Wir danken Anne-Marie Dion-Côte für Diskussionen, die zur Entwicklung der Methode. JK und RB FQNRT Doktor Gelehrten, ist ein M.-MG Vanier CIHR Gelehrter und J.-YM ist ein FRSQ Senior Researcher. Diese Arbeit wurde durch Mittel aus dem Natural Sciences and Engineering Research Council zu JY unterstützt. M.

1. Herstellung von rekombinantem Baculovirus Baculoviren werden unter Verwendung des Bac-to-Bac Baculovirus-Expressionssystem von Invitrogen hauptsächlich in Übereinstimmung mit dem Protokoll des Herstellers mit nur geringen Modifikationen erzeugt: <ol><li> Eine modifizierte pFastBac1 Vektor (Gibco, Leben) wurde mit der GST-und His-Tags (Abbildung 2) erstellt. Die Multiklonierungsstelle (MCS) von einem pET-52b (+)-Vektor (Novagen) und enthält einen 10xHis-Tag, wurde in die MCS eines pFastBac1 Vektor eingesetzt, um pFastBac1-Strep-10xHis generieren. Die pET-52b (+) MCS-Sequenz wurde PCR zwischen den XbaI und BlpI Restriktionsstellen verstärkt, indem eine BglII-Restriktionsstelle am 5&#39;-Ende und eine HindIII-Schnittstelle am 3 &#39;Ende. Das PCR-Produkt wurde dann zwischen BamHI und HindIII-Restriktionsstelle von pFastBac1 kloniert, mit der Kompatibilität der BamHI-und BglII-Restriktionssequenzen. Da das Ziel war, eine pFastBac1, die die Expression erzeugenrekombinante Protein mit Streptavidin-und 10xHis-Tags, die BamHI-Restriktionsstelle des pET-52b (+) MCS-Sequenz wurde nicht verwendet. Auch wurde die XbaI-Restriktionsstelle nicht verwendet werden, um die Redundanz von Restriktionsstellen zwischen den Standorten bereits in der MCS von pFastBac1 und diese mit der MCS von pET-52b eingesetzt (+) auftretenden vermeiden. Die GST kodierende Sequenz mit der PreScission Protease-Erkennungsstelle (LeuGluValLeuPheGln / GlyPro) wurde dann in pFastBac1-Strep-10xHis eingefügt. Die GST-cDNA-Sequenz von pGEX-6P-2 (GE Healthcare) wurde PCR amplifiziert mit Primern, welche NcoI und KpnI-Restriktionsstellen an den 5&#39;-und 3&#39;-Enden auf. Die NcoI-Stelle ermöglicht das Hinzufügen eines Start-Codon, sondern auch ein Alanin an der GST. Das PCR-Produkt wurde anschließend zwischen die NcoI und KpnI-Restriktionsstellen in pFastBac1-Strep-10xHis kloniert, was zu dem Löschen des Streptavidin-tag. Die so erhaltene neue Fusion Vektor wurde mit pFastBac1-GST-10xHis. </li><li> Klonen der cDNA-Kodierung für die protein von Interesse in das pFastBac-GST-10xHis Vektor zwischen dem GST (N-terminal) und dem His-Tag (C-terminal). Seien Sie vorsichtig, um die cDNA-Stop-Codon entfernen, um die Fusion mit der C-terminalen His-Tag zu ermöglichen. </li><li> Trans E. coli DH10Bac mit dem rekombinanten pFastBac (im Schritt 1.2) erhaltenen, die Bacmid erzeugen. Ermöglichen Kolonien über Nacht bei 37 ° C und mehrere Stunden bei 30 º C auf Platten, die Auswahl Bluo-gal und IPTG (10 ug / ml Tetracyclin, 50 &amp;mgr; g / ml Kanamycin, 7 &amp;mgr; g / ml Gentamycin, 100 &amp;mgr; g / ml Bluo-wachsen Gal, 40 ug / ml IPTG). </li><li> Pick und restreak 2 weiße Kolonien (aus Schritt 1.3) auf Selektionsplatten, um den weißen Phänotyp zu bestätigen. </li><li> Beimpfen von 2 ml LB, enthaltend 10 ug / ml Tetracyclin, 50 &amp;mgr; g / ml Kanamycin, 10 ug / ml Gentamycin mit den beiden weißen Kolonien aus Schritt 1.4 und ließ sie über Nacht unter Schütteln bei 37 ° C wachsen (250 rpm) </li><li> Um die Bacmid-DNA/CellFECTIN-Gemisch reinigen, zu pelletieren, die Bakterien von Schritt 1.5,Resuspendieren der Zellen in 300 ul Lösung I und 300 ul Lösung II (Maxiprep Kit von Qiagen). Inkubation für 5 min bei Raumtemperatur, fügen Sie 300 ul 3 M KOAc pH 5,5 und Inkubation für 10 min auf Eis. Zentrifugieren der Proben bei 4 ° C, maximale Geschwindigkeit für 10 min. In 800 ul von Isopropanol zu den Überständen und Inkubation für 10 min auf Eis. Zentrifugieren der Proben für 15 min bei 4 ° C und Waschen des Pellets mit 500 ul 70% Ethanol. Lassen Sie das Pellet zu resuspendieren es trocken und unter sterilen Bedingungen in 40 &amp;mgr; l Tris-EDTA pH 8,0. Die Bacmid-DNA bei 4 ° C gelagert werden </li><li> Generieren Baculoviren wie in dem Protokoll des Herstellers beschrieben. Baculoviren bei 4 ° C lichtgeschützt gelagert werden. </li></ol> 2. Rekombinante Proteinexpression <ol><li> Um die effizienteste Baculovirus zur Reinigung zu wählen und zu überwachen, wann der Spitzenwert der Proteinexpression zu erreichen, so wird ein Mini-Infektionstest eins folgt: Infect 2 × 10 7 Sf9-infizierten Zellen kultiviert bei 27 ° C in Grace-Medium (mit 10% FBS und 1% P / S ergänzt) mit 133 ul (1/150) des Baculovirus. Sammeln Aliquots von 1,5 ml bei 0, 24, 48 und 72 h nach der Infektion. Pelletierung der Zellen und Analyse der Expressionsspiegel des Proteins von Interesse durch Western-Blotting unter Verwendung von anti-Tag-Antikörper. </li><li> Verwenden die effizienteste Baculovirus von Schritt 2.1, eine Zentrifuge, die 1,0 × 10 6 Sf9-Zellen pro ml in 500 ml Medium in einem Verhältnis von 1/150 zwischen Virus und Medium zu infizieren. Lassen die infizierten Zellen wachsen in Suspension bei 27 ° C und Ernte der Zellen, wenn die maximale Protein-Expression (wie im Schritt 2.1 bestimmt wird) erreicht ist. Pelletierten Zellen können bei -80 ° C bis zur weiteren Verwendung gelagert werden. </li></ol> 3. Herstellung von löslichem Zelllysat Alle folgenden Inkubationsschritte werden bei 4 ° C unter leichten Drehgeführt. <ol><li> Lyse SF9 Zellen, die Protein von Interesse exprimieren, in ~ 20 ml GST-Bindungspuffer (150 mM NaCl, 1 mM EDTA, 0,05% Triton-X-100, 1 mM DTT in PBS1X für eine Endkonzentration von 250 mM NaCl und Proteaseinhibitor Cocktail (Roche). In einem Eis-Wasser-Bad, Dounce der Lösung 20x mit einem Dounce Homogenisator beschallen jeweils 3x 30 Sekunden (70% Leistung) und DOUNCE wieder 20x. </li><li> (Optional). Inkubieren der Gesamt Zelllysats mit 1 mM MgCl 2 und 7 Nuklease Benzonase (2,5 U / ml) für 30 min bei 4 ° C auf DNA-oder RNA-Kontamination zu entfernen. </li><li> Zentrifuge bei 18.000 rpm für 30 min bei 4 ° C. Halten Sie den Überstand. </li><li> (Optional). Zentrifuge der Überstand erneut für 30 min bei 18.000 rpm bei 4 ° C, um eine klare lösliche Lysat zu erhalten. Führen Sie den Überstand durch ein 0,2 oder 0,45 um Filter, um ein Verstopfen zu vermeiden. </li></ol> 4. Die Bindung von Protein an GST Perlen <ol><li> Inkubieren des löslichen Zell-Lysat mit 1 ml GST-Kügelchen (vorgewaschen zweimal mit10 ml GST-Bindungspuffer) für 1 h bei 4 º C unter leichter Rotation. </li></ol> Kommentar: Die Inkubationszeit muß nach der Stabilität des Proteins und Bindungseffizienz optimiert werden. <ol start="2"><li> Schnell Spin bei 700 rpm und Überstand entfernen ungebundenen Proteine ​​enthält (dies kann zur weiteren Analyse aufbewahrt werden). Waschen der gebundenen Proteine ​​GST-(3B, Spur 1) mit Waschpuffer GST (GST-Bindungspuffer mit 350 mM NaCl endgültig). </li><li> Wiederholen Sie Schritt 4.2 2x. Bei der letzten Waschung zu entfernen, so viel Überstand wie möglich. </li><li> Der Kügelchen mit 5 mM ATP und 15 mM MgCl 2 inkubieren (in 10 ml Bindungspuffer GST) für 1 h bei 4 ° C nicht-spezifische Bindung des Hitzeschock-Proteine ​​8 zu vermeiden. Dreimal mit Waschpuffer GST Waschen der Kügelchen. </li></ol> 5. PreScission Spaltung des GST <ol><li> Zentrifugieren Sie die GST-Perlen, den Überstand entfernen und waschen Sie die GST Perlen mit P5 buffer (50 mM NaHPO 4, pH 7,0, 500 mM NaCl, 10% Glycerin, 0,05% Triton-X-100, 5 mM Imidazol). </li><li> Die GST-Kügelchen mit PreScission Enzym in Puffer inkubieren P5 von 3 h bis über Nacht bei 4 ° C. (Teile die GST-Kügelchen in Fraktionen von 100 &amp;mgr; l hinzugefügt und 4-8 Einheiten (2-4 &amp;mgr; l) des Enzyms in 100 ul Puffer verdünnt, P5). </li></ol> Kommentar: Die Inkubationszeit der PreScission Schritt muss nach dem Molekulargewicht als auch die Stabilität des Proteins zu optimieren. Ein Abbau beobachtet, kann dieser Inkubationszeit auf ein Minimum von 2 Stunden statt über Nacht verringert werden. <ol start="3"><li> Schnell Spin und sammeln den Überstand. Wiederholen Sie diesen Schritt zwei Zeitpunkten nach Zugabe von 100 ul P5 Puffer zu den Perlen und bündeln alle Fraktionen (3B, Spur 2). </li></ol> Kommentar: Die restlichen Perlen können für die Analyse der Spaltungseffizienz verwendet werden (Abbildung 3B </strong&gt;, Spur 3). Üblicherweise wird 70-80% des Proteins abgespalten. 6. Protein-Bindung an TALON Metall Affinity Resin <ol><li> Teilen Sie die Elution von oben in 3 Fraktionen von 1 ml und Inkubation mit jeweils 100 ul Talon Metallaffinitätsharz Trockenperlen (zweimal mit Puffer P5 vorgewaschen) für 1 h unter Rotation. </li></ol> Kommentar: Die Aufteilung der Elution in mehrere Fraktionen erhöht die Bindung / Wascheffizienz. <ol start="2"><li> Mit P30-Puffer (P5 Puffer mit einer Endkonzentration von 30 mM Imidazol) Das Harz 5 min. </li><li> Wiederholen Sie Schritt 6,2 und 2x bündeln die Perlen in einer einzigen Röhre. Vor dem letzten Waschvorgang, zu entfernen, so viel Überstand wie möglich. </li></ol> Kommentar: Dies entspricht der TALON gebunden Probe (3B, Spur 4). 7. Elution des gereinigten Proteins <ol><li> Inkubation des Protein gebunden TALON Harz für 5 min unter Rotation mit P500 Puffer (P5 Puffer mit einer Endkonzentration von 500 mM Imidazol). Verwenden Sie ein Verhältnis zwischen Puffer und Perlen von 1:1 v / v (Zum Beispiel, wenn Sie 200 ul Trockenperlen genommen hatte, haben Sie zu 200 ul P500 hinzufügen). Wiederholen Sie diesen Schritt zweimal und halten jede eluierte Fraktion separat (benannt Elution 1 (E1), Elution 2 (E2) und Elution 3 (E3), 3B, Bahnen 5-7). </li></ol> Kommentar: Die restlichen Perlen können für die Analyse der Effizienz der Elution (3B, Spur 8) verwendet werden. Typischerweise wird 60-80% des Gesamtproteins in eluiert. <ol start="2"><li> Analysieren Sie die Menge und die Qualität der gereinigten Protein durch Laden ca. 20-40 ul jeder Fraktion mit einem Standard-BSA-Konzentration auf einem SDS-PAGE und Coomassie-Blau-Färbung mit (3B) oder SYPRO Protein-Färbung für die Visualisierung. </li></ol> Bemerkung: Das Protein von Interesse kann auch erkannt werden throughout des Reinigungsverfahrens unter Verwendung von Anti-GST-und Anti-His-Antikörper (Fig. 3C). 8. Lagerung des gereinigten Proteins <ol><li> Dialyse des gereinigten Proteins gegenüber einer entsprechenden Speicherpuffer (z. B. 20 mM Tris-Acetat pH 8,0, 200 mM KAc, 10% Glycerin, 1 mM EDTA, 0,5 mM DTT) bei 4 ° C. Es ist wichtig, zur Ausfällung des gereinigten Proteins während der Dialyse zu überprüfen. Wir dialysieren Regel 2x für 1 h bei 4 ° C unter Rühren Rotation. </li><li> Machen Sie kleine Aliquots des gereinigten Proteins (10-20 ul) und frieren auf Trockeneis für 30 min. Bewahren Sie die Aliquots bei -80 ° C und vermeiden viele Tau-/ Gefrierzyklen. </li></ol>

<ol>
	<li>Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J., Tan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+affinity+tags+for+protein+purification.">Comparison of affinity tags for protein purification.</a> Protein Expr. Purif. 41, 98-105 (2005).</li><li>Thain, A., Gaston, K., Jenkins, O., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+separation+of+GST+fusion+proteins+from+co-purifying+GroEL.">A method for the separation of GST fusion proteins from co-purifying GroEL.</a> Trends Genet. 12, 209-210 (1996).</li><li>Carr, S., 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=Expression+of+a+recombinant+form+of+the+V+antigen+of+Yersinia+pestis,+using+three+different+expression+systems.">Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems.</a> Vaccine. 18, 153-159 (1999).</li><li>Armisen, P., 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=Selective+adsorption+of+poly-His+tagged+glutaryl+acylase+on+tailor-made+metal+chelate+supports.">Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.</a> J. Chromatogr. A. 848, 61-70 (1999).</li><li>Kuhn, S., Zipfel, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+baculovirus+expression+vector+pBSV-8His+directs+secretion+of+histidine-tagged+proteins.">The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins.</a> Gene. 162, 225-229 (1995).</li><li>Buisson, R., 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=Cooperation+of+breast+cancer+proteins+PALB2+and+piccolo+BRCA2+in+stimulating+homologous+recombination.">Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.</a> Nat. Struct. Mol. Biol. 17, 1247-1254 (2010).</li><li>Filimonova, M. 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=Isoforms+of+Serratia+marcescens+nuclease.+The+role+of+Mg2++in+the+hydrolysis+mechanism.">Isoforms of Serratia marcescens nuclease. The role of Mg2+ in the hydrolysis mechanism.</a> Biochemistry. 62, 983-988 (1997).</li><li>Thorslund, T., 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=The+breast+cancer+tumor+suppressor+BRCA2+promotes+the+specific+targeting+of+RAD51+to+single-stranded+DNA.">The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA.</a> Nat. Struct. Mol. Biol. 17, 1263-1265 (2010).</li><li>Genois, M. 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=Interactions+between+BRCA2+and+RAD51+for+promoting+homologous+recombination+in+Leishmania+infantum.">Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.</a> Nucleic Acids Res. 40, 6570-6584 (2012).</li><li>Shrestha, B., Smee, C., Gileadi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+vector+system:+an+emerging+host+for+high-throughput+eukaryotic+protein+expression.">Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression.</a> Methods Mol. Biol. 439, 269-289 (2008).</li></ol>

Die hier vorgestellte GST-His Reinigungsprotokoll ist geeignet für die Reinigung von einer breiten Palette von Größen von rekombinanten Proteinen: Wir haben erfolgreich gereinigt Li RAD51 (41kDa), piBRCA2 (120kDa), PALB2 (130kDa) und eine instabile hochmolekularen Protein von Leishmania infantum: Li BRCA2 (125kDa) 6,9. Außerdem wurden die Proteine ​​mit diesem Protokoll gereinigt biochemisch aktiven 6. Der Erfolg dieser Methode hängt nur von der Expression, Löslichkeit u...

Die Reinigung von rekombinanten Proteinen ist entscheidend, um grundlegende Fragen in der Biochemie adressieren. Herkömmliche Wege zur Proteinreinigung, wie Ionenaustausch-Chromatographie und Größenausschluss-Chromatographie verlassen sich auf die physikalischen Eigenschaften des Zielproteins wie seinem isoelektrischen Punkt und Ladung oder Grße sind. Letztere Proteineigenschaften werden durch eine Vielzahl von Proteinen, die wesentlich die Wahrscheinlichkeit von verunreinigenden Proteinen in herkömmlichen Proteinreinigungsstrategien erhöht geteilt. Dieses Problem kann mit der Verwendung von mehreren Reinigungssäulen, die zeitaufwendig ist, umgangen werden. Zur gleichen Zeit werden die letzteren Chromatographie-Verfahren erfordern teure Versuchsaufbau. Affinitäts-Tag-Reinigung stark erhöht Zielspezifität, wie in den meisten Fällen der Tag einzigartig für das Protein von Interesse sein. In neueren Studien wurde Flag-oder HA-Affinitätsreinigung weit verbreitet. Im Gegensatz zu bestehenden recombinant Proteinreinigungsprotokolle, in denen einzelne Tags verwendet werden, haben wir die einzigartige Kombination von zwei Tags. Unser Verfahren umfaßt die Fusion eines GST-Tag am N-Terminus und ein His-tag am C-Terminus des Proteins von Interesse, für ein optimales Verhältnis zwischen der Menge und Reinheit des gewünschten Proteins. Die GST ist ein langer Tag (29 kDa), die hocheffiziente zur Reinigung an Glutathion-Sepharose-Kügelchen ist. Darüber hinaus unter Verwendung von GST garantiert Wirtschaftlichkeit unserer Methode 1. Die Möglichkeit, mit dem PreScission Enzym (mit Erkennungssequenz LeuGluValLeuPheGln / GlyPro, was die Zugabe von nur zwei Aminosäuren) Abspaltung der GST hat viele Vorteile, zum Beispiel, vermeidet diese Strategie Veränderungen der physiologischen Proteinfunktionen durch allosterische Hindernis. Die kleine His-Tag auf den anderen Protein fusioniert Extremität dient in einem zweiten Reinigungsschritt zur Proteinreinheit durch Abwaschen des gespaltenen GST, als auch abgebaut Proteine ​​und andere erhöhen<html> Verunreinigungen. Zusätzlich kann das Protokoll nicht eine Dialyseschritt, wenn von dem GST an den His-Reinigungsschritt Säule (TALON Harz) erforderlich. Gemeinsame Verunreinigungen in solchen Reinigungsverfahren sind Hitzeschockproteine ​​(HSP). Die Zugabe von einem Inkubationsschritt mit MgCl 2 und ATP ermöglicht die Entfernung dieser Verunreinigungen (Abbildung 1). Fast Protein Liquid Chromatography (FPLC) und High Performance Liquid Chromatography (HPLC) sind übliche Techniken, die auf teure Geräte abhängen hohe Ausbeute des gereinigten Proteins von Interesse zu erzeugen. Die Batch-Reinigungsprotokoll wir hier präsentieren, im Gegensatz dazu ist manuell und nicht eine teure Instrumental Setup erfordern. Eine hohe Ausbeute an Protein durch Aufstockung des Protokolls erreicht werden. Zur gleichen Zeit, mit der Chargenreinigungsprotokoll, das Elutionsvolumen kann, um die Proteinkonzentration, die nicht mit FPLC oder HPLC gegeben erweitern eingestellt werden. ntent &quot;&gt; Auch mit der Batch-GST-His Reinigungsprotokoll, Proteine ​​mit einem Größenbereich von ~ 10-300 kDa gereinigt werden. Einer der größten Vorteile ist durch die Tatsache gegeben, dass man mehrere Proteine ​​gleichzeitig zu reinigen beispielsweise sowohl ein Wildtyp und ihrer Mutanten-Protein. Der Erfolg der dargebotenen Protokoll hängt ausschließlich vom Expressionsniveau und die Löslichkeit des Proteins von Interesse.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>E.Coli DH10Bac (competent)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus Expression System</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Maxi Prep Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Solution I and II</td>
		</tr>
		<tr>
			<td>Ultra Pure Bluo-Gal</td>
			<td>Gibco (Life)</td>
			<td>15519028</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gibco (Life)</td>
			<td>15529019</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 infected cells</td>
			<td>ATCC</td>
			<td>CRL-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>PreScission enzyme</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#39;s infected medium supplemented</td>
			<td>Gibco (Life)</td>
			<td>11605-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum characterized</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>P/S (Penicillin-Streptomycin)</td>
			<td>Gibco (Life)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose resin</td>
			<td>GE bioscience</td>
			<td>17075605</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Talon resin</td>
			<td>Clontech</td>
			<td>635504</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>BioShop</td>
			<td>288324</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco (Life)</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal GST-antibody</td>
			<td>Production in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 6X-His-antibody</td>
			<td>Clontech</td>
			<td>631212</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dounce homogenizer (tight)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator (Fisher dismembrator)</td>
			<td>Fisher</td>
			<td>Model 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (50 mm)</td>
			<td>Fisher Scientific</td>
			<td>2115217</td>
			<td></td>
		</tr>
	</tbody>
</table>

gst-his purification: a two-step affinity purification protocol yielding full-length purified proteins

Schlüssel Assays in Enzymology für die biochemische Charakterisierung von Proteinen in vitro erfordern hohe Konzentrationen des gereinigten Proteins von Interesse. Proteinreinigung Protokolle sollten Effizienz, Einfachheit und Kosteneffektivität 1 kombinieren. Hier beschreiben wir die GST-His Verfahren nach einem neuen kleinen Affinitätsreinigungssystem für rekombinante Proteine, basierend auf einem N-terminalen Glutathion-Sepharose-Tag (GST) 2,3 und ein C-terminales 10xHis-Tag 4, der sowohl verschmolzen an das Protein von Interesse. Das letztere Konstrukt wird verwendet, um Baculoviren zu erzeugen, zur Infektion von Sf9-infizierten Zellen für die Proteinexpression 5. GST ist ein ziemlich langer Tag (29 kDa), die Reinigungsleistung zu gewährleisten dient. Allerdings könnte es physiologischen Eigenschaften des Proteins beeinflussen. Daher wird in der Folge aus dem Protein unter Verwendung des Enzyms PreScission 6 gespalten. Um maximale Reinheit zu gewährleisten und die gespaltenen GST entfernen, wirfügte eine zweite Affinitätsreinigungsschritt auf der Basis der vergleichsweise kleinen His-Tag. Wichtig ist, dass unser Verfahren auf zwei verschiedene Tags flankieren die beiden Enden des Proteins, das ein leistungsfähiges Werkzeug, um abgebaute Proteine ​​zu entfernen, und daher ist basiert, reichert Proteine ​​in voller Länge. Das hier vorgestellte Verfahren erfordert nicht eine teure instrumental Einrichtung, wie FPLC. Darüber hinaus haben wir MgCl 2 und ATP wäscht integriert, um Hitzeschockprotein Verunreinigungen und Nukleasebehandlung abzuschaffen kontaminierenden Nukleinsäuren zu entfernen. Zusammenfassend ist die Kombination von zwei verschiedenen Variablen flankieren die N-und C-terminalen und die Fähigkeit zur Abspaltung eines der Tags, garantiert die Rückgewinnung eines hochgereinigten und Volllängen-Protein von Interesse.

Um die Effizienz des GST-His-Reinigungsprotokolls veranschaulichen, reinigten wir Rec14, einen S. pombe Protein von 32,9 kDa. Die Rec14 cDNA wurde in unserer modifizierten pFastBac1 Vektor ermöglicht die Zugaben von GST-und His-Tags an der N-und C-Termini, die jeweils (1A) kloniert. Rekombinanten Baculovirus wurden hergestellt und verwendet, um Sf9-infizierten Zellen für die Proteinexpression zu infizieren. Die löslichen Zelllysate wurden mit GST inkubiert und Perlen-gebundenen Proteine ​?...

Watch this Scientific Journal Video about GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins at JoVE.com

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

In dem vorliegenden Protokoll, zeigen wir eine hocheffiziente und kostengünstige kleine Proteinreinigungsverfahren, die die Reinigung von rekombinanten Proteinen ermöglicht dank einer einzigartigen Kombination eine spaltbare GST-Tag und einen kleinen His-Tag.

GST-His-Reinigung: Ein Zwei-Schritt-Affinitätsreinigung Protokoll Nachgeben in voller Länge Gereinigte Proteine

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

gst-his-purification-two-step-affinity-purification-protocol-yielding

Research

JoVE Journal

Biology

41.3K Aufrufe.  Hôtel-Dieu de Québec.  In dem vorliegenden Protokoll, zeigen wir eine hocheffiziente und kostengünstige kleine Proteinreinigungsverfahren, die die Reinigung von rekombinanten Proteinen ermöglicht dank einer einzigartigen Kombination eine spaltbare GST-Tag und einen kleinen His-Tag. 

Serie: GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Forschung

Biologie

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Reinigung von Hsp104, ein Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Chromatographische Reinigung von hoch aktiven Hefe Ribosomen

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

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

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

Orthogonal Protein Purification von einer kleinen Bispezifische Affinity Tag Facilitated

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

Identification of Protein Interacting Partners Using Tandem Affinity Purification

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

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

Identifizierung von Protein Interaktionspartner mittels Tandem Affinity Purification

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

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

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

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

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

Identifizierung von Protein-Komplexen in<em&gt; Escherichia coli</em&gt; Mit Sequential Peptide Affinity Purification in Kombination mit Tandem-Massenspektrometrie

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

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

High-throughput Reinigung von Affinity-markierter rekombinanter Proteine

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Purification of Transcripts and Metabolites from Drosophila Heads

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

Purification of Transcripts and Metabolites from Drosophila Heads

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

Reinigung von Transcripts und Metaboliten aus<em&gt; Drosophila</em&gt; Köpfe

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

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

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

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

Protein Purification-freie Methode der Bindungsaffinität Bestimmung durch Microscale Thermophorese

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

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

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

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

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Identifizierung von Protein-Protein-Wechselwirkung in<em&gt; Drosophila</em&gt; Adult Heads von Tandem Affinity Purification (TAP)

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

GST-His-Reinigung: Ein Zwei-Schritt-Affinitätsreinigung Protokoll Nachgeben in voller Länge Gereinigte Proteine

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