The authors would like to acknowledge Natalie Doude and David Westerway (University of Alberta) for providing us Sho cDNA, Christophe Chevalier (INRA) for plasmid construction, Michel Br&#233;mont (INRA) for providing us BL21Sol bacteria; Christine Longin (INRA) for TEM assistance and Edith Pajot-Augy (INRA) for comments and proofreading.

1. Erzeugung von Plasmid und Expression Shadoo Hinweis: Das Gen, das murine Shadoo Protein (Shadoo 25-122), wurde in den Expressionsvektor pET-28 11 subkloniert. Dieser Klon wurde in den E. transformierten coli SoluBL21 Bakterienstamm, um Shadoo Protein mit einem N-terminalen Hexahistidin-Tag, (HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG) zum Ausdruck bringen, wie zuvor 11 erläutert. Das Plasmid kann von den Autoren angefragt werden. Das Molekulargewicht des Shadoo aus seiner Aminosäuresequenz berechneten 12,243.2 Da. Hinweis: Führen Sie alle Manipulationen mit Bakterien unter dem laminal Flow-Haube oder in der Nähe Bunsenflamme. <ol><li> Transformieren kompetenter SoluBL21 Bakterien mit dem pET-28-His 6 -Shadoo Plasmid, mit einem Standard-Hitzeschockprotokoll. Für diese Mischung 1 bis 5 &amp; mgr; l der Plasmid (typischerweise 10 bis 100 pgng) in 50 &amp; mgr; l der Bakterien in einem Rohr. Führen Sie den Hitzeschock bei 42 ° C für 45 sec. </li><li> Kultur transformierter Bakterien bei 37 ° C, bis ein 600 0,5-0,7 in Luria-Bertani-Medium mit 40 mg / ml Kanamycin (1B) ergänzt. </li><li> Induzieren Shadoo Expression O / N durch Zugabe von 240 &amp; mgr; g / ml Isopropyl-β-D-thiogalactopyranosid, ITPG, zu der Kultur. Kultur des Bakteriums unter Schütteln bei 150 Upm bei 37 ° C. Anmerkung: In der Regel reicht von 3 A 600 O / N-Bakterienkultur. Anmerkung: Die transformierten Bakterien können bei -80 ° C für zukünftige Proteinproduktion gespeichert werden. Dazu fügen Sie 10% -50% (v / v) steriles Glycerin in Bakterienkultur Aliquots und frieren Sie sie bei -80 ° C. </li></ol><img alt="Abbildung 1" src="/files/ftp_upload/53432/53432fig1.jpg" /> Abbildung 1. Schema der Shadoo Expressionskonstrukt, Bakterien Transformation und Induktion von Shadoo Ausdruck (wie in Schritt 1 beschrieben). </strong&gt; (A) Das Expressionskonstrukt für Shadoo codiert ein Hexahistidin-Tag an den N-Terminus des Proteins, Nt fusioniert. Das Schema zeigt, dass Shadoo Protein enthält grundlegende Arginin / Glycin-Repeats (grünen Zylinder), eine hydrophobe Domäne (HD) und eine einzige N-Glykosylierungsstelle (CHO), gefolgt von einem C-Terminus (Ct). (B) pET-28-His 6 -Shadoo Plasmid in SoluBL21 Bakterien durch Hitzeschock für 45 Sekunden bei 42 ° C umgesetzt. Transformierten Bakterien werden auf selektivem Agar, der 40 ug / ml Kanamycin ausplattiert. Eine einzelne Kolonie von der Platte verwendet, um eine Kultur in einem 1000-3000 ml-Schüttelkolben zu inokulieren. Die Expression des rekombinanten Proteins ist mit 1 mM IPTG induziert. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig1highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> 2. Reinigung von Shadoo Protein durch Niederdruck-Flüssigchromatographie (LPLC) <ol><li> Zell-Lyse und Inschluss Gremien Vorbereitung <ol><li> Sammeln Sie die Bakteriensuspension und übertragen sie in Zentrifugenröhrchen (Abbildung 2). </li><li> Schleudern bei 2.500 xg für 20 min bei 4 ° C. Überstand durch Dekantieren zu entfernen und resuspendieren Pellets in 15 ml Puffer (50 mM Tris, 10 mM EDTA, pH 8 mit Anti-Protease-Cocktail) für jeden Pellets aus 500 ml Zellkultur abgeleitet ist. Anmerkung: Die resuspendierten Pellets können eingefroren und bei -20 ° C für mehrere Tage gelagert werden. Freezing erleichtert Bakterienzellen-Lyse. </li><li> Überprüfen Sie die Überexpression des rekombinanten Proteins mit rohen Bakterien Pellets durch SDS-PAGE-Analyse und Coomassie-Färbung. <ol><li> Hierzu Zentrifugen 1 ml Bakterienkultur pelleZellen. 100 ul Laemmli Denaturierungslösung (277,8 mM Tris-HCl, pH 6,8, 4,4% SDS, 44,4% w / v Glycerol, 0,02% Bromphenolblau) pelletiert und Wärme bei 100 ° C für 5 min. </li><li> Last 10 ul der Probe auf einem 12% Acrylamidgel laufen SDS-PAGE und visualize getrennten Proteine ​​durch Coomassie-Färbung. </li></ol></li><li> Hinzufügen Triton X-100 auf 0,5% Endkonzentration in die vollständig resuspendierten Pellets 2.1.2). </li><li> Inkubieren der Suspension in einem Wasserbad bei 37 ° C für 30 min. </li><li> Beschallen der Suspension für 5 min bei 6-12 &amp; mgr; m Spitze-zu-Spitze-Amplitude in eiskaltem Wasser, um bakterielle Zellen zu lysieren. </li><li> Transfer beschallte Suspension in sauberen 50 ml Zentrifugenröhrchen. </li><li> Zentrifugenröhrchen bei 15.000 xg für 15 min bei 4 ° C. </li><li> Überstand entfernen durch Dekantieren und 5 ml des Bindungspuffers (20 mM Tris-HCl-Puffer, pH 7,4, 0,5 M NaCl, 8 M Harnstoff, 5 mM Imidazol) pro Röhrchen. </li><li> Die Röhrchen auf dem sich drehenden Rad O / N komplett resuspendieren Proteine ​​aus Einschlusskörpern. </li></ol><img alt="Figur 2" src="/files/ftp_upload/53432/53432fig2.jpg" /> Abbildung 2. Schema der Reinigung von EinschlussKörpern (wie in Schritt 2.1 beschrieben). Die transformierten Bakterien exprimiert Shadoo werden durch Zentrifugation geerntet und in Lysepuffer resuspendiert. Die Suspension wird bei 37 ° C für 30 min inkubiert und dann beschallt, um Bakterienzellen mechanisch zu brechen. Beschallung auf Eis durchgeführt, um Probenerwärmung zu verhindern. Gebrochenen Zellen werden durch Zentrifugation geerntet und in einem Puffer, der 8 M Harnstoff, um Proteine ​​aus Einschlusskörper solubilisiert resuspendiert. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig2highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> </li><li> Ni 2+ -Affinitätschromatographie Anmerkung: Die Chromatographie an FPLC-System unter Verwendung einer Ni 2+ -charged, 5 ml Sepharose chelatbildende Säule (3A) durchgeführt. Alle Lösungen werden auf die Säule mit einer Fließgeschwindigkeit von 1 ml / min geladen. <ol><li> Injizieren von 10 ml 0,2 M NiSO 4 Wasserlösung in die Säule Sepharose, um sie aufzuladenmit Ni 2+ -Ionen. </li><li> Äquilibrieren der Säule mit Ni 2+ -Ionen geladenen durch Injektion von 30-50 ml des Bindungspuffers, der eine niedrige Konzentration an Imidazol (20 mM Tris-HCl-Puffer, pH 7,4, 0,5 M NaCl, 8 M Harnstoff, 5 mM Imidazol). </li><li> Laden Sie die Harnstoff solubilisiert Proteine ​​mit einer 50 ml Injektionsschleife. </li><li> Wiederherstellung der Durchflussfraktion durch gründliches Waschen der Säule mit 10 ml niedrig Imidazol Bindungspuffer (20 mM Tris-HCl-Puffer, pH 7,4, 0,5 M NaCl, 8 M Harnstoff, 5 mM Imidazol), um ein Maximum von ungebundenen Proteine ​​zu entfernen von der Säule. </li><li> Mit 50-100 ml des Waschpuffers (20 mM Tris-HCl, pH 7,4, 0,5 M NaCl, 8 M Harnstoff, 80 mM Imidazol), Proteine ​​unspezifisch an die Säule gebunden eluieren Die Säule wird. </li><li> Anwenden eines 15 min Gradienten von 80-800 linearen mM Imidazol in Puffer, der 20 mM Tris-HCl, pH 7,4, 0,5 M NaCl, 8 M Harnstoff. Sammeln Sie 1-ml-Fraktionen während der Gradienten-Anwendung. Hinweis: His-Tag-Proteine ​​eluiert witha Gradienten von Imidazol enthaltenden Puffer (als Konkurrent verwendet). </li><li> Werfen Sie einen 8-ul-Aliquot jeder Fraktion und fügen Sie 4 ul 4x Laemmli Denaturierungslösung (siehe 2.1.3.1). Hitzelösungen bei 100 ° C für 5 min. </li><li> Last 10 ul Proteingrößenmarker und 10 ul jeder Probe auf den 12% igen Acrylamidgel laufen SDS-PAGE und sichtbar getrennten Proteine ​​durch Coomassie-Färbung. Hinweis: Das Molekulargewicht Shadoo berechnet bilden seine Aminosäuresequenz ist 12,243.2 Da. </li><li> Pool alle Fraktionen, die Shadoo. </li></ol><img alt="Figur 3" src="/files/ftp_upload/53432/53432fig3.jpg" /> Abbildung 3. Reinigungsverfahren (wie in den Schritten 2.2-2.4 beschrieben). (A) Ni 2+ -Ionen durch Histidinreste, die die selektive Reinigung von Polyhistidin-markierte Proteine ​​können koordiniert werden. Imidazol wird verwendet, um das Protein zu eluieren, durchseine Fähigkeit, mit der Ni 2+ -Ionen koordinieren und verdrängen das gebundene Protein. (B) Proteine ​​mit unterschiedlichen hydrodynamischen Radius, der auf der Ni 2+ -Säule auf einer Größenausschluss-Säule zurückgehalten wurden. Proteine ​​werden von der größten bis zur kleinsten, eluiert. (C) Vor dem Gebrauch enthaltenden Fraktionen werden vereinigt und Shadoo entsalzt Harnstoff, Imidazol und Salze zu entfernen. Qualitätskontrolle Tests umfassen SDS-PAGE / Coomassie-Färbung, Western-Blot. Gereinigtes Shadoo kann gefriergetrocknet werden. Lyophilisierte Shadoo ist über Monate stabil, wenn sie bei -20 ° C gelagert. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig3highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> </li><li> Größenausschlusschromatographie Hinweis: Eine zweite Reinigungsschritt besteht in der Größenausschluß-Chromatographie, die Shadoo Proteins von anderen Proteinen in der ersten Reinigungsstufe coeluierte trennt(3B). <ol><li> Äquilibrieren Superdex-Säule von 120 ml gesamten Bettvolumens durch die Injektion 250 ml 20 mM Tris-HCl-Puffer, pH 7,4, 0,5 M NaCl, 8 M Harnstoff mit einer Fließgeschwindigkeit von 1,5 ml / min. </li><li> Last vereinigten Fraktionen, Shadoo von 2.2.9 auf die äquilibrierte Säule. Sammeln 1 ml-Fraktionen nach der Säule-Hohlraumvolumen von 40 ml auf die konstante Strömung des obigen Puffers. </li><li> Nehmen Sie ein 10 ul-Aliquot jeder Fraktion und führen Sie eine SDS-PAGE-Analyse und Coomassie-Färbung wie oben beschrieben, um herauszufinden, welche Fraktionen enthalten Shadoo. </li><li> Pool Fraktionen, Shadoo. </li></ol></li><li> Entsalzung Anmerkung: Entsalzungsverfahren entfernt Salze, Imidazol und Harnstoff rekombinante Shadoo Protein in einer für die weitere biophysikalische und biochemische Untersuchungen (3C) Puffer erhalten. Das gleiche Ergebnis kann durch Dialysieren Proben gegen den Puffer erhalten werden. <ol><li> Äquilibrieren eine Entsalzungssäule von 53 ml mit 150 mlvon 10 mM Ammoniumacetat-Puffer, pH 5, mit einer Fließgeschwindigkeit von 5 ml / min. </li><li> Last gepoolt Shadoo Fraktionen auf die Säule. Bei Injektion des Ammoniumacetatpuffer sammeln manuell Fraktionen, die eine Erhöhung des Signals bei 280 nm am Ausgang des UV-Detektors zeigen. </li><li> Gefrierzutrocknen der entsalzten Shadoo und speichern Sie sie bei -20 ° C. </li><li> Aufzulösen lyophilisierten Shadoo in ausreichender Puffer, wie 10 mM Natriumacetatpuffer, pH 5, vor der Verwendung. Hinweis: Die Polyhistidin-Tag kann mit Enterokinase entfernt werden. </li></ol></li></ol> 3. In-vitro-Montage von Shadoo in Amyloidfibrillen bei physiologischen pH- Hinweis: Die Shadoo Proteinaggregate und Fibrillen bildet spontan bei pH ≥ 7 11. <img alt="Figur 4" src="/files/ftp_upload/53432/53432fig4.jpg" /> Abbildung 4. Schema der Shadoo Rückfaltung an Amyloidfibrillen (wie beschrieBett im Schritt 3). Gereinigtes Shadoo wandelt spontan an Amyloidfibrillen als in Puffer pH ≥ 7, gelöst in einer sauren Lösung, wandelt Shadoo an Amyloidfibrillen bei Inkubation mit 1 M Gdn-HCl unter Schütteln. Qualitätskontrolle Tests umfassen Elektronenmikroskopie und THT-Färbung. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig4highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <ol><li> Verdünnter Shadoo bis zu einer Endkonzentration von 2 &amp; mgr; M in 20 mM Tris-HCl-Puffer, pH 7,4. Messen der Konzentration durch Messen der optischen Dichte der Lösung bei 280 nm und unter Verwendung des Extinktionskoeffizienten von Shadoo Aminosäurezusammensetzung von 20.970 abgeleitet. </li><li> Inkubieren 500 ul der Lösung in einem konischen Kunststoffröhrchen bei 4 ° C für ein paar Wochen, um eine spontane Umwandlung Protein Amyloid-Strukturen (Figur 4) zu ermöglichen. </li><li> Hinzufügen frisch Thioflavin T (THT) bis zu einer endgültigen conce vorbereitetntration von 10 um bis 100 &amp; mgr; l Aliquot der Lösung Shadoo. Inkubieren der Lösung für 5 Minuten bei RT. Messung der Fluoreszenzemission von 460 bis 520 nm unter Verwendung einer Anregungswellenlänge von 435 nm, um das Vorhandensein von Amyloid-Strukturen 17 zu verifizieren. </li></ol> 4. In-vitro-Montage von Shadoo an Amyloidfibrillen bei sauren pH-Werten <ol><li> Verdünnter Shadoo in 1 M GdnHCl, 20 mM Na-Acetatpuffer, pH 5 zu einer Endkonzentration von 2 uM. </li><li> Inkubieren 500 ul dieser Lösung in einem konischen Röhrchen unter ständigem Schütteln bei 37 ° C für 3-7 Tage. </li><li> Prüfen Sie das Vorhandensein von Amyloidfibrillen durch Zugabe ThT zu einem aliquoten Teil der Lösung nach 3.3. </li><li> Dialyse erhaltene Suspension gegen 10 mM Na-Acetat-Puffer, pH 5,0, zu Shadoo Amyloidfibrillen zu reinigen. </li><li> Bewahren Sie gereinigte Fibrillen bei +4 ° C. </li></ol><img alt="Figur 5" src="/files/ftp_upload/53432/53432fig5.jpg"/&gt; Figur 5. Repräsentative Ergebnisse. Proteinfraktionen aus Ni 2+ eluiert -charged chelatbildende Säule (A) und Größenausschluss-Säule (B) wurden unter Verwendung von SDS-PAGE und Coomassie-Färbung analysiert. Bezeichnung des gereinigten Shadoo wurde mit SPRN Anti Shadoo Antikörper (C) bewertet. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig5large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

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The authors have nothing to disclose.

Ein einfaches und effizientes Protokoll sowohl für die Expression und Reinigung einer großen Menge an rekombinantem Maus Shadoo Protein präsentiert. Das beschriebene Verfahren ermöglicht erfolgreiche Solubilisierung und Reinigung von (His 6) -markierte rekombinanten Shadoo Protein. Es ist wichtig, wie in dem Dokument darauf hingewiesen, dass, wenn in einem prokaryontischen Bakteriums E. ausgedrückt coli Shadoo sammelt sich in Einschlusskörpern. Vor Shadoo Reinigung haben Bakterien, dur...

Prion neurodegenerativen Erkrankungen, die bovine spongiforme Enzephalopathie bei Rindern, Scrapie bei Schafen und Creutzfeldt-Jakob-Krankheit beim Menschen, sind tödlich und unheilbar. Prion-Krankheiten durch Konformationsänderungen des zellulären Prionproteins im Wesentlichen aus α-Helices in die Quer β-Faltblatt gekennzeichnet angereichert Amyloid Konformer 1,2. Cross-β-Strukturen enthalten dicht gepackt und hochgeordnete kristalline artigen β-sheets stabilisiert mit 3,4-Wasserstoffbrücken. Prion Amyloide sich selbst replizieren aufgrund der β-Stränge an die wachsende Kante, die eine Vorlage für die Rekrutierung geben und Umwandeln eines monomeren Proteineinheit. Nach Angaben der &quot;Nur-Protein-Hypothese&quot;, die Amyloid-Konformer des Prionprotein der einzige Infektionserreger. Jedoch einige andere Biomoleküle wurden vorgeschlagen wesentlich, Prion-Erkrankungen sein. Zum Beispiel werden die Zellmembranen gedacht, ein Ort, wo conversi seinauf erfolgt und einige negativ geladene Lipide sind gezeigt worden, um die Konvertierung zu 5,6 zu erhöhen. Darüber hinaus können einige Proteine ​​auch im Prion-Erkrankungen beteiligt. Insbesondere gibt es zwei weitere Mitglieder des Prionproteinfamilie: Doppel und Shadoo 7,8. Doppel weist eine relativ steife Struktur mit Di-Sulfidbrücken stabilisiert zu werden, keine spontan polymerisieren und Aggregat an Amyloidfibrillen 9. Im Gegensatz dazu, ähnlich wie bei Prion-Protein, Shadoo eine β-Faltblatt-angereicherten Struktur und Selbstassoziation in Amyloidfibrillen Strukturen erlassen. Es konnte gezeigt werden, dass Shadoo kann unter nativen Bedingungen oder bei der Bindung negativ geladenen Membranen 10,11 fibrillieren. Umwandlung von Shadoo in amyloidartigen Fasern können mit Krankheiten in Verbindung gebracht werden. Tatsächlich sind die Mechanismen, durch die Amyloid-ähnliche Strukturen Krankheiten verursachen kaum verstanden. Shadoo trägt eine hydrophobe Domäne (HD) und eine Reihe von Tandem Arg / Gly wiederholt ähnliche to den N-terminalen Teil des Prion-Proteins (1A). Als die N-terminalen Teil des Prionproteins wird Shadoo stark positiv und scheint eine nativ unstrukturierte Protein 11,12 sein. Shadoo scheint funktionell auf Prionen-Erkrankungen sein, da es direkt binden Prionprotein. Zusätzlich ist seine Expression unten während Prion Pathologie 13,14 geregelt. Jedoch ist die Rolle von Shadoo Prionenerkrankungen noch nicht etabliert. Wir entwickelten ein Plasmid, das die codierende Sequenz der Maus Shadoo Gens. Das Plasmid wurde verwendet, um E.-Transformation coli zur Herstellung von N-terminalen His-6-Fusionsprotein Shadoo. Dieses Expressionssystem ist in unserem Labor etabliert und wird häufig in unserer laufenden Projekte 6,15,16 verwendet. Ein wichtiges Thema für die Expression von Shadoo ist die Wahl des E. coli-Stamm. Während kompetente BL21 Bakterienstamm wird häufig für die Expression von rekombinanten Pro verwendetProteine ​​Shadoo wurde erfolgreich exprimiert und nur aus der transformierten SoluBL21 Bakterienstamm gereinigt. SoluBL21 kompetente E. coli ist eine verbesserte Mutante BL21 Wirtsstamm für die Produktion von Proteinen, deren Expression in der Mutter BL21 gab keinen nachweisbaren löslichen Produkt entwickelt. High-Level-Expression Shadoo in SoluBL21 E. coli führt zu einer Akkumulation des Proteins in Einschlusskörpern. Als eine allgemeine Funktion, wenn ein nicht-native Protein ist stark in E. ausgedrückt coli neigt dieses Protein in unlöslicher Einschlußkörper akkumulieren. Shadoo Aggregate vermutlich durch nicht-kovalente oder ionische Wechselwirkungen hydrophobe (oder einer Kombination von beiden), um hoch angereichertes dynamischen Strukturen durch das Protein in unterschiedlichem Maße gefaltet ist. Folglich umfasst die Reinigung wenigstens zwei Schritte: (i) eine selektive Trennung des Proteins von anderen Biomolekülen in einer Denaturierung Medium und (ii) eine Renaturierung des gereinigten Proteins mit in vitro FaltungsTechniken. Die selektive Abtrennung von Shadoo wurde in einer Pufferlösung, enthaltend 8 M Harnstoff (oder alternativ 6 M Guanidin-HCl) gelöst. (I) die Renaturierung in einem sauren pH-Lösung eine unstrukturierte monomeren Shadoo Protein oder (ii) die Renaturierung in einer Lösung mit pH ≥ 7 bis Shadoo polymerisierten erhalten: Die Harnstoffentfernung und die Renaturierung des Proteins kann durch Anwendung verschiedener Protokolle durchgeführt werden in stabile Amyloidfasern mit charakteristischen Quer β-Blatt-Motive.

<table>
	<tbody>
		<tr>
			<td>AKTA FPLC&#160;</td>
			<td>GE, Amersham</td>
			<td># 1363</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/60 Superdex</td>
			<td>GE Healthcare</td>
			<td>17-1069-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap IMAC</td>
			<td>GE Healthcare</td>
			<td>GE17-0920-05</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep26/10 Desalting column</td>
			<td>GE Healthcare</td>
			<td>GE17-5087-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SoluBL21 Competent E. coli</td>
			<td>Genlantis</td>
			<td>C700200</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-28b+</td>
			<td>Novogen</td>
			<td>69865-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Broth medium</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Biophotometar</td>
			<td>Eppendorf</td>
			<td>550507804</td>
			<td></td>
		</tr>
		<tr>
			<td>Trito X-100</td>
			<td>Life Technologies</td>
			<td>85111</td>
			<td></td>
		</tr>
		<tr>
			<td>NiSO4</td>
			<td>Sigma-Aldrich</td>
			<td>656895</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Gdn-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr>
			<td>protein size marker&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>26620</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli

Der Escherichia-coli-Expressionssystem ist ein leistungsfähiges Werkzeug für die Herstellung von rekombinanten eukaryotischen Proteinen. Wir verwenden es, um Shadoo, ein Protein, an das Prion-Familie gehören, zu erzeugen. Ein chromatographisches Verfahren zur Reinigung von (His) 6 -markierten rekombinanten Shadoo ausgedrückt als Einschlußkörper beschrieben. Die Einschlusskörper in 8 M Harnstoff solubilisiert und auf eine Ni 2+ -charged Säule gebunden zu Ionen-Affinitätschromatographie durchzuführen. Gebundenen Proteine ​​werden mit einem Gradienten von Imidazol eluiert. Fraktionen Shadoo Protein enthalten, werden Größenausschlußchromatographie unterzogen wird, eine hoch gereinigte Protein zu erhalten. Im letzten Schritt gereinigt Shadoo entsalzt, um Salze, Harnstoff und Imidazol zu entfernen. Rekombinante Shadoo Protein ist ein wichtiges Reagenz zur biophysikalischen und biochemischen Studien der Proteinkonformation Störungen bei Prionenerkrankungen auftreten. Viele Berichte gezeigt, dass neurodegenerative Erkrankungen Prion stammen aus der Ablagerung von Stichle, bestellt Amyloidfibrillen. Musterprotokolle beschreiben, wie Shadoo in Amyloidfibrillen bei sauren und neutralen fibrillieren / basischen pH-Werten dargestellt. Die Methoden, wie man produzieren und fibrillieren Shadoo kann Forschung in Labors arbeiten an Prionenerkrankungen zu erleichtern, da es für die Produktion von großen Mengen an Protein in eine schnelle und kostengünstige Weise.

Etwa 5-10 mg gereinigtes Shadoo Protein pro Liter Bakterienkultur erhalten. Ein Zwei-Schritt-Reinigung ist notwendig, um rekombinante Shadoo hoher Reinheit zu erhalten. Der erste Schritt ist durch Affinitätschromatographie mit einem Ni 2+ -charged Spalte, die His-Tag-Proteine ​​behält geführt. Eluierten Fraktionen werden der SDS-PAGE unterworfen und mit Coomassie Brilliant Blue (5A) angefärbt. Fraktionen Shadoo Protein enthalten, werden vereinigt und weiter durch Grßenausschlußchrom...

Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Reinigung und Rückfaltung an Amyloidfibrillen von (His)<sub&gt; 6</sub&gt; -markierte Recombinant Protein Shadoo als Einschlusskörper in Ausgedrückt<em&gt; E. coli</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

purification-refolding-to-amyloid-fibrils-his6-tagged-recombinant

Research

JoVE Journal

Biology

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

14.1K Aufrufe.  INRA. A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures. 

Serie: Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

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

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagenese und Functional Selection Protocols for Directed Evolution von Proteinen in E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

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 ...

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 ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Gekoppelt Assays zur Überwachung Proteinrückfaltung in<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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

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

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

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

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

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

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Hochdurchsatz-Screening Quantitative Expression und Aufreinigung rekombinanter zu Disulfid-reichen Proteine ​​Venom Produziert in Applied<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Nicht-chromatographischen Reinigung von rekombinantem Elastin-ähnliche Polypeptide und deren Fusionen mit Peptiden und Proteinen aus<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.