This work was supported by NIH R01-HL119813 (to T.J.B.), American Lung Association RG-196051 (to T.J.B.), a CIMED Pilot and Feasibility grant (to T.J.B.), American Heart Association Predoctoral Fellowships 14PRE19970008 (to Z.Y.) and 15PRE22110004 (to D.L.K.).

1. Herstellung von Milligramm-Mengen von Plasmid-DNA für eine groß angelegte Transiente Transfektion <ol><li> Klonierung des Proteins von Interesse in einer hohen Kopienzahl Säuger-Expressionsvektor unter Verwendung von Restriktionsstelle Klonierung oder andere geeignete Technik. <ol><li> Für optimale Ergebnisse verwenden pHLsec 7 Vektor, der hat einen eingebauten in C-terminal 6His-tag, ein starker Promotor Kozak-Sequenz und eine optimierte Sekretionssignal. </li></ol></li><li> Verwandeln Sie das Plasmid auf kompetente Zellen. <ol><li> Mit 20 ul kompetenter E. coli-Zellen auf 1 &amp; mgr; g Plasmid-DNA und Inkubieren auf Eis für 30 min. </li><li> Hitzeschock-Zellen bei 42 ° C für 35 sec, dann auf Eis für 2 min inkubiert. </li><li> Werden 300 &amp; mgr; l mikrobielle Wachstumsmedium (SOC) und bei 37 ° C für 45 Minuten, bei 220 UpM schüttelnd. </li><li> Platte Zellen auf Agar-Platte mit geeigneten Antibiotika-Selektion. <ol><li> Verwenden von 100 &amp; mgr; g / ml Carbenicillin, wenn das Plasmid in der pHLsec vector. </li></ol></li></ol></li><li> Kultur Kolonien in 250 ml Luria Broth (LB) Medium, ergänzt mit 100 ug / ml Antibiotikum (Carbenicillin) O / N bei 37 ° C, bei 220 Upm Schütteln. </li><li> Reinigen DNA aus der Kultur unter Verwendung von Hallo-Geschwindigkeit Plasmid Maxi Kit nach Herstellerprotokoll. <ol><li> Eluieren der DNA in Puffer EB (10 mM Tris-Cl, pH 8,5) anstelle von Puffer TE. </li><li> Aliquots des gereinigten Plasmids an Menge für die Transfektion und Speicherung erforderlich bei -20 ° C. </li></ol></li></ol> 2. Großflächige, Kultur und transiente Transfektion von Zellen 293F <ol><li> Supplement 1 L 293F Medien mit 10 ml Glutamin und 5 ml Pen / Strep (beide 100x). Lagerung bei 4 ° C. 5 ml Pen / Strep ist eine ausreichende Festigkeit in serumfreien Bedingungen und das reduzierte antibiotische Konzentration verbessert die Lebensfähigkeit der Zellen während der Transfektion, die Proteinausbeute verbessert. </li><li> Kultur 293F-Zellen in 300 ml Medium in 1 L Polycarbonat Erlenmeyer-Kolben mit belüfteten Kappes bei 37 ° C und 8% CO 2, während sie in einem Standard-Gewebekultur-Inkubator Schütteln. </li><li> Verdünnte Zellen bis 5 x 10 5 / ml Dichte einen Tag vor der Transfektion. </li><li> Am Tag der Transfektion Ergänzung Kulturmedium durch Zugabe von 10% Volumen von 2% w / v Cell Boost in 293F Medien. <ol><li> Messung der Glucosekonzentration unter Verwendung einer Glukoseüberwachungsvorrichtung gemß den Anweisungen des Herstellers und die Verwendung Ergänzungsmittel erforderlich, um eine Glukosekonzentration von 500 mg / dl zu erreichen. </li></ol></li><li> Hinzufügen kifunensine (1 ug / ml Endkonzentration) bei diesem Schritt zu Proteinglykosylierung steuern. </li><li> Berechnung des Volumens von DNA 1 &amp; mgr; g Plasmid pro 1 x 10 6 Zellen benötigt. Unter sterilen Bedingungen, verdünnte DNA in 5 ml serumfreiem Medium. </li><li> Berechnen Sie Volumen von Transfektionsreagenz für 1 &amp; mgr; g Plasmid pro 2 ul Transfektionsreagenz erforderlich. Unter sterilen Bedingungen, verdünnten Transfektionsreagenz (PEI-TMC-25) in 5 ml serumfreiem Medium. </li><li> HinzufügenTransfektionsreagenz in die DNA-Lösung in 1 ml-Schritten, Mischen sanft. Inkubation 30 min bei RT Reagenz-DNA-Komplexe zu bilden. Dann fügen Sie die Lösung auf die Zellen in einer tropfenweise Mode. </li><li> Erlauben transfizierten Zellen an Protein für 72 bis 96 h zum Ausdruck bringen. Ergänzung mit ~ 10% Volumen Cell Boost Medien täglich, oder wie erforderlich, zu halten Glukosemesswert von 400 bis 600 mg / dl. </li></ol> 3. Reinigung <ol><li> Dekantier-Kultur in ein Zentrifugenflasche, Zentrifuge für 20 min bei 1.300 × g Zellen zu pelletieren und zu sammeln, dann den Überstand. Wenn nötig, drehen ein zweites Mal und / oder verwenden 0,22 um Filter, um Überstand zu klären. </li><li> Zugabe von 10% Volumen 10x Ni-NTA-Bindungspuffer (1,5 M NaCl, 0,5 MK 2 HPO 4, 0,1 M Tris pH 8,5, 50 mM Imidazol). </li><li> Vorbereitung einer Schwerkraftsäule durch Zugabe von 2 ml von Ni-NTA-Aufschlämmung in einer Spalte und Äquilibrieren mit 10 Säulenvolumina (CV) des 1x Bindungspuffer. Wenn möglich, alle Spalten Schritte in einem 4 ° C Raum. AlternativEly, Chill-Protein und alle Puffer auf Eis vor der Spalte Schritt, und halten Protein und gesammelt Durchfluss auf Eis. Anmerkung: Ni-NTA-Aufschlämmung beträgt 50% Harz nach Volumen und angegebenen Bindungskapazität des Herstellers beträgt 50 mg / ml. Für mehrfachen Gebrauch Ni-NTA-Perlen kann wieder aufgeladen werden </li><li> Fließt der Überstand über das Harz und sammeln Durchfluss. Wiederholen Sie diesen Schritt. </li><li> Waschen mit 10 CV Waschpuffer (300 mM NaCl, 50 mM K 2 HPO 4, 20 mM Imidazol, pH 8). </li><li> Eluieren des Proteins in 5 CV Elutionspuffer (300 mM NaCl, 50 mM K 2 HPO 4, 250 mM Imidazol, pH 8). </li><li> Wenn Deglykosylierung ist erforderlich: <ol><li> Bis zu einem Endvolumen von 0,5 ml Konzentrat Eluat 0,43 ml unter Verwendung einer Zentrifugation Konzentrator. Wenn Niederschläge gebildet, pelle jede Trümmer durch Zentrifugation bei 16.000 × g und 4 ° C. </li><li> In 50 ul 500 mM Na-Citrat pH 5,5. </li><li> Mit 20 ul EndoHf (1 x 10 &amp; sup6; U / ml). Inkubation bei RT für 2 h. NichtE: Das Enzym arbeitet optimal bei 37 ° C, was dazu führen kann die konzentrierte Protein zu aggregieren. Verlängern Sie die RT Inkubation wenn Deglykosylierung, durch SDS-PAGE und Immunoblotting untersucht, ist unvollständig. Das Enzym muss nicht Aktivität bei 4 ° C. </li><li> Um EndoHf entfernen: Wash Amyloseharz 3x in phosphatgepufferter Salzlösung (PBS) oder Endlagerung Puffer. Inkubieren Protein mit Harz für 1 h bei 4 ° C. Spin 5 min bei 1000 xg zu pelletieren Perlen und sammeln Sie den Überstand. </li><li> Konzentrat Protein unter Verwendung geeigneter Molekulargewichtsgrenze Zentrifugation Filter und Pufferaustausch in Lagerungspuffer (150 mM NaCl und 20 mM HEPES pH 7.5). </li></ol></li></ol>

<ol>
	<li>Meyer, 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=Multi-host+expression+system+for+recombinant+production+of+challenging+proteins.">Multi-host expression system for recombinant production of challenging proteins.</a> PLoS One. 8, e68674 (2013).</li><li>Chang, V. 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=Glycoprotein+structural+genomics:+solving+the+glycosylation+problem.">Glycoprotein structural genomics: solving the glycosylation problem.</a> Structure. 15, 267-273 (2007).</li><li>Rich, J. R., Withers, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+methods+for+the+production+of+homogeneous+human+glycoproteins.">Emerging methods for the production of homogeneous human glycoproteins.</a> Nat Chem Biol. 5, 206-215 (2009).</li><li>Wu, K., 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=TREM-2+promotes+macrophage+survival+and+lung+disease+after+respiratory+viral+infection.">TREM-2 promotes macrophage survival and lung disease after respiratory viral infection.</a> J Exp Med. 212, 681-697 (2015).</li><li>Yang, C., 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=Mitigated+cytotoxicity+and+tremendously+enhanced+gene+transfection+efficiency+of+PEI+through+facile+one-step+carbamate+modification.">Mitigated cytotoxicity and tremendously enhanced gene transfection efficiency of PEI through facile one-step carbamate modification.</a> Adv Healthc Mater. 2, 1304-1308 (2013).</li><li>Elbein, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosidase+inhibitors:+inhibitors+of+N-linked+oligosaccharide+processing.">Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.</a> FASEB J. 5, 3055-3063 (1991).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta Crystallogr D Biol Crystallogr. 62, 1243-1250 (2006).</li><li>Kelker, M. S., Debler, E. W., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+mouse+triggering+receptor+expressed+on+myeloid+cells+1+(TREM-1)+at+1.76+A.">Crystal structure of mouse triggering receptor expressed on myeloid cells 1 (TREM-1) at 1.76 A.</a> J Mol Biol. 344, 1175-1181 (2004).</li><li>Kober, D. L., 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=Preparation,+crystallization,+and+preliminary+crystallographic+analysis+of+wild-type+and+mutant+human+TREM-2+ectodomains+linked+to+neurodegenerative+and+inflammatory+diseases.">Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases.</a> Protein Expr Purif. 96, 32-38 (2014).</li><li>Yurtsever, Z., 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=Self-cleavage+of+human+CLCA1+protein+by+a+novel+internal+metalloprotease+domain+controls+calcium-activated+chloride+channel+activation.">Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.</a> J Biol Chem. 287, 42138-42149 (2012).</li><li>Sala-Rabanal, M., Yurtsever, Z., Nichols, C. G., Brett, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+CLCA1+modulates+TMEM16A+to+activate+Ca-dependent+chloride+currents+in+human+cells.">Secreted CLCA1 modulates TMEM16A to activate Ca-dependent chloride currents in human cells.</a> Elife. 4, (2015).</li><li>Niesen, F. H., Berglund, H., Vedadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+differential+scanning+fluorimetry+to+detect+ligand+interactions+that+promote+protein+stability.">The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.</a> Nat Protoc. 2, 2212-2221 (2007).</li></ol>

The authors declare that they have no competing financial interests.

HEK 293F-Zellen bieten robuste Produktion von Proteinen erfordert post-translationale Modifikationen. Dieses System ermöglicht eine schnelle und skalierbare Ausdruck der nativ gefalteten Proteine ​​enthalten, Disulfide, Glykosylierung und Phosphorylierung, die sonst mit mehr Routine Expression Tools abwesend sein würde. Darüber hinaus kann dieses System für die Expression und Reinigung von Multiproteinkomplexen einfach durch Co-Transfektion von mehreren Plasmiden verwendet werden. Neben TREM2 Dieses System wurde...

Das Verständnis von Proteinstrukturen auf atomarer Ebene ist der Schlüssel zur Aufdeckung der molekularen Grundlagen der biologischen Wege und Krankheiten. Röntgen Proteinkristallographie ist das am häufigsten verwendete / anwendbares Verfahren zur Bestimmung von hochmolekularen Strukturen. Die größte Herausforderung bei diesem Verfahren ist, ausreichende Mengen an korrekt gefalteten, reines Protein. Dies wird ein Problem besonders bei der Arbeit mit sezerniertes Säugerproteinen, die bestimmte post-translationale Modifikationen unterzogen werden. Bakteriell exprimierten Proteine ​​sind die Hauptquelle für kristallisierte Proteine ​​in der Proteindatenbank 1 abgeschieden. Bakterielle Expressionssysteme sind weitgehend bevorzugt, da sie schnell, kostengünstig und erzeugen typischerweise hohe Ausbeuten an Protein. Jedoch extrazellulären Domänen von Säugerproteinen in Bakterien exprimiert werden oftmals nicht richtig gefaltet sind, zum Erhalten homogen fo in welchem ​​Fall die Rückfaltung und aufwendige Reinigungsschritte erforderlich sindlded Protein. Darüber hinaus haben viele Säugetierproteinen erfordern posttranslationale Glykosylierung, um die richtige Faltung 2 zu erzielen. Obwohl die Expression und Glycosylierung in Hefe oder Insektenzellen können die Falte Problem zu überwinden, post-translationale Modifikationen einschließlich Glykosylierung, unterscheiden sich erheblich von denen von Säugetierzellen 3, wodurch Proteine, die mit fehlerhaften oder nicht homogenen Modifikationen. Säugerzellen alle erforderlichen molekularen Maschinerie zum richtigen post-translationalen Modifikationen und Faltung zu gewährleisten; aber diese Expressionssysteme sind in der Regel nicht von den meisten Labors bevorzugt, aufgrund der begrenzten Ausbeuten und hohen Kosten für Reagenzien und Verbrauchsmaterialien. Polyethylenimin (PEI), ist ein Standard-Transfektionsreagenz relativ billig, aber erlegt beträchtliche Zytotoxizität und niedrige Transfektionseffizienz, was zu erhöhten Kosten in Zellmedien, DNA, und Kultivieren Ausrüstung. Viele Alternativen zu PEI sind unerschwinglich teuer. Wir sprechenDiese Probleme mit der Beschreibung einer Kombination von verbesserten Zellkulturwerkzeuge und chemisch modifizierte PEI für die schnelle und relativ kostengünstige Methode für die Expression von sezernierten Säugetierproteinen, gefolgt von der Einzelschritt-Reinigung. Das robuste Verfahren ergibt ausreichende Ausbeuten zur funktionellen und biochemischen Untersuchungen 4 und in einigen Fällen führt Protein zugänglich, um die Kristallisation ohne weitere Reinigung. Dieses Protokoll beschreibt verschiedene Techniken, um die Expression zu maximieren und die Ausbeute für sezernierte Säugetierproteinen in humanen embryonalen Nieren (HEK) 293F Zellen in Suspension gezüchtet. Transfektionseffizienz (und Kosten), Protein-Produktion und Reinigung sind alle stark von folgenden dieses Protokoll erweitert. PEI durch Zugabe von Carbamaten durch eine Einzelschritt-Ringöffnungsreaktion modifiziert (PEI-TMC-25 Synthese und Eigenschaften im Detail in Lit. 5 beschrieben) verbessert Transfektionseffizienz, verringert die Zytotoxizität von kationischenMembranzerstörung und entsprechend reduziert Experiment Kosten. Weiterhin werden die Lebensfähigkeit der Zellen und die Proteinexpression stark mit der Zugabe der Kultur Ergänzungen Glucose und Vitamine zuzuführen verbessert. Wichtig ist für die Herstellung von glycosylierten Proteinen, Behandlung mit kifunensine, eine ungiftige chemische Inhibitor von Mannosidase I, produziert Proteine ​​mit definierten, unreife Glycane, die durch die Endoglycosidase EndoHf entfernt werden, um Proteine ​​mit einer einzelnen N-Acetylglucosamin anstelle von zu ergeben ein voller Länge N-gebundene Glycan 6. Schließlich wird die Sekretion von Proteinen in einem serumfreien, chemisch definierten Medium ermöglicht eine schnelle und leichte Reinigung für strukturelle und biochemische Untersuchungen. Einzelschritt-Nickel-Nitrilotriessigsäure (Ni-NTA-Harz) Reinigung entfernt die meisten verunreinigenden Spezies im Überstand und in einigen Fällen kann Protein mit ausreichender Reinheit für eine Kristallisation ergeben.

<table>
	<tbody>
		<tr>
			<td>Culture Flasks</td>
			<td>GeneMate</td>
			<td>F-5909B</td>
			<td></td>
		</tr>
		<tr>
			<td>293 Freestyle Media</td>
			<td>Gibco/Life Technologies</td>
			<td>12338-018</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco/Life Technologies</td>
			<td>35050-061</td>
			<td>Use in place of Glutamine</td>
		</tr>
		<tr>
			<td>Hype 5 transfection reagent</td>
			<td>Oz Biosciences</td>
			<td>HY01500</td>
			<td></td>
		</tr>
		<tr>
			<td>293fectin transfection reagent</td>
			<td>Life Technologies</td>
			<td>12347019</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI transfection reagent</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep Kit</td>
			<td>Qiagen</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow&#160;</td>
			<td>Qiagen</td>
			<td>30430</td>
			<td></td>
		</tr>
		<tr>
			<td>Endo Hf</td>
			<td>NEB</td>
			<td>P0703L</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose Resin</td>
			<td>NEB</td>
			<td>E8021S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Boost R05.2</td>
			<td>HyClone</td>
			<td>SH30584.02</td>
			<td>Cell Culture Supplement</td>
		</tr>
		<tr>
			<td>GlucCell</td>
			<td>CESCO Bioengineering</td>
			<td>DG2032</td>
			<td>Glucose Monitoring System</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>519850.91</td>
			<td>Serum Free Medium for DNA transfection</td>
		</tr>
		<tr>
			<td>Luria Broth (LB Media)</td>
			<td>Life Technologies</td>
			<td>10855-001</td>
			<td></td>
		</tr>
		<tr>
			<td>GC10 Competent Cells</td>
			<td>Sigma-Aldrich</td>
			<td>G2919</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient mammalian cell expression and single-step purification of extracellular glycoproteins for crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

Hierin folgt die Ergebnisse dieser Expressionssystem zu einem sekretierten 13 kDa-Immunglobulin (Ig) -Domäne von menschlichem Protein Auslöse Rezeptor auf myeloide Zellen 2 (hTREM2, Reste 19 bis 132) angegeben angewendet wird. TREM2 ist ein Typ I Transmembranprotein, das eine einzelne extrazelluläre Ig-Domäne, die zwei Disulfidbrücken und zwei N-verknüpfte Glykosylierungsstellen aufweist. Im Gegensatz zu anderen Ig-Domänenproteine ​​8 wurde TREM2 nicht zugänglich Rückfaltung aus bakteriellen Eins...

Watch this Scientific Journal Video about Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization at JoVE.com

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Effiziente Mammalian Cell Expression und Single-step Reinigung der extrazellulären Glykoproteinen für die Kristallisation

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

efficient-mammalian-cell-expression-single-step-purification

Research

JoVE Journal

Biology

13.7K Aufrufe.  Washington University School of Medicine. This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Serie: Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Forschung

Biologie

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 ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Pulse-Chase-Analyse von N-verknüpften Zuckerketten von Glykoproteinen in Säugerzellen

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

MISSION esiRNA für RNAi-Screening in Säugerzellen

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Expressionsanalyse von Säugetier-Linker-Histon-Subtypen

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Effiziente Agroinfiltration von Pflanzen für High-Level-Transient Expression von rekombinanten Proteinen

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Effizienten Herstellung und Reinigung von rekombinantem murinem Kindlin-3 von Insektenzellen für biophysikalische Studien

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse der globalen RNA-Synthese auf der Einzelzellebene folgende Hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA Screening zu Ubiquitin und Ubiquitin-ähnlichen System Regler der biologischen Stoffwechselwege in kultivierten Säugerzellen identifizieren

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...