Wir danken den National Institutes of Health (1R21CA138373 und Stand Up to Cancer (SU2C) für die finanzielle Unterstützung dieser Arbeit. HY ist dankbar für das Geschäftsjahr 2009 Elion Award von der American Association of Cancer Research, ein Kimmel Scholar Award der Sidney Kimmel Stiftung für Cancer Research (SKF-08 bis 101) und der National Science Foundation Fakultät Early Career Award (NSF0954819).

1. Cloning Überlegungen <ol><li> Kommerziell hergestellte Oligonukleotide, die die TMD von Interesse durch NheI und BamHI Restriktionsstellen und 5&#39;-phosphorylierten flankiert können in pTox7 (modified in unserem Labor durch Insertion von einem Basenpaar direkt nach dem BamHI 14) (Abbildung 3) verdaut nacheinander ligiert werden mit BamHI und NheI. Ein Beispiel Oligonukleotid ist unten dargestellt: 5&#39;ctagcTMDSEQUENCEg3 &quot; 3 &#39;gTMDSEQUENCEcctag5 &quot; </li></ol> Die TMD-Sequenz sollte 12-24 Rückstände (kürzere Sequenzen wird vermutlich durch den Vektor kodierten hydrophobe Reste verlängert werden). Um die Schnittstelle zu untersuchen, sollten vier Varianten des TMD-Design erstellt, wo sequenzielles Rest Insertionen und Deletionen gleichzeitige Rückstände Ergebnis in Rotation der TMD bezogen auf ToxR. 15,16 Schließlich wird die Arabinose-Konzentration zwischen 0,001 und 0,01% variiert werden sollten (w / v) zu Ermittlung der Konzentration, wo maximale Unterschiede in β-Galactosidase-Signale zwischen verschiedenen TMD Sequenzen beobachtet werden; Testen verschiedener Expression wird empfohlen, unter welchen Bedingungen unterschiedliche Affinitäten am besten unterschieden werden identifizieren können. Neben Arabinose und Antibiotika, können 0,4 mM IPTG verwendet werden, um Unterschiede in der Affinität zwischen verschiedenen TMDs zu verbessern. Die ToxR Messung sollten mindestens in vierfacher Ausfertigung durchgeführt werden. Der ganze Vorgang sollte mindestens dreimal mit verschiedenen Plasmid-Transformationen wiederholt werden. 2. Das Wachstum von Bakterienkulturen <ol><li> Vorsichtig auftauen FHK12 kompetente Zellen (200 ul) auf Eis und Überführung in eine 15 ml Kultur Röhre. Add Plasmid-DNA (200 ng) und inkubieren Sie die Zellen auf Eis für 30 min. </li><li> Hitze-Schock-Zellen durch Inkubation für 90 s bei 42 ° C, durch Inkubation auf Eis für 2 min folgte. </li><li> Add SOC Media (800 ul) und inkubieren Sie die Proben bei 37 ° C unter Schütteln (300 rpm) für eine h. </li><li> Impfen 5 ml LB-Medium mit Chloramphenicol (30 ug / ml) und Arabinose (0,0025% w / v) mit 50 ul der Transformation Mischung in 15 ml Kulturröhrchen in dreifacher Ausfertigung. Inkubieren Proben bei 37 ° C unter Schütteln (300 rpm) für 20 h. (Alternativ 5 ul der Kultur kann zu 100 ul Medium in einer 96-Well-Platte zu impfen. Diese Methode ist nützlich, wenn es mit einer großen Anzahl von Proben, obwohl Fehler wird etwas höher sein. Um die Verdunstung, die führen würde vermeiden um Fehler, füllen Sie die äußersten Brunnen mit Medien, sondern verwenden sie nicht für die Proben. Schließlich, Doppel-wrap die Verbindung zwischen dem Deckel und die Platte mit Parafilm). </li></ol> 3. Die Messung der β-Galactosidase-Aktivität <ol><li> Heizen Sie den Platten-Leser bis 28 ° C. </li><li> Übertragen Sie die Z-Buffer in ein Reservoir mit einem großen Pipettenspitze, achten Sie darauf, nehmen nur die obere (wässrige Schicht). Transfer 100 ul frisch zubereitet Z-buffer/chloroform in die Vertiefungen einer 96-Well-Platte. Transfer-5 ul jeder Kultur in die Vertiefungen der Platte in vierfacher Ausfertigung. Lassen Sie die Kultur von vier Brunnen, die als leere dienen wird. </li><li> Messen Sie die OD 595 von der Platte auf die Zelldichte zu bestimmen. </li><li> Add 50 ul Z-buffer/SDS in alle Vertiefungen der Platte. Schütteln Sie die Platte in der Platte-Reader für 10 min auf die Zellen zu lysieren. Sicherstellen, dass die Zell-Suspensionen sind nach der Lyse klar und wiederholen das Schütteln Schritt, wenn erforderlich. Unvollständige Lyse schlägt die Z-buffer/chloroform nicht frisch zubereitet. </li><li> Dann werden 50 ul frisch zubereiteten Z-buffer/ONPG in alle Vertiefungen und kehren die Platte auf die Platte Leser und messen OD 405 alle 30 s für 20 min. </li><li> Berechnen Sie β-Galactosidase-Aktivität mit der folgenden Gleichung (wir erinnern uns, um die leeren subtrahieren). Das Verhältnis von OD 405 / min sollte anhand aller Datenpunkte in der OD 405 Bereich von 0,0 bis 1,0 unter Verwendung eines linearen Modells angepasst werden. <img src="files/ftp_upload/2721/2721eq1.jpg" alt="Gleichung 1" /> Miller Einheiten unterscheiden sich manchmal, wenn an verschiedenen Tagen aufgenommen. Deshalb sollte eine Referenz zu konstruieren, wie GpA in jeder Prüfung zu messen. Die Werte können für die Normalisierung der ToxR Werte verwendet werden. </li></ol> 4. Steuerung für die Proteinexpression <ol><li> Führen Western-Blot, auch Protein-Expression von Konstrukten zu überprüfen. Kombinieren Sie 50 ul des dreifachen Kulturen und Zentrifuge (2000 rpm, 4 min) in einer Mikrozentrifuge. Entfernen Sie den Überstand mit einer Pipette resuspendieren und die verbleibende Pellet in 2 x Probe Ladepuffer. </li><li> Last 7,5 ul auf einem Standard 8% Gel und Durchführung Elektrophorese bei 125 V für 1 Stunde 5 min. Nach der Übertragung mit anti-MBP HRP-konjugierten Antikörper inkubiert und zu visualisieren, die chimäre Protein ist bei ca. 70 kDa mit einigen Abbauprodukte manchmal um 48 kDa zu sehen beobachtet. Endogene MBP ist auch bei 45 kDa beobachtet (siehe Abbildung 5). </li></ol>5. Control für Proper Membraninsertion Eine Zelllinie Mangel an Maltose bindendes Protein verwendet wird, um eine ordnungsgemäße Membraninsertion des chimären TMD konstruieren zu beurteilen. Wenn auf Minimalmedium mit Maltose gewachsen als alleinige Kohlenstoff-Quelle, Ausdruck nur Zellen eine Membran-Integral-Ausdruck Produkt mit Maltose-Bindungsprotein richtig in das Periplasma befindet sind in der Lage zu wachsen. <ol><li> Transform PD28-Zellen (wie bei FHK12 Zellen beschrieben) und impfen 2 ml LB-Medium. Wachsen die Zellen bei 37 ° C unter Schütteln (300 rpm) über Nacht. </li><li> Pellet die Zellen durch Zentrifugation bei 3500 rpm, 10 min, 4 ° C und Waschen durch Resuspendieren in PBS (2 ml) durch vorsichtiges Pipettieren mit einer großen Spitze oder sanfte Vortexen. Pellet den Zellen (wie oben), mit PBS waschen ein zweites Mal, Pellet-und schließlich in PBS (1 ml) resuspendieren. </li><li> Verwenden Sie 25 ul der resuspendierten Zellen in 5 ml Minimalmedium in dreifacher Ausfertigung und bei 37 zu impfen ° C unter Schütteln (300 rpm). Nehmen OD 595 Lesungen zwischen 15-25 h, ca. alle 2 Stunden durch die Übertragung von 200 ul von jeder Probe in eine 96-Well-Platte und das Lesen mit dem Platten-Leser. </li></ol> 6. Repräsentative Ergebnisse: Ein Beispiel für die Verwendung des ToxR transkriptionelle Reporter-Assay auf die Oligomerisierung Neigung von Transmembrandomänen in Abbildung 4 zu analysieren. Bisher haben wir die Oligomerisierung von Transmembrandomänen von untersuchten die multispanning Membran-Integral-Protein latent membrane protein-1 (LMP-1) durch verschiedene Techniken, darunter ToxR 14 Transmembrandomäne fünf (TM5) wurde gezeigt, dass eine starke Neigung zur Oligomerisierung aufweisen.; Diese zeichnet sich durch hohe Miller Units, vergleichbar mit der positiven Kontrolle, GPA, eine gut etablierte Dimerisierung Sequenz gezeigt. Eine Mutation in TM5, D150A, reduziert die Fähigkeit der Sequenz oligomerisieren. LMP-1 TM1 nicht wesentlich oligomerisieren und weist einen sehr niedrigen Miller Einheit Signal, gerade über das Signal für leere, nicht-transformierten Zellen FHK12. <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Cartoon Darstellung der ToxR Reporter-Assay. Transmembrandomäne (TMD) angetrieben Oligomerisierung Ergebnisse in Dimerisierung von ToxR und Aktivierung von LacZ Transkription. Das Genprodukt von LacZ, können β-Galactosidase als Maß für die Neigung einer TMD zu oligomerisieren quantifiziert werden. <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Die hydrolytische Spaltung von ONPG von β-Galactosidase Ergebnisse in die Produktion von dem Licht absorbierenden Spezies o-nitrophenolat (ONP). <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Plasmidkarte von pToxR7. <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Representative ToxR transkriptionelle Reporter-Assay Analyse der Oligomerisierung Neigung der latent membrane protein-1 Transmembrandomänen. Transmembrandomäne 5 (TM5) oligomerisiert stark, während Transmembrandomäne 1 (TM1) zeigt nur eine schwache Wechselwirkung. Mutation D150A in TM5 reduziert seine Fähigkeit zur Oligomerisierung. GPA ist als eine positive Kontrolle Sequenz für starke Dimerisierung enthalten. Blank stellt transformierten FHK12 Zellen. <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Western Blot für die Proteinexpression. <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="Abbildung 6" /> Abbildung 6. PD28 Komplementation Test auf korrekte Membraninsertion in das Periplasma zu kontrollieren. Negative Kontrolle stellt ein Konstrukt Mangel an Maltose bindendes Protein.

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Die ToxR transkriptionelle Reporter-Assay ist eine einfache Möglichkeit, transmembrane Sequenzen mit dem Potenzial, oligomerisieren identifizieren. Da die Wechselwirkungen innerhalb der bakteriellen inneren Membran stattfindet, umgeht diesen Test die Probleme mit der Gültigkeit der Untersuchung von Systemen in Membran-Mimetikum-Umgebungen. Da das Klonen von mehreren TMDs in einem einzigen Plasmid leicht können parallel ausgeführt werden und der gesamte Test kann in 96-Well-Platte-Format durchgeführt werden, kann di...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> BamHI Restriktionsenzym </td><td> Invitrogen </td><td> 15201023 </td><td> Invitrogen Enzyme erwiesen sich als effizienter als alternative Lieferanten </td></tr><tr><td> NheI Restriktionsenzym </td><td> Invitrogen </td><td> 15444011 </td><td> Invitrogen Enzyme erwiesen sich als effizienter als alternative Lieferanten </td></tr><tr><td> 15 mL Kulturröhrchen </td><td> Fisher Scientific </td><td> 14 bis 956-1J </td><td></td></tr><tr><td> SOC-Medium </td><td> Teknova </td><td> S0225 </td><td> Bis das entsprechende Volumen auf und sterilisiert durch Autoklavieren. </td></tr><tr><td> LB-Medium </td><td> Sigma-Aldrich </td><td> L7275 </td><td> Bis das entsprechende Volumen auf und sterilisiert durch Autoklavieren. </td></tr><tr><td> Chloramphenicol </td><td> Sigma-Aldrich </td><td> CO 3 78 </td><td> Stammlösung von 30 mg / mL in Ethanol in Gefrierschrank gelagert </td></tr><tr><td> Arabinose </td><td> Fluka </td><td> 10839 </td><td> Stammlösung von 2,5% (w / v) in Wasser im Gefrierschrank gelagert </td></tr><tr><td> Na 2 HPO 4 </td><td> Sigma-Aldrich </td><td> S9390 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> Sigma-Aldrich </td><td> S9638 </td><td></td></tr><tr><td> KCl </td><td> Mallinckrodt Chemicals </td><td> 6858-06 </td><td></td></tr><tr><td> MgSO 4 .7 H 2 O </td><td> Sigma-Aldrich </td><td> 63138 </td><td></td></tr><tr><td> Natriumdodecylsulfat (SDS) </td><td> Sigma-Aldrich </td><td> L6026 </td><td></td></tr><tr><td> 2-Nitrophenyl β-D-galactopyranosid (ONPG) </td><td> Sigma-Aldrich </td><td> 73660 </td><td></td></tr><tr><td> Z-Puffer </td><td></td><td></td><td> 16,1 g Na 2 HPO 4 5.5g NaH 2 PO 4 0,75 g KCl 0.246g MgSO 4 Machen Sie bis zu 1 l, pH 7,0 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 mL β-Mercaptoethanol, 2 mL Chloroform, machen bis zu 20 ml mit Z-Buffer. Vortex für 1 min, Zentrifuge für 1 min bei 800 Umdrehungen pro Minute. Machen Sie für jede Platte frisch. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 mg SDS in 10 mL Z-Puffer </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40 mg ONPG in 10 mL Z-Buffer. Machen Sie für jede Platte frischer </td></tr><tr><td> β-Mercaptoethanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Anti-MBP monoklonalen Antikörper (HRP-konjugiert) </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td> Minimal-Medien mit Maltose </td><td></td><td></td><td> 1 x M9-Salze, 0,4% Maltose, 1 mg / mL Thiamin, 2 mM MgSO 4 </td></tr><tr><td> 96-well Flachboden Platte </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> Plate-Reader </td><td> Beckman Coulter </td><td> DTX880 Multimode-Detektor </td><td></td></tr><tr><td> Wasserbad </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> Schüttelinkubator </td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

Die vereinfachte Sicht von Protein Transmembrandomänen lediglich als Anker in Phospholipid-Doppelschichten ist längst widerlegt. In vielen Fällen membrandurchspannende Proteine ​​hochentwickelten Wirkmechanismen entwickelt. 1-3 Ein Weg in die Membranproteine ​​modulieren ihre Strukturen und Funktionen können durch direkten und konkreten Kontakt der hydrophoben Helices bilden strukturierte transmembrane Oligomere. 4,5 Großteil der jüngsten Arbeit über die Verteilung der Aminosäuren bevorzugt in der Membran-Umgebung im Vergleich zu wässrigen Lösung und die verschiedenen intermolekularen Kräfte, die Fahrt Protein-Assoziation. 6,7 Trotzdem Studien der molekularen Erkennung an der Transmembrandomäne von Proteinen immer noch hinter denen der Schwerpunkt gefunden wasserlöslichen Regionen. Eine große Hürde bleibt: Trotz der bemerkenswerten Spezifität und Affinität, die transmembrane Oligomerisierung erreichen können, 8 direkte Messung ihrer Vereinigung ist eine Herausforderung. Traditionelle Methoden angewendet, um das Studium integrale Membranprotein-Funktion kann durch die inhärenten Unlöslichkeit der Sequenzen unter Prüfung behindert werden. Biophysikalische Erkenntnisse aus dem Studium synthetischer Peptide, die Transmembrandomänen gewonnen werden können nützliche strukturelle Einsicht. Allerdings ist die biologische Relevanz des Waschmittels mizellaren oder Liposomen-Systeme in diesen Studien verwendet werden, um Zellmembranen imitieren oft in Frage gestellt; tun Peptide Annahme eines nativen Struktur unter diesen Bedingungen und hat ihre funktionale Verhalten wirklich die Wirkungsweise in einer nativen Membran ? Um die Wechselwirkungen von Transmembran-Sequenzen in natürlichen Phospholipid-Doppelschichten Studie entwickelten die Langosch Labor ToxR transkriptionelle Reporter-Assays. 9 Die Transmembrandomäne von Interesse, wie eines chimären Proteins mit Maltose-Bindungsprotein für die Lage auf dem Periplasma und ToxR ausgedrückt wird, um einen Bericht zu der Grad der Oligomerisierung (Abbildung 1). In den letzten zehn Jahren mehrere andere Gruppen (zB Engelman, DeGrado, Shai) weiter optimiert und wandte diese ToxR Reporter-Assay. 10-13 Die verschiedenen ToxR Assays haben sich zu einem Goldstandard, um Protein-Protein-Interaktionen in Zellmembranen zu testen. Wir hier zeigen einen typischen experimentellen Betrieb in unserem Labor, die vor allem folgende Protokolle, die von Langosch entwickelt durchgeführt. Diese allgemein anwendbare Methode für die Analyse der Transmembrandomäne Selbstassoziation in E. sinnvoll coli, wo β-Galaktosidase Produktion verwendet werden, um die TMD Oligomerisierung Neigung zu beurteilen ist. Bei TMD-induzierte Dimerisierung bindet ToxR die ctx-Promotor verursacht up-Regulierung des LacZ Gen für β-Galactosidase. Eine kolorimetrische Ablesung wird durch Zugabe von ONPG zu lysierten Zellen erhalten. Hydrolytische Spaltung von ONPG von β-Galactosidase Ergebnisse in die Produktion von dem Licht absorbierenden Spezies o-nitrophenolat (ONP) (Abbildung 2).

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

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Ein effizientes Verfahren zur Oligomerisierung Neigung der Single-Pass-Transmembrandomänen (TMD) zu beurteilen ist beschrieben. Chimäre Proteine, bestehend aus dem TMD fusioniert ToxR sind in einer E. coli-Stamm-Reporter zum Ausdruck gebracht. TMD-induzierte Oligomerisierung Ursachen Dimerisierung von ToxR, Aktivierung der Transkription und Produktion von der Reporter-Proteins,-Galactosidase.

Transmembrandomäne Oligomerisierung Anschaffungsneigung durch ToxR Assay ermittelt

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

transmembrane-domain-oligomerization-propensity-determined-toxr

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JoVE Journal

Biology

15.1K Aufrufe.  University of Colorado at Boulder. Ein effizientes Verfahren zur Oligomerisierung Neigung der Single-Pass-Transmembrandomänen (TMD) zu beurteilen ist beschrieben. Chimäre Proteine, bestehend aus dem TMD fusioniert ToxR sind in einer E. coli-Stamm-Reporter zum Ausdruck gebracht. TMD-induzierte Oligomerisierung Ursachen Dimerisierung von ToxR, Aktivierung der Transkription und Produktion von der Reporter-Proteins,-Galactosidase.

Serie: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

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

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

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

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

Expression und Reinigung des Cystic Fibrosis Transmembrane Conductance Regulatorprotein in Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Forschung

Biologie

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

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

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

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Die Reinigung des Cystic Fibrosis Transmembran Regulator-Protein in Ausgedrückt<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

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

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

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

Charakterisierung von G-Protein gekoppelten Rezeptoren durch eine Fluoreszenz-basierte Calciummobilisierungsassay

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

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

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

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

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

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

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

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

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

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

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

Genomweite Protein-Protein-Wechselwirkungen durch Screening Protein-Fragment Komplementierungsassay (PKA) in lebenden Zellen

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

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

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

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

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 ...

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

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

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

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

Optische Kartierung der Intra-Retikulum Ca<sup&gt; 2+</sup&gt; Und Transmembranpotential in der Langendorff-perfundierten Kaninchen Herz

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

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

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

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

Fecal Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in Equids

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

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

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

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

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

Genetische und biochemische Ansätze für<em&gt; In Vivo</em&gt; und<em&gt; In Vitro</em&gt; Beurteilung von Protein Oligomerisierung: Der Ryanodinrezeptor Case Study

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

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

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

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

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

Studie des endoplasmatischen Reticulum und Mitochondria Wechselwirkungen durch<em&gt; In Situ</em&gt; Nähe Ligatur Assay in fixierten Zellen

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