung-Methode durch eine Transcriptionally geregelten Schönheit Schlafsystem verpackt in eine Integration defekt Lentivirale Vektor

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: Ein Gewebe Clearing-Methode für die dreidimensionale Darstellung von Fettgewebe

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.

Ein Luft-Flüssig-Interface-Bronchial-Epithelmodell für realistische, wiederholte Inhalationsexposition gegenüber Luftpartikeln für Toxizitätstests

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