Wir danken dem Mitarbeiter im Institut für Humangenetik für NGS Bibliothek Vorbereitung und Sequenzierung. Wir danken für ihre Expertise bei der Vorbereitung genomische Bibliothek Fragmente für die Y2H-Plasmid-Bibliothek hier Einat Snir. Diese Arbeit wurde vom National Institutes of Health unterstützt: NIH R21 EB021870-01A1 und durch NSF Research Project Grant: 1517110.

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Protein-Protein+Interactome+Networks+Using+Yeast+Two-Hybrid+Screens.">Mapping the Protein-Protein Interactome Networks Using Yeast Two-Hybrid Screens.</a> Advances in Experimental Medicine and Biology. 883, 187-214 (2015).</li><li>Weimann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Y2H-seq+approach+defines+the+human+protein+methyltransferase+interactome.">A Y2H-seq approach defines the human protein methyltransferase interactome.</a> Nature Methods. 10 (4), 339-342 (2013).</li><li>Yachie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pooled-matrix+protein+interaction+screens+using+Barcode+Fusion+Genetics.">Pooled-matrix protein interaction screens using Barcode Fusion Genetics.</a> Molecular Systems Biology. 12 (4), 863 (2016).</li><li>Trigg, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrY2H-seq:+a+massively+multiplexed+assay+for+deep-coverage+interactome+mapping.">CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.</a> Nature Methods. , (2017).</li><li>Estojak, J., Brent, R., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+two-hybrid+affinity+data+with+in+vitro+measurements.">Correlation of two-hybrid affinity data with in vitro measurements.</a> Molecular and Cellular Biology. 15 (10), 5820-5829 (1995).</li><li>Rajagopala, S. V., Hughes, K. T., Uetz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+yeast+two-hybrid+systems+using+the+interactions+of+bacterial+motility+proteins.">Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins.</a> Proteomics. 9 (23), 5296-5302 (2009).</li><li>James, P., Halladay, J., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+libraries+and+a+host+strain+designed+for+highly+efficient+two-hybrid+selection+in+yeast.">Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.</a> Genetics. 144 (4), 1425-1436 (1996).</li><li>Barbas, C. F., Burton, D. R., Scott, J. K., Silverman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+DNA+and+RNA.">Quantitation of DNA and RNA.</a> CSH Protoc. 2007, pdb ip47 (2007).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS-Polyacrylamide+Gel+Electrophoresis+of+Proteins.">SDS-Polyacrylamide Gel Electrophoresis of Proteins.</a> Cold Spring Harbor Protocols. 2006 (4), (2006).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences USA. 76 (9), 4350-4354 (1979).</li><li>Alegria-Schaffer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting+using+chemiluminescent+substrates.">Western blotting using chemiluminescent substrates.</a> Methods in Enzymology. 541, 251-259 (2014).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of Visualized Experiments. (62), (2012).</li><li>Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p21+Cdk-interacting+protein+Cip1+is+a+potent+inhibitor+of+G1+cyclin-dependent+kinases.">The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.</a> Cell. 75 (4), 805-816 (1993).</li></ol>

Die Autoren haben nichts preisgeben

Hier bieten wir Ihnen eine Anleitung für Y2H-Assays im Batch mit optimierten Methoden durchführen. Es gibt ein paar wichtige Schritte im Verfahren um sicherzustellen, dass die Bevölkerung von Hefe, die Auswahl unterstellt würde Vertreter der Start-Bibliothek ist, genug der Ausgangspunkt Hefe Bevölkerung dient zur Auswahl um Variabilität zu begrenzen zu unterziehen . Diese Benchmarks sind relativ leicht zu erreichen, neben der Anpassung der Methoden und Materialien für einen traditionellen Y2H-Assay, wodurch dieser Ansatz für die meisten Labors ausgestattet für standard Molekularbiologie zugänglich. DEEPN erlaubt die Auswahl aus der gleichen Beute Bibliothek Bevölkerung während der Verwendung verschiedener Köder Plasmide. Daher kann der Satz der interagierenden Kandidaten, die eine Y2H Interaktion mit einem Köder vs. andere produzieren direkt verglichen werden. Da Tiefe Sequenzierung verwendet wird, kann die Start Bibliothek Komposition für jeden Köder-spezifische Hefe Bevölkerung zu überprüfen und folgen die Bereicherung jeder Kandidat Beute Gens in der Bibliothek unabhängig. Batch-Verarbeitung ermöglicht Abfragen derselben Bibliothek gegen verschiedene Köder in einer semi-quantitative Weise, die dann erlaubt, eine statistische Rang4zu berechnen.
Es gibt mehrere Schritte in diesem Protokoll, die entscheidend für den Erfolg sind. Ist, dass eine große Anzahl von diploiden eingeholt werden muss, um sicherzustellen, dass angemessene Vertretung der Y2H Beute Bibliothek mit jeder Köder Plasmid von Interesse gemischt wird. Die Paarung hier beschriebene Vorgehensweise wurde durch Variation verschiedener Parameter optimiert. Wir fanden, dass einige Stämme, die Haus kommerzielle Y2H Bibliotheken aber schlecht im allgemeinen Paaren, die Paarung hier beschriebene Vorgehensweise 2 x 106- 2 x 107 diploiden als folgte, generieren kann angemessene und reproduzierbare Übertragung der der Y2H ermöglichen Bibliothek in der Bevölkerung. Ein weiterer kritischer Aspekt des Verfahrens soll sicherstellen, dass die Köder-Plasmid des Interesses keine positive Y2H-Interaktion auf eigene schaffen oder mit leeren Gal4-Aktivierungsdomäne Plasmid Beute. Die Methode zur Auswahl einer Y2H-Interaktion ist Wachstum in Ermangelung von Histidin, wobei eine positive Interaktion Y2H Transkription von HIS3 induziert damit Zellen werden, anspruchsvoll sein +. Während routinemäßig die traditionellen Y2H-Assays kompetitiver Inhibitor 3AT hinzufügen können, um Hintergrund Wachstum verringern oder verwenden andere Reporter3 , das Verfahren funktioniert am besten, wenn Zellen mit schwachen Y2H Interaktionen wachsen können und wodurch sich die Stringenz für Wachstum und die Auswahl nur für Zellen mit starken Y2H Interaktionen Grenzen des Repertoires der Beute Plasmide, die identifiziert werden kann. Ein weiterer wichtiger Aspekt ist es, sicherzustellen, dass eine große Menge an Start diploiden Bevölkerung verwendet, selektive und nicht-selektiven Bedingungen, evolutionäre Engpässe von sampling Error4 zu vermeiden zu wachsen. Im Anschluss an die Kultur Volumen und Zelle hilft hier die angegebenen Nummern Reproduzierbarkeit zu gewährleisten und Lärm zu vermindern.
Einer der Gründe ist der DEEPN Ansatz mächtig ist, dass es umfassend alle Plasmide in einer bestimmten Beute-Bibliothek für ihre Fähigkeit zur Interaktion mit den Köder von Interesse folgen kann. So ist eine Einschränkung des DEEPN die Komplexität der Bibliothek verwendet. Für einige der kommerziellen Bibliotheken wie wir hier, wir fanden, dass die Anzahl der Plasmide, die bona-fide Fragmente von cDNA kodieren ORF, die in der gleichen Leseraster des Gal4 Aktivierungsdomäne zwischen 3 und 6 x 10 ist4 mit Darstellung der ~ 6.000 - 8,00 0 verschiedene Gene. Wir fanden, dass fast 75 % der diese Bibliotheken enthaltenen Fragmente cDNA-Regionen, die 3' die kodierenden DNA-Sequenz (CDS/ORF) wurden ausschließlich entspricht. Darüber hinaus hatte fast ein Drittel der Gene die Fragmente in der Bibliothek hatte keine, die Regionen im ORF oder oberhalb des ORF entsprach.
Wir haben drei andere Änderungen der DEEPN-Methode. Ein neuer MATalpha-Stamm, der tragen kann "Beute" Bibliotheken in ein Plasmid TRP1 und die Kumpels weit besser als manche handelsüblichen Bibliothek-haltige Stämme wie Y187 untergebracht ist. Dadurch wird gewährleistet vollständige Übertragung der Bibliothek Bevölkerung für jeden Köder von Interesse. Wir haben auch eine neue "Köder" Ausdruck Plasmid, das unterscheidet sich von der zuvor beschriebenen Plasmide, die eine Variable Kopie 2µ-basierten Backbone10verwenden. Hier beschreiben wir eine Low-Kopie CEN Plasmid (pTEF-GBD) und finden, die eine einzelne Runde selektives Wachstum ist ausreichend für interagierenden Beute Plasmide zu bereichern. Zu guter Letzt wir bauen einen stromlinienförmigen "Beute" Bibliothek Vektor und zur Herstellung einer High-Density-random-Fragment genomic Y2H Bibliothek für Saccharomces Cerevisiae, was in der Zukunft für die Entdeckung und Charakterisierung von Interaktionen Amonst hilfreich sein sollte Hefe-Proteinen. Insgesamt machen die Verbesserungen bei Materialien und Bioinformatik (beschrieben in der begleitenden Arbeit) die DEEPN eine zugänglich, praktikable und effiziente Möglichkeit für die Durchführung umfassender und vergleichende Y2H-Bildschirme zu nähern.

Ein vollständiges Verständnis der biologischen Zellprozesse stützt sich auf die Suche nach der Protein-Interaktion-Netzwerke, die ihre molekularen Mechanismen zugrunde liegen. Ein Ansatz zur Protein-Interaktionen zu identifizieren ist die Hefe 2-Hybrid (Y2H) Assay, die Arbeiten durch einen funktionierenden Chimären Transkriptionsfaktor zusammenfügen, sobald zwei Protein Domänen von Interesse an einander1binden. Ein typische Y2H-Bildschirm erfolgt durch die Schaffung einer Bevölkerung von Hefe, die beide eine Bibliothek von Plasmiden Codierung interagierenden Proteine verschmolzen zu einem transcriptional Aktivator beherbergt (zB., "Beute" Schmelzverfahren Protein) und ein bestimmtes "Köder" Plasmid bestehend aus dem Protein von Interesse verschmolzen zu einem DNA-bindende Domäne (z.B. die Gal4 DNA-bindende Domäne, die an die Gal4-upstream aktivierende Sequenz bindet). Einer der Hauptvorteile des Y2H-Ansatzes ist, dass es relativ einfach ist und kostengünstig durchzuführen in einem typischen Labor für Routinearbeiten molekularen biologischen2 ausgestattet. Jedoch wenn traditionell durchgeführt, Proben ein Benutzer einzelne Kolonien, die bei der Auswahl für eine positive Y2H-Interaktion entstehen. Dies schränkt die Anzahl der Bibliothek "Beute"-Klone, die befragt werden können. Dieses Problem wird verstärkt, wenn die Fülle an eine bestimmte interagierenden Beute sehr hoch im Vergleich zu anderen, verringern die Chance Interaktion von niedrigen Fülle Beute Plasmide zu erkennen ist.
Eine Lösung für die Verwendung der Y2H-Prinzip in umfassende Abdeckung des Proteoms ist die Verwendung eines Matrix-formatierte Ansatzes, wobei ein Array mit bekannten individuelle Beute Plasmide Digital abgefragt werden kann. Ein solches Vorgehen erfordert jedoch eine Infrastruktur, die nicht ohne weiteres zugänglich oder kostengünstig an die einzelnen Ermittler ist, die bei der Festlegung der Interaktom einer kleinen Anzahl von Proteinen oder Domänen3interessiert sind. Darüber hinaus würde sehr komplexe Beute-Bibliotheken, die mehrere Fragmente von interagierenden Proteine kodieren können solche Matrix Arrays zu unpraktisch Größen vergrößern. Eine Alternative ist Assays mit komplexen Bibliotheken in Chargen und das Vorhandensein von interagierenden Klone mit massiv parallelen Hochdurchsatz-Sequenzierung4zu bewerten. Dies kann angewendet werden, um das Vorhandensein von Beute Plasmide assay, die in mehrere Kolonien mit einem typischen entstehen Y2H formatiert Ansatz in der Hefe Zellen Gehäuse interagierenden Pair von Fusionsproteinen dürfen auf einer Platte5,6wachsen. Diese allgemeine Idee kann akzentuiert werden, zur Steigerung der Abfrage der beiden mehrere Köder und Komponenten in der gleichen Zeit7,8zum Opfer.
Dennoch, viele Untersuchungen erfordern, dass ein einfacher noch mehr Aufwand auf nur wenige Protein "Köder" konzentriert und profitieren mehr von einer erschöpfenden und semi-quantitativen Abfrage einer einzigen komplexen Beute-Bibliothek. Wir haben entwickelt und validiert ein Konzept für die breit angelegte Protein Wechselwirkungsstudien mit einem Y2H-Prinzip in Batch Format4durchführen. Dies nutzt das Wachstumstempo von einem bestimmten Beute Plasmid als Proxy für die relative Stärke der Y2H Interaktion9. Tiefe Sequenzierung aller Plasmide innerhalb einer Population normales Wachstum ausgesetzt oder selektive Wachstumsbedingungen produziert eine vollständige Karte der Klone, die starke und schwache Y2H Interaktionen ergeben. Das Repertoire der interagierenden werden erhalten und direkt über mehrere Köder Plasmide verglichen. Die resultierende Workflow bezeichnet DEEPN (dynamische Bereicherung für Evaluation of Protein Networks) kann so zum differenziellen Interactomes aus den gleichen Beute Bibliotheken, Proteine, Vergleich zwischen ein Protein vs. anderen zu identifizieren identifizieren.
Hier zeigen wir DEEPN und Verbesserungen in die Labormethoden, die seine Verwendung zu erleichtern, die in Abbildung 1aufgeführt sind. Signifikante Verbesserungen umfassen:
Generation der Beute Hefe Bevölkerung. Eine der wichtigsten Voraussetzungen der DEEPN erzeugt Populationen von Hefe mit verschiedenen Köder Plasmide, die die gleiche Verteilung der Plasmid-Beute-Bibliotheken haben. Gleichwertige Grundlinie Populationen der Beute Plasmid Bibliothek sind essentiell für genaue Vergleiche zwischen den Interactomes von unterschiedlichen Ködern. Dies geschieht am besten, wenn ein Bibliothek-Plasmid sich bereits in einer Population von haploiden Hefe befindet und Umzug eines bestimmten Köder-Plasmid in dieser Population erreicht wird, indem die Paarung um einen diploiden zu produzieren. Hier bieten wir einen klaren Leitfaden wie man solche Populationen mit kommerziellen Bibliotheken in haploiden Hefe untergebracht. Obwohl wir Methoden, die eine hohe Anzahl von diploiden zu erzeugen gefunden, war die Paarung Gesamteffizienz des diese kommerzielle Bibliothek-haltigen Hefestämme gering. Daher haben wir eine neue Sorte, die Beute Bibliotheken aufnehmen kann, die weit mehr diploiden pro Paarung Reaktion ergibt.
New set Köder Plasmide. Viele aktuelle Plasmide, die "Köder" Schmelzverfahren Proteine enthalten des Proteins des Interesses und eine DNA-bindende Domäne Ausdrücken basieren auf 2µ, so dass sie ihre Kopienzahl verstärken. Diese Kopienzahl kann sehr variabel in der Bevölkerung und führen zu Schwankungen in der Y2H transcriptional Antwort. Dies könnte wiederum die Fähigkeit zu beurteilen, die Stärke einer bestimmten Protein Interaktion anhand der Antwort Wachstum von Zellen unter Auswahl verzerren. Dies kann teilweise Adresse sein, mit einem niedrigen Kopie Plasmid, von denen einige wie die im Handel erhältlichen pDEST3210zuvor beschrieben wurden. Wir konstruierten eine neue Köder-Plasmid (pTEF-GBD), das produziert Gal4 DNA-bindende Domäne Fusionsproteine innerhalb einer TRP1 Zentromer-basierte niedrigen Kopie Plasmid trägt die Kanr-Resistenz-Gen, die auch ermöglicht, Klonen von Köder Fragmente sowohl stromaufwärts und unterhalb des Gal4 DNA-bindende Domäne.
Neue High-Density Y2H fragmentbibliothek. Wir ein neues Plasmid, Haus Y2H Beute Bibliotheken gebaut und benutzte es, um eine hochkomplexe Y2H-Bibliothek nach dem Zufallsprinzip geschert Bruchstücke von genomischer DNA aus Saccharomyces Cerevisiaegemacht zu bauen. Sequenzanalyse zeigte, dass diese Bibliothek mehr als 1 Million verschiedene Elemente, die weitaus komplexer als die zuvor beschriebenen Hefe genomische Y2H Plasmid Bibliotheken11. Mit dieser neuen Bibliothek konnten wir zeigen, dass der DEEPN-Workflow robust genug ist, um komplexe Bibliotheken mit vielen unterschiedlichen Plasmide in einer Art und Weise aufzunehmen, die zuverlässig und reproduzierbar ist.

<table>
	<tbody>
		<tr>
			<td>Illumina HiSeq 4000</td>
			<td>Illumina</td>
			<td></td>
			<td>deep sequencing platform</td>
		</tr>
		<tr>
			<td>Monoclonal anti-HA antibodies</td>
			<td>Biolegend</td>
			<td>901514</td>
			<td>Primary Antibody to detect expression of HA in pGal4AD constructs</td>
		</tr>
		<tr>
			<td>Polyclonal anti-myc antibodies</td>
			<td>QED Biosciences Inc</td>
			<td>18826</td>
			<td>Primary Antibody to detect expression of MYC in pTEF-GBD constructs</td>
		</tr>
		<tr>
			<td>NarI</td>
			<td>New England BioLabs</td>
			<td>R0191S</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF</td>
			<td>New England BioLabs</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI-HF</td>
			<td>New England BioLabs</td>
			<td>R3236S</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>New England BioLabs</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 3350, powder</td>
			<td>J.T. Baker</td>
			<td>U2211-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Trevigen, Inc sold by Fisher Scientific</td>
			<td>50-948-286</td>
			<td>carrier DNA for yeast transformation section 3.2.1.</td>
		</tr>
		<tr>
			<td>Kanamycin Monosulfate</td>
			<td>Research Products International</td>
			<td>K22000</td>
			<td></td>
		</tr>
		<tr>
			<td>LE Agarose</td>
			<td>GeneMate</td>
			<td>E-3120-500</td>
			<td>used for making DNA agarose gels</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Research Products International</td>
			<td>S23025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Research Products International</td>
			<td>T60060</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Research Products International</td>
			<td>S23080</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate Dihydrate</td>
			<td>MP Biomedicals</td>
			<td>155256</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>ThermoFisher</td>
			<td>C79</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Sodium Salt</td>
			<td>Research Products International</td>
			<td>E57020</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Powder</td>
			<td>Research Products International</td>
			<td>Y20020</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base (ammonium sulfate) w/o amino acids</td>
			<td>Research Products International</td>
			<td>Y20040</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Leu+40ADE</td>
			<td>Formedium</td>
			<td>DCS0789</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Leu-His+40ADE</td>
			<td>Formedium</td>
			<td>DCS1169</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Leu-Met</td>
			<td>Formedium</td>
			<td>DCS0549</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Met</td>
			<td>Bio 101, Inc</td>
			<td>4520-922</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Formedium</td>
			<td>DOC0168</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Research Products International</td>
			<td>A11500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Research Products International</td>
			<td>G32045</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>used for making media plates in section 1</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Research Products International</td>
			<td>P20240</td>
			<td></td>
		</tr>
		<tr>
			<td>3-amino-1,2,4 Triazole</td>
			<td>Sigma</td>
			<td>A8056</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoehanol (BME)</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 100T</td>
			<td>USBiological</td>
			<td>Z1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma</td>
			<td>P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:IAA</td>
			<td>Ambion</td>
			<td>AM9732</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>238074</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories, INC</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse A</td>
			<td>ThermoFisher</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Research Products International</td>
			<td>U20200</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Research Products International</td>
			<td>L22010</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Gibco</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Amresco</td>
			<td>449</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson Assembly Cloning Kit</td>
			<td>New England Biolabs</td>
			<td>E5510S</td>
			<td>Rapid assembly method for cloning of plasmids in section 2</td>
		</tr>
		<tr>
			<td>NEBNext High-Fidelity 2x PCR Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0541S</td>
			<td>Used for amplification of products for Gibson Assembly in Section 2.3 as well assample preparation for DEEPN deep sequencing in section 6.2.1</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Amresco</td>
			<td>0492-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Used for purification of pcr products in section 6.2.3</td>
		</tr>
		<tr>
			<td>Qiaquick DNA Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Used for purification of digested pTEF-GBD in section 2.1</td>
		</tr>
		<tr>
			<td>KAPA Hyper Prep kit</td>
			<td>KAPA Biosystems</td>
			<td>KK8500</td>
			<td>preparation kit for deep sequencing</td>
		</tr>
		<tr>
			<td>Codon optimization</td>
			<td>http://www.jcat.de</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Codon optimization</td>
			<td>https://www.idtdna.com/CodonOpt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gBlocks</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>DNA fragments used for cloning in Section 2.2</td>
		</tr>
		<tr>
			<td>Strings</td>
			<td>Thermofisher</td>
			<td></td>
			<td>DNA fragments used for cloning in Section 2.2</td>
		</tr>
		<tr>
			<td>GenCatch Plasmid DNA mini-prep Kit</td>
			<td>EPOCH Life Sciences</td>
			<td></td>
			<td>Used to prepare quantities of DNA in Section 2.3</td>
		</tr>
		<tr>
			<td>Covaris E220</td>
			<td>Covaris</td>
			<td></td>
			<td>high performance ultra-sonicator in section 7</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;- CGGTCTT 
			CAATTTCTCAAGTTTCAG -3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 5&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-GAGTAACG 
			ACATTCCCAGTTGTTC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 5&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-CACCGTAT 
			TTCTGCCACCTCTTCC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 3&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-GCAACCGC 
			ACTATTTGGAGCGCTG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 3&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligonucleotide 5&#8217;-GTTCCGATG 
			CCTCTGCGAGTG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>5&#39; Pimer used for insert amplification of pGAL4AD</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;-GCACATGCT 
			AGCGTCAAATACC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>3&#39; Pimer used for insert amplification of pGAL4AD</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;-ACCCAAGCA 
			GTGGTATCAACG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>5&#39; Pimer used for insert amplification of pGADT7</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;- TATTTAGA 
			AGTGTCAACAACGTA -3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>3&#39; Pimer used for insert amplification of pGADT7</td>
		</tr>
		<tr>
			<td>PJ69-4A MatA yeast strain</td>
			<td>http://depts.washington.edu/yeastrc/ James P, Halladay J, Craig EA: Genomic Libraries and a host strain designed for highly efficient two-hybrid selection in yeast. GENETICS 1996 144:1425-1436</td>
			<td></td>
			<td>MATA leu2-3,112 ura3-52 trp1-901 his3-200 gal4D, gal80D, GAL-ADE2 lys2::GAL1-HIS3 met2::GAL7</td>
		</tr>
		<tr>
			<td>pTEF-GBD</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>Gal4-DNA binding doimain expression plasmid</td>
		</tr>
		<tr>
			<td>PLY5725 MATalpha yeast strain</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>MATalpha his3&#8710;1 leu2&#8710;0 lys2&#8710;0 ura3&#8710;0 gal4&#8710; trp1&#8710; Gal80&#8710;</td>
		</tr>
		<tr>
			<td>pGal4AD (pPL6343)</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>Gal4-activation domain expression plasmid</td>
		</tr>
		<tr>
			<td>100 mm petri dishes</td>
			<td>Kord-Vallmark sold by VWR</td>
			<td>2900</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63270</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63271</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63274</td>
			<td></td>
		</tr>
		<tr>
			<td>2,000 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63275</td>
			<td></td>
		</tr>
		<tr>
			<td>20 X 150 mm Disposable Culture Tube</td>
			<td>Thermofisher</td>
			<td>14-961-33</td>
			<td></td>
		</tr>
		<tr>
			<td>pipet-aid</td>
			<td>Drummond</td>
			<td>4-000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7127</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7128</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7129</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Griffin Beaker</td>
			<td>Fisher Scientific</td>
			<td>02-540P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Reuasable Media Storage Bottle</td>
			<td>Fisher Scientific</td>
			<td>06-414-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL graduated cylinder</td>
			<td>Fisher Scientific</td>
			<td>08-572-6G</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax 190</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>used to measure the Optical Density of cells</td>
		</tr>
		<tr>
			<td>NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>Spectrophotometer used to quantify amount of DNA</td>
		</tr>
		<tr>
			<td>Electronic UV transilluminator</td>
			<td>Ultra Lum</td>
			<td>MEB 20</td>
			<td>used to visualize DNA in an Ethidium Bromide agarose gel</td>
		</tr>
		<tr>
			<td>P1000 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123601G</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123600G</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F144802G</td>
			<td></td>
		</tr>
		<tr>
			<td>1250 &#181;L Low Retention Pipette Tips</td>
			<td>GeneMate</td>
			<td>P-1236-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;LLow Retention Pipette Tips</td>
			<td>VWR</td>
			<td>10017-044</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L XL Low Retention Pipette Tips</td>
			<td>VWR</td>
			<td>10017-042</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>VWR</td>
			<td>490001-627</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>VWR</td>
			<td>490001-621</td>
			<td></td>
		</tr>
		<tr>
			<td>cell scraper</td>
			<td>Denville Scientific</td>
			<td>TC9310</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fluka Analytical</td>
			<td>318949-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>J.T. Baker</td>
			<td>5674-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden applicators</td>
			<td>Solon Care</td>
			<td>55900</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge 5424</td>
			<td>Fisher Scientific</td>
			<td>05-400-005</td>
			<td>microcentrifuge</td>
		</tr>
		<tr>
			<td>Sorvall ST16R</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004381</td>
			<td>benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey)</td>
			<td>GE Healthcare</td>
			<td>NA934-1ML</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep)</td>
			<td>GE Healthcare</td>
			<td>NA931-1ML</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34080</td>
			<td>ECL detection solution</td>
		</tr>
		<tr>
			<td>Isotemp Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Incubator</td>
		</tr>
		<tr>
			<td>Mutitron 2</td>
			<td>INFORS HT</td>
			<td></td>
			<td>Shaking incubator</td>
		</tr>
		<tr>
			<td>Isotemp Digital-Control Water Bath Model 205</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>water bath</td>
		</tr>
		<tr>
			<td>Y2H mouse cDNA library in Y187 (pan tissue)</td>
			<td>Clontech</td>
			<td>630482</td>
			<td>commercially available cDNA Library</td>
		</tr>
		<tr>
			<td>Y2H mouse cDNA library in Y187 (mouse brain)</td>
			<td>Clontech</td>
			<td>630488</td>
			<td>commercially available cDNA Library</td>
		</tr>
		<tr>
			<td>pGADT7 AD Vector</td>
			<td>Clontech</td>
			<td>630442</td>
			<td>commercially available AD Vector housing many cDNA libraries</td>
		</tr>
		<tr>
			<td>pGBKT7 DNA-BD Vector</td>
			<td>Clontech</td>
			<td>630443</td>
			<td>commercially available DNA-BD Vector</td>
		</tr>
		<tr>
			<td>Biolase DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21042</td>
			<td>DNA polymerase used for section 2.4</td>
		</tr>
		<tr>
			<td>GeneMate GCL-60 Thermal Cycler</td>
			<td>BioExpress</td>
			<td>P-6050-60</td>
			<td>pcr machine</td>
		</tr>
		<tr>
			<td>TempAssure 0.5 mL PCR tubes</td>
			<td>USA Scientific</td>
			<td>1405-8100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a yeast 2-hybrid screen in batch to compare protein interactions

Screening für Protein-Protein-Interaktionen mit der Hefe-2-Hybrid-Assay ist seit langem ein wirksames Instrument, aber seine Verwendung wurde weitgehend auf die Entdeckung der hohen Affinität Interaktoren, die in der Bibliothek der interagierenden Kandidaten hochangereichertes sind begrenzt. In einem traditionellen Format kann Hefe 2-Hybrid Assay zu viele Kolonien zu analysieren, wenn bei niedrigen Stringenz durchgeführt ergeben, wo geringe Affinität Interaktoren gefunden werden könnte. Darüber hinaus kann nicht ohne eine umfassende und vollständige Befragung derselben Bibliothek gegen verschiedene Köder Plasmide, eine vergleichende Analyse erreicht werden. Obwohl einige dieser Probleme behoben werden kann, angeordneten Beute Bibliotheken verwenden, können die Kosten und die erforderliche Infrastruktur für solche Bildschirme betreiben unerschwinglich sein. Als Alternative haben wir die Hefe 2-Hybrid Assay um gleichzeitig entdecken Sie Dutzende von Transienten angepasst und statische Protein-Interaktionen in einem einzigen Bildschirm mit Hilfe einer Strategie bezeichnet DEEPN (dynamische Bereicherung für Evaluation of Protein Networks), die enthält Hochdurchsatz-DNA-Sequenzierung und Berechnung, mit der die Entwicklung einer Population von Plasmiden folgen, die Interaktionspartnern zu kodieren. Hier beschreiben wir maßgeschneiderte Reagenzien und Protokolle, die einen DEEPN Bildschirm einfach und kostengünstig ausgeführt werden können.

Watch this Scientific Journal Video about A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions at JoVE.com

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Eine Hefe 2-Hybrid Bildschirm im Batch Proteininteraktionen vergleichen

Batch-Verarbeitung von Hefe-2-Hybrid-Bildschirme ermöglicht direkten Vergleich der Interaktion profile mehrere Köder Proteine mit einem hoch komplexen Satz von Fusionsproteinen Beute. Hier beschreiben wir verfeinerten Methoden, neue Reagenzien und ihre Verwendung für solche Bildschirme zu implementieren.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

a-yeast-2-hybrid-screen-in-batch-to-compare-protein-interactions

Nanyang Technological University

Research

JoVE Journal

Biochemistry

13.2K Aufrufe.  University of Iowa. Batch-Verarbeitung von Hefe-2-Hybrid-Bildschirme ermöglicht direkten Vergleich der Interaktion profile mehrere Köder Proteine mit einem hoch komplexen Satz von Fusionsproteinen Beute. Hier beschreiben wir verfeinerten Methoden, neue Reagenzien und ihre Verwendung für solche Bildschirme zu implementieren.

Serie: A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Fluoreszenzanisotropiemessungen als Werkzeug Protein-Protein-Interaktionen zu untersuchen

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Forschung

Biochemie

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Scaffold Liposomen zu rekonstituieren Lipid-proximalen Protein-Protein-Wechselwirkungen<em&gt; In Vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Zeitaufgelöste Elektrospray-Ionisations Wasserstoff-Deuterium-Austausch-Massenspektrometrie zur Struktur Protein Studium und Dynamik

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

Ein modifiziertes Hefe-ein-Hybrid-System für Heteromere Protein-Komplex-DNA-Interaktionsstudien

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Biotinylierte Zelle eindringen Peptide, intrazelluläre Protein-Protein-Wechselwirkungen zu studieren

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Eine Fluoreszenz Fluktuation Spektroskopie Assay von Protein-Protein-Wechselwirkungen an den Zell-Zell-Kontakten

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Ein Fluorogenic Peptid Spaltung Assay zum Bildschirm für die proteolytische Aktivität: Anwendungen für Coronavirus spike Protein-Aktivierung

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Analyse der Proteinfaltung, Transport und Abbau in lebenden Zellen durch radioaktive Puls Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

Anwendung der Biolayer-Interferometrie (BLI) zur Untersuchung von Protein-Protein-Wechselwirkungen bei der Transkription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.