Nous remercions Olivier Brüstle et tous les membres du groupe d&#39;ingénierie de cellules souches de l&#39;Université de Bonn, de soutien et de discussions fructueuses. Nous remercions Sabine Schenk pour la préparation du SDS-PAGE et le soutien continu tout au long du projet. Nicole Russ et Magerhans Anna fourni un excellent soutien technique. Par ailleurs, nous tenons à remercier Andreas Bär et Mertens Sheila pour la production du film. Ce travail a été soutenu par des subventions de la Fondation Volkswagen (Az I/77864) et le ministère allemand de l&#39;Education et la Recherche (BMBF, 01 GN 0813).

Construction du vecteur d&#39;expression et d&#39;expression: Le vecteur d&#39;expression pSESAME-Cre a été construit en insérant un fragment de Cre-encodage dans pSESAME via des sites de restriction Avril et Nhel utilisant des méthodes de clonage standard. pSESAME code pour une protéine de fusion constituée d&#39;une histidine-tag, TAT-domaine, séquence NLS et la CRE, en abrégé HTNCre. Pour l&#39;expression de HTNCre pSESAME-Cre a été transformé en TUNER (DE3) Placi et utilisés pour préparer un stock de glycérol. <ol><li> Une culture durant la nuit a été inoculé avec une pointe de pipette enduits de bactéries transformées par le stock de glycérol. La culture du jour au lendemain se composait de milieu LB supplémenté en glucose 0,5% [v / v] et carbénicilline à une concentration finale de 50 pg / ml et a été autorisé à croître à 37 ° C pendant 16 heures. </li><li> Le lendemain, le plus densément cultivée nuit de la culture a été utilisée pour inoculer la culture d&#39;expression à un ratio de 1 à 40 et a été mis dans un incubateur à 37 ° C. Expression de la culture se composait des médias tuberculose complété par du glucose à 0,5% [v / v] et l&#39;ampicilline à une concentration finale de 100 pg / mL. </li><li> A une DO 595 de 1,5 à la culture d&#39;expression a été induite avec 0,5 mM d&#39;IPTG pendant 1 h. </li><li> Par la suite les bactéries ont été recueillies par centrifugation à 5000 rpm pendant 10 minutes dans un rotor SLA3000. </li><li> Les bactéries ont été granulés stockés à -20 ° C jusqu&#39;à la purification. </li></ol> Purification des cellules perméables protéines: <ol><li> Frozen granulés bactéries ont été remises en suspension dans 10 ml de tampon de lyse par culture flacon litre pendant 15 minutes à température ambiante. </li><li> La suspension a été ensuite incubés avec 1 mg / ml de lysozyme pour d&#39;autres 15 minutes tout en mélangeant à température ambiante. </li><li> 25 U / mL a été ajouté par la suite benzonase et incubé tout en mélangeant pendant 15 minutes à température ambiante. </li><li> Après sonication sur glace pendant 1,5 minutes avec des impulsions s 0,5 à 45% de la puissance, 1 ml de tampon froid sel tartrique (BST) par ml de suspension a été soigneusement ajouté tout en mélangeant et incubé pendant 5 min sur la glace. SDS-PAGE de la fraction de l&#39;échantillon lysat (L) a été prise. </li><li> Lysat clarifié a été obtenu par centrifugation à 4 ° C pendant 30 min à 30000 g. SDS-PAGE des échantillons de fractions solubles (S) et insoluble (I) ont été prises. </li><li> Le surnageant a été transféré dans de nouveaux tubes de 50 ml Falcon et a ensuite été délicatement mélangés pendant 1 h à 4 ° C avec 2 ml de 50% de Ni-NTA boue par litre de culture d&#39;expression initiale. </li><li> La suspension a été emballé dans une colonne à écoulement par gravité EconoPac (SDS-PAGE de l&#39;échantillon de l&#39;écoulement à travers la fraction (FT) a été prise) et lavé deux fois avec 5 volumes de lit de tampon de lavage. SDS-PAGE des échantillons des deux fractions de lavage (W1 et W2) ont été collectés. </li><li> HTNCre fractions contenant ont été élues avec 3 lits volumes de tampon d&#39;élution et l&#39;échantillon de la fraction d&#39;éluat (E) pour analyse SDS-PAGE a été prise. </li><li> Imidazole a été éliminée par dialyse contre le tampon d&#39;élution fraction élevée de sel à deux reprises. </li><li> La solution de protéine a été encore concentré par dialyse contre du tampon glycérol deux fois. Dans toutes les étapes de dialyse le ratio de tampon d&#39;échantillon a été d&#39;au moins 50. Cette procédure a abouti à une solution stock de glycérol contenant HTNCre à une concentration d&#39;habitude entre 200 et 450 uM, soit 1 litre de culture d&#39;expression se traduira par environ 12 mg de protéine. Exemple de stock glycérol (GS) pour analyse SDS-PAGE ont été recueillis. Solution stock HTNCre peuvent être stockés à moins 20 ° C. </li></ol><img border="0" src="/files/ftp_upload/1627/1627fig1tmb.jpg" alt="Figure 1" /> Figure 1: analyse SDS-PAGE des échantillons prélevés pendant le processus de purification de recombinase Cre. L&#39;induction de l&#39;expression de Cre est indiquée par la bande dominante de la fraction lysat. Bien qu&#39;une partie de la protéine est insoluble la protéine Cre peut encore être enrichi comme on le voit dans l&#39;éluat et les fractions d&#39;actions de glycérol. L: lysat, I: Insoluble, S: surnageant, FT: Flow-through, W: linge, E: éluat, GS: Stock Gylcerol. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1627/1627fig1.jpg">cliquez ici</a> pour voir une version agrandie de la figure 1. Transduction des protéines dans murin souches embryonnaires (ES) cellules: <ol><li> Les cellules ES portant une conditionnelle β-galactosidase journaliste de 5 construire ont été ensemencées en utilisant des cellules individuelles TrypLE ™ Express pour la dissociation des cellules adhérentes. Après 4 à 6 heures, les cellules ont re-joint et le milieu a été enlevé. </li><li> Par la suite les cellules ES ont été incubées avec un milieu contenant du HTNCre pendant 16 heures. <ul><li> Une quantité appropriée de protéines HTNCre (correspondant à 10 pM) sur le stock de glycérol a été dilué dans le milieu de ES et filtré par la suite stériles (0,22 m). </li></ul></li><li> Après moyennes de transduction de protéines a été modifié pour revenir à un milieu de croissance normal. </li><li> Après deux jours cellules ont été lavées avec du PBS et fixées avec du paraformaldéhyde 4% (PFA) pendant 10 minutes. </li><li> Deux suppléinternationale étapes de lavage avec du PBS ont été exécutés avant X-Gal coloration a été réalisée. </li><li> Les cellules fixées ont été couverts d&#39;une couche de solution de coloration X-Gal 6 et incubée pendant la nuit à 37 ° C. </li></ol> Les résultats représentatifs: Suivant une solution de coloration X-Gal jour a été aspiré et les cellules ont été recouvertes d&#39;une couche de PBS pour l&#39;analyse microscopique. 80 à 100% de cellules recombinées pu être observé dans les cellules ES murines jugés par β-galactosidase.

<ol>
	<li>Gump, J. M., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAT+transduction:+the+molecular+mechanism+and+therapeutic+prospects.">TAT transduction: the molecular mechanism and therapeutic prospects.</a> Trends Mol Med. 13 (10), 443-448 (2007).</li><li>Edenhofer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+transduction+revisited:+novel+insights+into+the+mechanism+underlying+intracellular+delivery+of+proteins.">Protein transduction revisited: novel insights into the mechanism underlying intracellular delivery of proteins.</a> Curr Pharm Des. 14 (34), 3628-3636 (2008).</li><li>Branda, C. S., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Talking+about+a+revolution:+The+impact+of+site-specific+recombinases+on+genetic+analyses+in+mice.">Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice.</a> Dev Cell. 6 (1), 7-28 (2004).</li><li>Nolden, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+recombination+in+human+embryonic+stem+cells+induced+by+cell-permeant+Cre+recombinase.">Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase.</a> Nat Methods. 3 (6), 461-467 (2006).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+site-directed+recombination+in+mouse+embryonic+stem+cells.">Inducible site-directed recombination in mouse embryonic stem cells.</a> Nucleic Acids Res. 24 (4), 543-548 (1996).</li><li>Peitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purification+of+cell-permeant+Cre+and+germline+transmission+after+transduction+into+mouse+embryonic+stem+cells.">Enhanced purification of cell-permeant Cre and germline transmission after transduction into mouse embryonic stem cells.</a> Genesis. 45 (8), 508-517 (2007).</li><li>Bosnali, M., Edenhofer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transducible+versions+of+transcription+factors+Oct4+and+Sox2.">Generation of transducible versions of transcription factors Oct4 and Sox2.</a> Biol Chem. 389 (7), 851-861 (2008).</li><li>Landry, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx+genes+are+direct+targets+of+Scl/Tal1+in+the+yolk+sac+and+fetal+liver.">Runx genes are direct targets of Scl/Tal1 in the yolk sac and fetal liver.</a> Blood. 111 (6), 3005-3014 (2008).</li></ol>

Pendant le processus de purification de la protéine fusion Cre il est important de ne pas omettre l&#39;ajout de la glace froide tampon TBS avant la centrifugation. Sinon recombinase Cre tend à précipiter dans la mémoire tampon glycérol. Si la fraction de l&#39;éluat semble devenir trouble dû à la forte concentration de protéine de fusion élution tampon supplémentaire doit être ajouté jusqu&#39;à ce que la solution a autorisé à nouveau. L&#39;application de 10 uM de la protéine fusion Cre entraîne habituellement une efficacité de recombinaison de 80 à 100%. Sérum de veau foetal (FCS) étant une composante importante du milieu de cellules ES inhibe fortement la transduction des protéines. Par conséquent une forte concentration de la recombinase Cre a dû être utilisé. Lorsque vous travaillez dans un milieu sans sérum moins de protéines (0,5 - 2 M) peut être utilisé pour réaliser des économies recombinaison similaires. Avec le système vectoriel pSESAME à portée de main, on peut appliquer la technique de transduction protéique à d&#39;autres protéines, y compris les facteurs de transcription tels que Oct4 et Sox2 7 et 8 Scl/Tal1.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>TUNER (DE3) pLacI</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70625</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3783.2</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>T876.1</td>
<td>&nbsp;</td>
<tr>
<td>Trizma Base</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>HCl</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>4625.1</td>
<td>&nbsp;</td>
<tr>
<td>Imidazol</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>X998.4</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>9265.2</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>2363.4</td>
<td>&nbsp;</td>
<tr>
<td>Trypton/Pepton</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>8952.4</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4 </td>
<td>&nbsp;</td>
<td>Roth</td>
<td>P749.2</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>3904.1</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9518</td>
<td>&nbsp;</td>
<tr>
<td>Carbenicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>6344.2</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-Tartaric acid, disodium salt </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50% Ni-NTA slurry</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>R901-15</td>
<td>&nbsp;</td>
<tr>
<td>EconoPac columns</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>732-1010</td>
<td>&nbsp;</td>
<tr>
<td>Sterile filter 0,22&mu;m</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (PFA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Yeast extract, Trypton/Pepton, NaCl</td>
</tr>
<tr>
<td>TB medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Yeast extract, Trypton/Pepton, Glycerol, K2HPO4, KH2PO4</td>
</tr>
<tr>
<td>Lysis Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50 mM Na2HPO4, 5 mM Tris, pH 7.8</td>
</tr>
<tr>
<td>Tartaric Salt Buffer (TSB)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM Imidazol</td>
</tr>
<tr>
<td>Washing Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB, 500 mM NaCl, 15 mM Imidazol</td>
</tr>
<tr>
<td>Elution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB, 500 mM NaCl, 250 mM Imidazol</td>
</tr>
<tr>
<td>High Salt Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>600 mM NaCl, 20 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>Gylcerol Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50% glycerol, 500 mM NaCl, 20 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>TrypLE&trade; Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ESGRO (LIF)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NEAA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11140035</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030024</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>31350010</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960044</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>PAA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal staining solution:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4 mM K3(FeIII(CN)6), 4 mM K4(FeII(CN)6), 2mM MgCl2 0.4 mg/mL X-Gal solved in PBS</td>
</tr>
<tr>
<td>K3(FeIII(CN)6)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-3367</td>
<td>&nbsp;</td>
<tr>
<td>K4 (FeII(CN)6)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-9387</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B4252</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

engineering cell-permeable protein

La technique de transduction de protéines permet la livraison directe du matériel biologiquement active dans les cellules de mammifères [pour revue, voir 1,2]. Pour cela, on peut faire usage de la capacité translocation des peptides cellule dite pénétrante (CPP), également désigné comme domaines de transduction protéique (DAP). Le TAT-RPC dérivés du type de virus de l&#39;immunodéficience humaine 1 (VIH-1) Tat (trans-activateur de la transcription) des protéines a été largement utilisé. Les chargés positivement TAT favorise la perméabilité des cellules permettant ainsi de surmonter les barrières de la membrane cellulaire par endocytose et / ou la pénétration de membrane directe 2. En combinaison avec un signal de localisation nucléaire (NLS) des protéines de fusion sont capables d&#39;entrer dans le noyau de fonctionnalité exposantes. Notre vidéo de présentation démontre, comme une ampliation de l&#39;ingénierie des protéines cellulaires perméables, la construction, la production et l&#39;application d&#39;une version de cellules-perméable de la CRE ADN modification enzymatique. Cre est une recombinase site-spécifique qui est capable de reconnaître et de se recombiner 34 sites loxP dans paires de base des cellules de mammifères in vitro et in vivo. Par conséquent, le système Cre / loxP est largement utilisé pour induire des mutations conditionnellement dans le génome de 3,4 cellules vivantes. La livraison des actifs de la recombinase Cre dans les cellules, cependant, représente une limitation. Nous décrivons le système vectoriel pSESAME, ce qui permet une insertion directe du gène d&#39;intérêt et fournit une plateforme pour cloner rapidement les différents domaines et les tags utilisés dans le vecteur d&#39;une manière commode et standardisés. Réorganisation des balises différentes a été montré pour modifier les propriétés biochimiques des protéines de fusion offrant une possibilité d&#39;atteindre un rendement supérieur et une meilleure solubilité. Nous démontrons comment exprimer et de purifier les cellules recombinantes perméants protéines à l&#39;intérieur et de E. coli. La fonctionnalité de la protéine recombinante Cre est finalement validé en culture cellulaire en évaluant son activité recombinase intracellulaire.

Watch this Scientific Journal Video about Engineering Cell-permeable Protein at JoVE.com

Engineering Cell-permeable Protein

Transduction des protéines permet la livraison directe de protéines biologiquement actives dans les cellules. Contrairement aux méthodes conventionnelles telles que la transfection d&#39;ADN ou de transduction virale ce paradigme non invasive permet une manipulation très efficace cellulaires de façon titrable contourner la toxicité cellulaire et le risque de transformation oncogénique par modification génétique permanente.

Ingénierie cellulaire perméable protéines

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Research

JoVE Journal

Biology

14.4K vues.  University of Bonn - Life & Brain Center and Hertie Foundation. Transduction des protéines permet la livraison directe de protéines biologiquement actives dans les cellules. Contrairement aux méthodes conventionnelles telles que la transfection d'ADN ou de transduction virale ce paradigme non invasive permet une manipulation très efficace cellulaires de façon titrable contourner la toxicité cellulaire et le risque de transformation oncogénique par modification génétique permanente.

Vidéo: Engineering Cell-permeable Protein

Experimental Approaches to Tissue Engineering

Approches expérimentales pour le génie tissulaire

Recherche

Biologie

Micro-scale Engineering for Cell Biology

Micro-échelle d&#39;ingénierie de biologie cellulaire

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 ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Encapsulation Cellulaire dans les hydrogels 3D pour l&#39;ingénierie tissulaire

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utiliser le système de fractionnement GELFREE 8100 pour les Poids Moléculaire à base de fractionnement avec récupération de la phase liquide

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Automatisé d&#39;interaction hydrophobe Chromatographie sur colonne de sélection pour l&#39;utilisation dans la purification des protéines

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

Protéines SAGESSE: un plan de travail pour<em&gt; In silico</em&gt;<em&gt; De novo</em&gt; Conception de biomolécules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Souris ingénierie des génomes Utilisation nucléases de créateurs

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

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 base Expression dépistage des protéines membranaires dans<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 ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Protein livraison directe aux cellules de mammifères en utilisant la cellule perméable Cys<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt; Domaines doigt de zinc

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...