Wir danken Oliver Brüstle und alle Mitglieder des Stem Cell Engineering Group, Universität Bonn, für die Unterstützung und wertvolle Diskussionen. Wir bedanken uns bei Sabine Schenk für die Vorbereitung des SDS-PAGE und dauerhafte Unterstützung während des gesamten Projekts. Nicole Russ und Anna Magerhans vorgesehen ausgezeichneten technischen Support. Darüber hinaus möchten wir Andreas Bär und Sheila Mertens für die Produktion des Films zu danken. Diese Arbeit wurde durch Zuschüsse aus dem Volkswagen-Stiftung (Az I/77864) und dem deutschen Bundesministerium für Bildung und Forschung (BMBF, 01 GN 0813) unterstützt.

Bau von Expressionsvektor und Ausdruck: Die pSESAME-Cre Expressionsvektor wurde durch Einfügen eines Cre-encoding-Fragment in pSESAME über AvrII und NheI-Restriktionsstellen mit Standard-Klonierungsverfahren konstruiert. pSESAME kodiert für ein Fusionsprotein, bestehend aus einem Histidin-Tag, TAT-Domäne, NLS-Sequenz und Cre, abgekürzt HTNCre. Für die Expression von HTNCre die pSESAME-Cre wurde in TUNER (DE3) Placi umgewandelt und verwendet, um eine Glycerin Lager vorzubereiten. <ol><li> Eine Über-Nacht-Kultur beimpft mit einer Pipettenspitze mit transformierten Bakterien aus dem Glycerin Lager beschichtet. Die Über-Nacht-Kultur bestand aus LB-Medium mit 0,5% Glucose [v / v] und Carbenicillin in einer Endkonzentration von 50 ug / mL und durfte bei 37 ° C wachsen für 16 Stunden. </li><li> Am nächsten Tag dicht gewachsen über Nacht Kultur wurde verwendet, um den Ausdruck Kultur bei einem Verhältnis von 1 bis 40 zu impfen und wurde in einem Brutschrank bei 37 ° C stellen Expression Kultur bestand aus TB-Medien mit 0,5% Glucose [v / v] und Ampicillin in einer Endkonzentration von 100 ug / ml ergänzt. </li><li> Bei einer OD 595 von 1.5 die Expression Kultur wurde mit 0,5 mM IPTG für 1 h induzierten </li><li> Anschließend wurden die Bakterien durch Zentrifugation bei 5000 rpm für 10 Minuten in einem SLA3000 Rotor gesammelt. </li><li> Bakterien-Pellets wurden bei minus 20 ° C bis zur Reinigung gespeichert. </li></ol> Reinigung von zellpermeablen Protein: <ol><li> Gefrorene Bakterien-Pellets wurden in 10 mL Lysispuffer pro Liter Flasche Kultur für 15 Minuten bei Raumtemperatur resuspendiert. </li><li> Suspension wurde dann mit 1 mg / ml Lysozym für weitere 15 Minuten inkubiert beim Mischen bei Raumtemperatur. </li><li> 25 U / ml Benzonase wurde später und fügte hinzu, inkubiert beim Mischen für 15 Minuten bei Raumtemperatur. </li><li> Nach Ultraschallbehandlung auf Eis für 1,5 min mit 0,5 s Impulse bei 45% der Leistung, 1 ml kaltem Wein-Salz-Puffer (TSB) pro ml Suspension wurde sorgfältig während zugegeben, die Vermischung und inkubiert für 5 min auf Eis. SDS-PAGE-Probenpuffer Lysat Fraktion (L) aufgenommen wurde. </li><li> Cleared Lysat wurde durch Zentrifugation bei 4 ° C für 30 min bei 30.000 g erhalten SDS-PAGE-Proben von löslichen (S) und unlöslichen Fraktionen (I) aufgenommen wurden. </li><li> Der Überstand wurde in frische 50 ml-Falcon-Röhrchen überführt und wurde dann vorsichtig für 1 h bei 4 ° C gemischt mit 2 ml 50% Ni-NTA Gülle pro Liter ersten Ausdruck Kultur. </li><li> Die Suspension wurde in ein Fließ EconoPac Spalte (SDS-PAGE Stichprobe von Flow-Through-Fraktion (FT) aufgenommen wurde) verpackt und zweimal mit 5-Bett-Waschpuffer. SDS-PAGE-Proben der beiden Waschfraktionen (W1 &amp; W2) gesammelt wurden. </li><li> HTNCre-haltigen Fraktionen wurden mit 3-Bett-Elutionspuffer und Probe Eluatfraktion (E) für die SDS-PAGE-Analyse eluiert wurde. </li><li> Imidazol wurde durch Dialyse Elutionsfraktion gegen hohe Salz-Puffer zweimal entfernt. </li><li> Die Protein-Lösung wurde durch Dialyse gegen Glycerin-Puffer zweimal konzentriert. In aller Dialysepatienten Schritte das Verhältnis von Puffer zu Probe wurde mindestens 50. Dieses Verfahren führte zu einem Glycerin-Stammlösung enthält HTNCre in einer üblichen Konzentration zwischen 200 und 450 uM, dh 1 Liter Ausdruck Kultur in ~ 12 mg des Proteins führen. Beispiel Glycerin Lager (GS) für die SDS-PAGE-Analyse gesammelt. HTNCre Stammlösung kann bei minus 20 ° C gelagert werden </li></ol><img border="0" src="/files/ftp_upload/1627/1627fig1tmb.jpg" alt="Abbildung 1" /> Abbildung 1: SDS-PAGE-Analyse der Proben während des Reinigungsprozesses der Rekombinase Cre gesammelt. Induktion der Cre Expression wird durch dominante Bande in Lysat Bruchteil angezeigt. Obwohl ein Teil des Proteins ist unlöslich das Cre-Protein kann weiter angereichert werden, wie in Eluat und Glycerin Lager Fraktionen gesehen. L: Lysate, I: Unlöslich, S: Überstand, FT: Flow-through, W: Waschen, E: Eluat, GS: Gylcerol Lager. Bitte <a href="https://www.jove.com/files/ftp_upload/1627/1627fig1.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 1 zu sehen. Protein Transduktion in murine embryonale Stammzellen (ES-Zellen): <ol><li> ES-Zellen, die eine bedingte β-Galactosidase-Reporter 5. Konstruieren Sie wurden als einzelne Zellen mit Hilfe TrypLE ™ Express für die Dissoziation von anhaftenden Zellen ausgesät. Nach 4 bis 6 Stunden wurden die Zellen hatten wieder angebracht und das Medium wurde entfernt. </li><li> Anschließend ES-Zellen wurden mit HTNCre-haltigem Medium für 16 Stunden inkubiert. <ul><li> Eine entsprechende Menge HTNCre Protein (entspricht 10 nM) aus dem Glycerin Lager wurde in ES-Medium und anschließend sterilfiltriert (0,22 um) verdünnt. </li></ul></li><li> Nach Proteintransduktionsdomäne Medium wurde wieder auf das normale Wachstum Medium gewechselt. </li><li> Nach zwei Tagen wurden die Zellen mit PBS gewaschen und mit 4% Paraformaldehyd (PFA) für 10 Minuten. </li><li> Zwei zusätzlichezusätzliche Waschschritte mit PBS wurden hingerichtet, bevor X-Gal-Färbung durchgeführt wurde. </li><li> Fixierten Zellen wurden mit einer Schicht von X-Gal Färbelösung 6 abgedeckt und inkubiert über Nacht bei 37 ° C. </li></ol> Repräsentative Ergebnisse: Am nächsten Tag X-Gal Färbelösung wurde angestrebt und die Zellen wurden mit einer Schicht von PBS für die Mikroskopie Analyse abgedeckt. 80 bis 100% der rekombinierten Zellen konnten in den murinen ES-Zellen durch β-Galactosidase-Aktivität gemessen beobachtet werden.

<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>

Während des Reinigungsprozesses des Cre-Fusionsprotein ist es wichtig, nicht auf die Zugabe von eiskaltem TBS-Puffer vor dem Zentrifugieren weglassen. Ansonsten Rekombinase Cre tendenziell Niederschlag in der Glycerin-Puffer. Wenn die Eluatfraktion scheint trüb aufgrund der hohen Konzentration des Fusionsproteins zusätzliche Elutionspuffer sollte hinzugefügt werden, bis die Lösung wieder gelöscht hat. Die Anwendung von 10 pM Cre-Fusionsprotein führt in der Regel eine Rekombination Effizienz von 80 bis 100%. Fötalem Kälberserum (FCS), ein wichtiger Bestandteil der ES-Zell-Medium stark hemmt Proteintransduktionsdomäne. Deshalb hohe Konzentration von Cre-Rekombinase eingesetzt werden mussten. Bei der Arbeit in serum-freien Bedingungen weniger Protein (0,5 bis 2 M) kann verwendet werden, um ähnliche Rekombination Wirkungsgrade zu erreichen. Mit dem pSESAME Vektor-System zur Hand kann man die Technik der Proteintransduktionsdomäne an andere Proteine ​​einschließlich Transkriptionsfaktoren wie Oct4 und Sox2 7 und 8 gelten 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

Das Protein Transduktion Technik ermöglicht die direkte Abgabe von biologisch aktiven Materials in Säugerzellen [zur Übersicht siehe 1,2]. Für diese Verwendung des translozierenden Fähigkeit der sogenannten Zell-penetrierende Peptide (CPP) machen kann, auch als Proteintransduktionsdomänen (PTDs) bezeichnet. Das TAT-CPP aus dem humanen Immundefizienz-Virus Typ 1 (HIV-1) Tat (trans-Aktivator der Transkription)-Protein wurde in großem Umfang eingesetzt abgeleitet. Die positiv geladenen TAT fördert Zellpermeabilität damit die Überwindung der Barrieren der Zellmembran durch Endozytose und / oder direkte Membranpenetration 2. In Kombination mit einem Kernlokalisierungssignal (NLS) Fusionsproteine ​​sind in der Lage, den Kern aufweisen Funktionalität geben. Unsere Video-Präsentation demonstriert, wie eine Veranschaulichung für das Engineering von zellpermeablen Proteine, die Konstruktion, Produktion und Anwendung eines zellpermeablen Version des DNA-modifizierende Enzyme Cre. Cre ist eine ortsspezifische Rekombinase, die in der Lage zu erkennen und zu rekombinieren 34 Basenpaar loxP-Stellen in Säugetierzellen in vitro und in vivo ist. Deshalb ist die Cre / loxP-System ist weit verbreitet bedingt induzieren Mutationen im Genom lebender Zellen 3,4. Die Lieferung von aktiven Cre-Rekombinase zu Zellen, stellt jedoch eine Einschränkung. Wir beschreiben die pSESAME Vektor-System, das eine direkte Insertion des Gens-of-Interest können und bietet eine Plattform, um schnell Klon unterschiedlichen Domänen und-Tags innerhalb des Vektors in eine komfortable und standardisierte Art und Weise verwendet. Umstellen der verschiedenen tags wurde gezeigt, dass die biochemischen Eigenschaften der Fusionsproteine ​​bietet die Möglichkeit, höhere Ausbeute und bessere Löslichkeit zu erreichen ändern. Wir zeigen, wie zu äußern und zu reinigen rekombinanten Zell-Permeationsmittel Proteine ​​in und aus E. coli. Die Funktionalität des rekombinanten Proteins ist Cre schließlich in der Zellkultur durch die Bewertung ihrer intrazellulären Rekombinase-Aktivität geprüft.

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

Engineering Cell-permeable Protein

Protein Transduktion ermöglicht die direkte Abgabe von biologisch aktiven Proteinen in Zellen. Im Gegensatz zu herkömmlichen Methoden wie DNA-Transfektion oder virale Transduktion dieser nicht-invasiven Paradigma ermöglicht hoch effiziente zelluläre Manipulation in eine titrierbare Weise umgehen zelluläre Toxizität und das Risiko der onkogenen Transformation durch permanente genetische Veränderung.

Engineering zellpermeablen Protein

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 Aufrufe.  University of Bonn - Life & Brain Center and Hertie Foundation. Protein Transduktion ermöglicht die direkte Abgabe von biologisch aktiven Proteinen in Zellen. Im Gegensatz zu herkömmlichen Methoden wie DNA-Transfektion oder virale Transduktion dieser nicht-invasiven Paradigma ermöglicht hoch effiziente zelluläre Manipulation in eine titrierbare Weise umgehen zelluläre Toxizität und das Risiko der onkogenen Transformation durch permanente genetische Veränderung.

Serie: Engineering Cell-permeable Protein

Experimental Approaches to Tissue Engineering

Experimentelle Ansätze zur Tissue Engineering

Forschung

Biologie

Micro-scale Engineering for Cell Biology

Micro-Maßstab Engineering for Cell Biology

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 ...

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.

Cellular Encapsulation in 3D Hydrogele für Tissue Engineering

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.

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

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.

Automatisierte Hydrophobe Interaktions-Chromatographie Spaltenauswahl für den Einsatz in Protein Purification

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.

Protein WISDOM: A Workbench für<em&gt; In silico</em&gt;<em&gt; De novo</em&gt; Aufbau von Biomolekülen

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.

Maus-Genom-Engineering mit Designer Nukleasen

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

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

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.

Direkter Protein Lieferung Säugerzellen mit Zellpermeable Cys<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt; Zink-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 ...