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. production de quantités milligramme d&#39;ADN plasmidique à grande échelle pour la transfection transitoire <ol><li> Cloner la protéine d&#39;intérêt dans un certain nombre vecteur d&#39;expression mammifère élevé de copies en utilisant le site de restriction clonage, ou une autre technique appropriée. <ol><li> Pour des résultats optimaux, utilisez pHLsec 7 vecteur, qui a un haut-C-terminal 6His-tag, une séquence forte Kozak promoteur et un signal de sécrétion optimisé. </li></ol></li><li> Transformer le plasmide sur des cellules compétentes. <ol><li> Ajouter 20 ul de cellules compétentes de E. coli sur 1 ug d&#39;ADN de plasmide et incuber sur de la glace pendant 30 min. </li><li> Des cellules de choc thermique à 42 ° C pendant 35 sec, puis incuber sur de la glace pendant 2 min. </li><li> Ajouter 300 pi de milieu de croissance microbienne (SOC) et incuber à 37 ° C pendant 45 minutes, en agitant à 220 rpm. </li><li> Les cellules de plaque sur plaque de gélose avec la sélection antibiotique approprié. <ol><li> Utilisez 100 pg / ml de carbénicilline si le plasmide est dans le vecteur pHLsec. </li></ol></li></ol></li><li> Les colonies de la culture dans 250 ml de bouillon de Luria (LB) additionné de médias 100 ug / antibiotique (carbénicilline) O / N ml à 37 ° C, sous agitation à 220 tours par minute. </li><li> On purifie l&#39;ADN à partir de la culture en utilisant un kit Salut débit Plasmid Maxi selon le protocole du fabricant. <ol><li> Eluer ADN dans un tampon EB (10 mM de Tris-Cl, pH 8,5), au lieu de tampon TE. </li><li> Aliquoter le plasmide purifié au montant nécessaire pour la transfection et conserver à -20 ° C. </li></ol></li></ol> 2. grande échelle Culture et transfection transitoire de cellules 293F <ol><li> Supplément médias 1 L 293F avec 10 ml de glutamine et 5 ml Pen / Strep (deux 100x). Conserver à 4 ° C. 5 ml Pen / Strep est une résistance suffisante dans des conditions sans sérum et la concentration en antibiotique réduite améliore la viabilité des cellules au cours de la transfection, ce qui améliore les rendements en protéines. </li><li> Culture cellules 293F dans 300 ml médias dans 1 L polycarbonate déconcertés flacons Erlenmeyer avec bouchon à évents à 37 ° C avec 8% de CO2, sous agitation dans un incubateur de culture de tissu standard. </li><li> Diluer les cellules à 5 x 10 5 / ml densité une veille de la transfection. </li><li> Le jour de la transfection, le milieu de culture par addition de supplément de volume de 10% à 2% en poids / volume dans 293F cellulaire Boost médias. <ol><li> Mesurer la concentration de glucose en utilisant un moniteur de glucose selon les instructions du fabricant et les suppléments d&#39;utilisation selon les besoins pour obtenir une concentration en glucose de 500 mg / dL. </li></ol></li><li> Ajouter kifunensine (1 pg / ml de concentration finale) à cette étape pour contrôler la glycosylation des protéines. </li><li> Calculer le volume de l&#39;ADN requis pour une pg de plasmide pour 1 x 10 6 cellules. Dans des conditions stériles, diluer l&#39;ADN dans un milieu exempt de sérum de 5 ml. </li><li> Calculer le volume de réactif de transfection requis pour une pg de plasmide par 2 ul de réactif de transfection. Dans des conditions stériles, diluer le réactif de transfection (PEI-TMC-25) dans du milieu exempt de sérum de 5 ml. </li><li> Ajouterréactif de transfection d&#39;ADN en solution dans 1 ml incréments, en mélangeant doucement. Incuber pendant 30 min à température ambiante pour les complexes ADN-réactif de se former. Puis ajouter la solution sur les cellules dans un mode goutte à goutte. </li><li> Laisser les cellules transfectées pour exprimer la protéine de 72-96 h. Supplément avec ~ 10% du volume cellulaire Boost médias quotidiens, ou au besoin pour maintenir le glucose lecture 400-600 mg / dl. </li></ol> 3. Purification <ol><li> Décanter la culture dans un flacon de centrifugeuse, centrifugeuse pendant 20 min à 1300 x g pour sédimenter les cellules, puis recueillir le surnageant. Si nécessaire, faites tourner une deuxième fois et / ou utiliser le filtre de 0,22 um pour clarifier surnageant. </li><li> Ajouter tampon de liaison de 10% du volume 10x Ni-NTA (1,5 M de NaCl, 0,5 MK 2 HPO 4, Tris 0,1 M pH 8,5 mM imidazole 50). </li><li> Préparer une colonne de gravité par addition de 2 ml de suspension épaisse de Ni-NTA dans une colonne d&#39;équilibrage et avec 10 volumes de colonne (CV) de tampon de liaison 1x. Si possible, faire toutes les étapes de colonne dans une chambre de 4 C °. AlternativEly, la protéine de refroidissement et tous les tampons sur la glace avant l&#39;étape de la colonne, et garder la protéine et recueilli accréditives sur la glace. Remarque: Ni-NTA suspension est une résine de 50% en volume et de capacité de liaison déclaré de la fabrication est de 50 mg / ml. Ni-NTA beads peuvent être rechargées pour des usages multiples </li><li> Écouler le surnageant sur la résine et de recueillir accréditives. Répétez cette étape. </li><li> Laver avec 10 CV de tampon de lavage (300 mM de NaCl, 50 mM de K 2 HPO 4, 20 mM d&#39;imidazole pH 8). </li><li> Eluer la protéine en 5 CV de tampon d&#39;élution (300 mM NaCl, 50 mM de K 2 HPO 4, imidazole 250 mM, pH 8). </li><li> Si déglycosylation est nécessaire: <ol><li> Pour un volume final de 0,5 ml, on concentre l&#39;éluat à 0,43 ml en utilisant un concentrateur de centrifugation. Si des précipités se forment, sédimenter les débris par centrifugation à 16.000 xg et 4 ° C. </li><li> Ajouter 50 pi de 500 mM Na-Citrate pH 5,5. </li><li> Ajouter 20 ul de EndoHf (1 x 10 6 U / ml). Incuber à température ambiante pendant 2 h. ne pase: L&#39;enzyme fonctionne de manière optimale à 37 ° C, ce qui peut provoquer le concentré de protéine agrégée. Prolonger l&#39;incubation RT, si déglycosylation, évaluée par SDS-PAGE ou immunoblotting, est incomplète. L&#39;enzyme n&#39;a pas d&#39;activité à 4 ° C. </li><li> Pour supprimer EndoHf: Laver amylose Résine 3x dans un tampon phosphate salin (PBS) ou un tampon de stockage final. Incuber protéines avec de la résine pendant 1 h à 4 ° C. Spin 5 min à 1000 x g pour sédimenter perles et de recueillir le surnageant. </li><li> Concentrez-protéine en utilisant un filtre approprié de centrifugation poids de coupure moléculaire et échange de tampon dans le tampon de stockage (NaCl 150 mM et HEPES 20 mM 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.

Cellules HEK 293F offrent une forte production de protéines nécessitant modifications post-traductionnelles. Ce système permet l&#39;expression rapide et évolutive des protéines pliées contenant nativement disulfures, la glycosylation, la phosphorylation et qui, autrement, être absent en utilisant plus d&#39;outils d&#39;expression de routine. En outre, ce système peut être utilisé pour l&#39;expression et la purification de complexes multi-protéines simplement par co-transfection de plusieurs plasmides. Outr...

Comprendre la structure des protéines à un niveau atomique est clé pour découvrir la base moléculaire des voies biologiques et de maladies. X-ray cristallographie des protéines est la méthode la plus largement utilisée / applicable pour la détermination des structures macromoléculaires. Le principal défi de cette méthode est d&#39;obtenir des quantités suffisantes de bien plié, protéine pure. Cela devient un problème particulier lorsque l&#39;on travaille avec des protéines sécrétées mammifères, qui subissent des modifications post-traductionnelles spécifiques. Protéines bactérienne exprimées sont la principale source de protéines cristallisées déposés dans la Protein Data Bank 1. Des systèmes d&#39;expression bactériens sont largement préférés parce qu&#39;ils sont rapides, peu coûteux et généralement produire des rendements élevés de protéine. Cependant, domaines extracellulaires des protéines de mammifère exprimées dans des bactéries sont souvent pas correctement repliés, dans ce cas, repliement et purification extensive étapes sont nécessaires pour obtenir de façon homogène foLDED protéines. En outre, de nombreuses protéines de mammifères exigent glycosylation post-traductionnelle pour atteindre un repliement approprié 2. Bien que l&#39;expression et la glycosylation dans les cellules de levure ou d&#39;insectes peuvent surmonter le problème de pliage, modifications post-traductionnelles, y compris la glycosylation, diffèrent considérablement de celles des cellules de mammifères 3, produisant des protéines avec des modifications incorrectes ou non-homogènes. Les cellules de mammifères expriment toute la machinerie moléculaire nécessaire pour assurer modifications et pliage de post-traductionnelle appropriés; Toutefois, ces systèmes d&#39;expression ne sont pas généralement préféré par la plupart des laboratoires, en raison de rendements limités et des coûts élevés de réactifs et de consommables. Polyéthylèneimine (PEI), un réactif de transfection norme est relativement pas cher mais impose cytotoxicité considérable et une faible efficacité de transfection, résultant en une augmentation des coûts dans les médias de la cellule, l&#39;ADN, et de l&#39;équipement en culture. Beaucoup de solutions de rechange aux PEI sont prohibitifs. Nous nous adressonsces questions en décrivant une combinaison de l&#39;amélioration des outils de culture cellulaire et chimiquement modifiée PEI pour la méthode rapide et relativement peu coûteux pour l&#39;expression de protéines de mammifères sécrétées, suivie d&#39;une purification en une seule étape. Ce procédé donne des rendements suffisants robuste pour des études fonctionnelles et biochimiques 4, et dans certains cas, se traduit par une protéine se prêtent à une cristallisation sans autre purification. Ce protocole décrit plusieurs techniques pour maximiser l&#39;expression et le rendement pour les protéines de mammifères sécrété dans le rein embryonnaire humain (HEK) 293F cellules cultivées en suspension. L&#39;efficacité de transfection (et le coût), la production et la purification de la protéine sont grandement améliorées tout en suivant le protocole. PEI modifié par l&#39;addition de carbamates par réaction d&#39;ouverture de cycle en une seule étape (PEI-TMC-25, synthèse et les propriétés décrites en détail dans la référence 5) améliore grandement l&#39;efficacité de transfection, réduit la cytotoxicité de cationiquerupture de la membrane et par conséquent de réduire les coûts d&#39;expérimentation. En outre, la viabilité des cellules et l&#39;expression des protéines sont grandement améliorées grâce à l&#39;ajout de compléments de culture à fournir du glucose et des vitamines. Il est important pour la production de protéines glycosylées, le traitement par la kifunensine, un inhibiteur chimique non-toxique de la mannosidase I, produit des protéines avec définis glycanes immatures, qui peuvent être éliminés par la EndoHf d&#39;endoglycosidase pour produire des protéines avec un seul N-acétylglucosamine à la place du une pleine longueur glycane 6 N-liée. Enfin, la sécrétion de protéines dans un milieu défini chimiquement sans sérum permet une purification rapide et facile pour les études structurales et biochimiques. Single-step nickel-acide nitrilotriacétique de (Ni-NTA) purification de résine élimine la majorité des espèces contaminantes dans le surnageant et, dans certains cas, peut donner une protéine d&#39;une pureté suffisante pour la cristallisation.

<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.

Ici suit les résultats de ce système d&#39;expression appliquée à une immunoglobuline sécrétée 13 kDa (Ig) le domaine de récepteur de protéine humaine exprimée de déclenchement sur les cellules myéloïdes (2, résidus 19-132 hTREM2). TREM2 est une protéine transmembranaire de type I contenant un seul domaine de type Ig extracellulaire qui dispose de deux ponts disulfure et deux sites de glycosylation N-liés. Contrairement à de nombreux autres domaines Ig protéines 8, TREM2 ne se prêtaient pas...

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.

Une expression efficace de cellules de mammifères et en une seule étape de purification extracellulaire glycoprotéines à la cristallisation

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

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13.7K vues.  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.

Vidéo: 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.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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

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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.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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 des chaînes de sucre N-glycoprotéines liées à des cellules de mammifères

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.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules Namalwa

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.

EsiRNA MISSION pour le dépistage de l&#39;ARNi dans les cellules de mammifères

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.

Analyse de l&#39;expression de mammifères Linker-histones sous-types

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.

Agroinfiltration efficace des plantes pour l&#39;expression transitoire de haut niveau de protéines recombinantes

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.

La production efficace et purification de recombinant murin Kindlin-3 à partir de cellules d&#39;insectes pour les études biophysiques

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 de Global ARN de synthèse à la Cellule Plain suivante 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 contrôle pour la ubiquitine et les régulateurs du système ubiquitine-comme des voies biologiques dans des cellules de mammifères en culture

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