Diese Arbeit wurde von den National Institutes of Health (DP1CA174426 Carlos F. Barbas) und ShanghaiTech Universität, Shanghai, China (JL) unterstützt. Molekulare Grafiken wurden mit PyMol erzeugt.

1. Klonierung <ol><li> Erhalten Alanin-substituierten Zwei-Finger-ZiF Domänen, die in den pET-28 Expressionsvektorsystem subkloniert haben und sind auf Anfrage erhältlich (pET-2F-ZiF). 34 </li><li> PCR zu amplifizieren EmGFP aus dem Plasmid pBAD-Smaragd mit den Primern 5 &#39;XmaI-EmGFP (5&#39;-GGAAATTG CCCGGG ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#39;; XmaI Website in fett) und 3&#39;-SacI-EmGFP (5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3 &#39; ; SacI-Stelle fett gedruckt). <ol><li> Verwenden 5 ng Template-DNA, 10 ul 10x-Polymerasepuffer, 1-Einheiten (U) Taq DNA-Polymerase, 0,2 mM von jedem dNTP und 0,2 &amp; mgr; M von jedem Primer in 100 &amp; mgr; l Lösung mit dem Restvolumen bis destilliertem / deionisiertem Wasser . Verwenden Sie die Zyklusbedingungen: 95 ° C für 5 min; 30 Zyklen von 95 ° C für 30 sec, 55 ° C für 30 sec und 72 ° C für 1 min; 72 ° C für 10 min. Ein Reinigen des PCR-Produkts durch Gel extractiauf und bestimmen, DNA-Konzentration unter Verwendung eines Spektrophotometers Mess Abs 260 x 50 &amp; mgr; g / ml. </li></ol></li><li> Digest pET-2F ZIF und das Insert EmGFP mit den Restriktionsenzymen Xma kodiert I und Sac I in empfohlenen Puffer für 3 h bei 37 ° C unter Verwendung von 10 U Enzym pro 1 ug DNA. Visualisieren DNA durch Agarose-Gelelektrophorese unter Verwendung eines fluoreszierenden interkalierenden Farbstoff wie Ethidiumbromid. </li><li> Reinige den verdauten DNA durch Gelextraktionskit und bestimmen, DNA-Konzentration mit einem Spektrophotometer Mess Abs 260 x 50 &amp; mgr; g / ml. </li><li> Ligation der gereinigten EmGFP kodierenden DNA in 50 bis 100 ng pET-2F-ZiF mit 1 U T4-DNA-Ligase für mindestens 1 h bei RT. Die besten Ergebnisse erzielen, führen Sie die Ligationsreaktion mit einem 6: Einsatz-zu-Vektor-Molverhältnis 1. </li><li> Thaw 50 ul aller chemisch kompetente XL-1 Blue E. coli Zellen auf Eis geben und vorsichtig mischen mit 10-20 ng ligierte pET-2F-ZiF-EmGFP. </li><li> Halten Sie auf Eis für 30 min. Hitzeschock die Mischung bei 42 ° C für 90 sec und erholen sich die Zellen in 2 ml SOB-Medium mit Katabolitrepression (SOC) für 1 Stunde bei 37 ° C unter Schütteln. </li><li> Verbreiten Sie 100 ul der Erholung Kultur auf einem LB-Medium (LB) Agar-Platte mit 50 ug / ml Kanamycin und Inkubation O / N bei 37 ° C. </li><li> Am folgenden Tag inokulieren 6 ml Super Broth (SB) oder LB-Kultur, die 50 ug / ml Kanamycin mit einer Kolonie von der LB-Agar-Platte und Kultur O / N bei 37 ° C. Hinweis: Kolonie-PCR unter Verwendung der Primer 5 &#39;XmaI-EmGFP und 3&#39;-SacI-EmGFP könnte verwendet werden, um positive Klone zu identifizieren, bevor Miniprep werden. </li><li> Reinige pET-2F ZIF-EmGFP durch Miniprep und Plasmid bestätigt durch DNA-Sequenzierung mit T7-Promotors (5&#39;-TAATACGACTCACTATAGGG-3 &#39;). </li></ol> 2. Expression und Reinigung <ol><li> Auftauen 50 ul chemisch kompetente BL21 E. coli-Zellen auf Eis geben und vorsichtig mischen mit 100 ng pET-2F-ZiF-EmGFP plasmid. Transform wie in Schritten von 1,7 bis 1,8 beschrieben. </li><li> Am folgenden Tag impfen 10 ml LB-Medium, das 50 ug / ml Kanamycin mit einer Einzelkolonie und wachsen O / N bei 37 ° C unter Schütteln. </li><li> Am folgenden Tag, verdünnt den 10 ml des O / N-Kultur in 1 l LB-Medium mit 50 ug / ml Kanamycin, 0,2% Glucose und 100 &amp; mgr; M ZnCl 2 ergänzt. Die Kultur bei 37 ° C unter Schütteln bis zu einer optischen Dichte bei 600 nm (OD 600) von 0,8 und induzieren Proteinexpression mit 2 mM Isopropyl-β-D-1-thiogalactopyranosid (IPTG). Nach 6 Stunden Wachstum bei 37 ° C, Ernten der Zellen durch Zentrifugation bei 5.000 × g für 10 min bei 4 ° C. Anmerkung: Induktionsbedingungen sind sehr variabel und hängt von der Stabilität der Proteine ​​exprimiert. Überwachen der OD 600 alle 30 Minuten, bis eine OD 600 von 0,8 erreicht wird. </li><li> Zellpellet in 5 ml Lysepuffer (50 mM Tris-HCl, 500 mM NaCl, 100 &amp; mgr; M ZnCl 2, 1 mM Dithiothreitol (DTT), 1 mM MgCl 2, 1 mM Phenylmethylsulfonylfluorid (PMSF) und 10 mM Imidazol, pH 8,0). Lyse der Zellen auf Eis durch Ultraschallbehandlung mit folgender Einstellung: 50% der Leistung, 4 min Prozesszeit mit 5 s an / 10 sec aus Intervallen. Vermeiden Sie eine Überhitzung der Lösung. </li><li> Zentrifugieren Sie das Zelllysat bei 25.000 · g für 30 min bei 4 ° C und den Überstand in ein frisches Collection Tube. Die besten Ergebnisse erzielen, führen Sie alle folgenden Schritte bei 4 ° C. </li><li> Führen des Überstandes durch eine Säule mit 1 ml äquilibriert Ni-NTA Aufschlämmung vorverpackt. Mit 20 ml Waschpuffer (50 mM Tris-HCl, 500 mM NaCl, 100 &amp; mgr; M ZnCl 2, 1 mM DTT, 1 mM MgCl 2 und 30 mM Imidazol, pH 8,0) Waschen des Harzes. </li><li> Eluieren des Proteins mit 5 ml Elutionspuffer (50 mM Tris-HCl, 500 mM NaCl, 100 &amp; mgr; M ZnCl 2, 1 mM DTT, 1 mM MgCl 2 und 300 mM Imidazol, pH 8,0). </li><li> Buffer tauschen die eluierte Protein mit Speicherpuffer (50 mMTris-HCl, 500 mM NaCl, 100 &amp; mgr; M ZnCl 2, 1 mM DTT, 1 mM MgCl 2 und 10% Glycerin, pH 8,0) und konzentriere das Protein zu mindestens 40 um unter Verwendung einer Spin-Konzentrator durch Zentrifugation gemäß den Anweisungen des Herstellers. </li><li> Bestimmen der Proteinkonzentration von BCA oder Bradford-Test. Mix 2 ul gereinigte Proteine ​​mit 2 &amp; mgr; l 2 × SDS-PAGE-Ladepuffer, Kochen bei 95 ° C für 10 min und dann auf 4% zu lösen -20% Tris-Glycin-SDS-PAGE, um die Proteinreinheit (2) zu bewerten. </li></ol> 3. Protein Lagerung <ol><li> Aliquot des konzentrierten Protein, Flash Einfrieren in flüssigem Stickstoff und bei -80 ° C. Für EmGFP Fusionsproteine ​​umfassen das Röhrchen mit Aluminiumfolie, um das Protein vor Licht zu schützen. </li><li> Vermeiden Sie wiederholtes Einfrieren und Auftauen der Proteinlösung. Hinweis: ZiF-Fusionsproteine ​​sind für mindestens 1 Monat unter diesen Bedingungen stabil. Brachte oder&gt; 4 Monate Lagerung führen kannum Proteinfällung oder Bleichen von EmGFP. </li></ol> 4. Protein Transduction <ol><li> Pflegen HeLa-Zellen in Dulbeccos modifiziertem Eagle-Medium (DMEM), das 10% (v / v) fötalem Rinderserum (FBS) und 1% Antibiotikum-Antimykotikum bei 37 ° C in einer vollständig befeuchteten Atmosphäre mit 5% CO 2. Anmerkung: Die Zellen passagiert mehr als 30 Mal werden für Proteintransduktionsdomäne empfohlen. </li><li> Vorbeschichtung eine Platte mit 24 Vertiefungen mit 500 ul von 50 ug / ml poly-Lysin für 30 bis 60 min bei 25 ° C. Saatgutzellen auf eine Platte mit 24 Vertiefungen in einer Dichte von 2 × 10 5 Zellen pro Vertiefung. Bei 24 Stunden nach dem Aussäen oder einmal Zellen zwischen 80% -90% konfluent, entfernen Medien aus jeder Vertiefung und Waschen mit 500 ul vorgewärmtem serumfreiem DMEM (SFM). </li><li> Zu jeder Vertiefung, fügen Sie 250 ul SFM mit 2 uM ZiF-EmGFP Protein und 100 uM ZnCl 2. Zellen bei 37 ° C für 1 Stunde. Hinweis: ZiF Domains in die Zellen primarily durch Makropinozytose, 34, die eine energieabhängige Mechanismus ist. Daher müssen die Zellen bei 37 ° C für eine effiziente Protein Internalisierung inkubiert werden. </li><li> Entfernen Medien von Zellen, und dreimal mit 500 ul calcium- und magnesiumfreien Dulbeccos phosphatgepufferter Salzlösung (DPBS) mit 0.5 mg / ml Heparin ergänzt. Hinweis: Heparin ist notwendig, um oberflächengebundene Protein, das Downstream-Analysen erschweren könnten, zu entfernen. </li><li> (Optional) Wiederholen Sie die Behandlung bis zu dreimal für erhöhte Auslieferung. </li><li> Isolieren behandelten Zellen unmittelbar nach Heparin Wasch durch Verdau mit 0,05% Trypsin-EDTA bei 37 ° C für 2 min. Resuspendieren abgelösten Zellen in 0,5 ml DPBS mit 1% FBS ergänzt. </li><li> Messen der Fluoreszenzintensität jeder Probe durch Flusszytometrie 35 mit der Fluorescein-Isothiocyanat (FITC) Kanal (Abbildung 3). Stellen Sie die Vorwärtsstreuung (FSC) und Seitwärtsstreuung (SSC), die Bevölkerung zu platzierenInteresse auf der Skala, um sicherzustellen, dass Populationen mit unterschiedlichen Eigenschaften voneinander gelöst. </li><li> Sammle 10.000 Live-Veranstaltungen für jede Probe und die Daten mit Hilfe der Durchflusszytometrie Datenanalyse-Software. 36 Normalisieren Fluoreszenz von HeLa-Zellen mit ZiF-EmGFP auf diese Zellen nur mit SFM behandelten analysieren. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Aart,+a+designed+six-finger+zinc+finger+peptide,+bound+to+DNA.">Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA.</a> J. Mol. Biol. 363, 405-421 (2006).</li></ol>

Die Autoren haben nichts zu offenbaren.

Hier wird ein Schritt-für-Schritt-Protokoll für Proteinabgabe mit zellpermeablen Zinkfinger (ZIF) Domänen dargestellt. Das ZiF Domain nicht senken die Aktivität verschmolzen enzymatische Fracht 34; erlaubt die Herstellung und Reinigung von Proteinen in Ausbeuten fast identisch mit denen mit unmodifizierten Protein beobachtet; und kann Proteine ​​und Enzyme mit Wirkungsgraden, die traditionelle zellpenetrierenden Peptid oder Proteintransduktionsdomäne Systeme übersteigen, in einer Vielzahl von Zellty...

Hoch effizient und vielseitig Protein Lieferstrategien sind entscheidend für viele der Grundlagenforschung und therapeutische Anwendungen. Die direkte Lieferung von gereinigten Proteinen in Zellen ist eine der sichersten und einfachsten Methoden, um dies zu erreichen. Im Gegensatz zu 1,2-Strategien, die auf die Genexpression von Nukleinsäuren beruhen, stellt 3-5 Protein Liefer kein Risiko einer Insertionsmutagenese, ist unabhängig von der zelluläre Transkriptions / Translations-Maschinen und ermöglicht eine sofortige Wirkung. Jedoch das Fehlen einfacher und verallgemeinerbar Methoden auszustatten zellpenetrierenden Aktivität an Proteine ​​routine confounds ihrer direkten Eintritt in die Zellen. Aktuelle Verfahren zur Erleichterung der intrazellulären Proteinabgabe auf der Verwendung von natürlich vorkommenden oder 6-8 ausgebildet zellpenetrierenden Peptide 9-12 aufgeladenen Transduktion Domänen 13,14 Nanopartikel 15 und Liposomen, 16 virusähnlichen Partikeln 17,18 basierend </sup&gt; und polymere Mikromaterialien. 19. Leider sind viele dieser Ansätze zeichnen sich durch geringe zelluläre Aufnahme Tarife, 20,21 schlechte Stabilität, 22 unbeabsichtigte Zelltyp-Spezifität, 23 Low endosomalen Flucht Objekte 24 und 25 neben Toxizität. behindert, viele Protein Übertragungstechnologien reduzieren die Bioaktivität der Proteine ​​geliefert. 14 In unserem Labor wurde gezeigt, dass Zinkfinger-Nuclease (ZFN) Proteine ​​- chimären Restriktionsendonukleasen aus einem programmierbaren Cys 2 -His 2 Zinkfinger-DNA-Bindungsprotein und die Spaltdomäne des FokI Restriktionsendonuklease 26-28 - inhärent zell- durchlässig. Es wurde gezeigt, 29 Diese überraschende zellpenetrierenden Aktivität eine intrinsische Eigenschaft des kundenspezifischen Zinkfinger-Domäne, einer DNA-bindenden Plattform, die als ein leistungsfähiges Werkzeug für die gezielte Genom en hervorgegangen sein,Engineering, 30-32 und als das Ergebnis der Konstellation von sechs positiv geladene Reste auf der Proteinoberfläche sein. Tatsächlich haben mehrere DNA-bindenden Proteinen, einschließlich c-Jun und N-DEK wurde gezeigt, dass eine angeborene Fähigkeit, Zellmembranen zu durchqueren besitzen. 33 In jüngerer Zeit haben unser Labor auf diese Ergebnisse und zeigten, dass die Zellen eindringende Aktivität zink- Finger (ZiF) Domains können für den intrazellulären Proteintransport genutzt werden. Genetische Fusion von entweder einem oder zwei Finger ZiF Domänen an spezifische Proteinfracht, ergab einen Wirkungsgrad, die viele herkömmliche zellpenetrierende Peptid Abgabesysteme überschritten Aufnahme. 34 Vor allem ZiF-vermittelte Abgabe nicht beeinträchtigen die Aktivität des fusionierten enzymatische Ladung und erleichtert hohe cytosolische Lieferung. Zusammen zeigen diese Ergebnisse zeigen das Potenzial der ZiF Domäne zur Erleichterung der effizienten und einfachen Verabreichung von Proteinen und möglicherweise mehrere verschiedene Arten von MakroMoleküle, in Zellen. Hier wird eine detaillierte Schritt-für-Schritt-Protokoll zur Implementierung ZiF Technologie für Proteintransport in Säugerzellen präsentiert. Wir zuvor konstruierten eine Reihe von ein-, zwei-, drei-, vier-, fünf- und sechs Finger ZiF Domänen, die die Fähigkeit zur Bindung von DNA, durch Substitution von jeder der α-helicalen DNA-bindenden Reste fehlen, aber fähig sind, liefert Proteine ​​in Zellen 34 (Abbildung 1). Die Herstellung und die Transduktion von Smaragd GFP (EmGFP) in HeLa-Zellen unter Verwendung eines Zwei-Finger-Domäne ZiF beschrieben. Dieses Protokoll ist erweiterbar, um fast jedes Protein in der Lage, lösliche Expression in Escherichia coli und fast jedem Säugerzelltyp. Erwartete Ergebnisse werden bereitgestellt und Strategien für die Maximierung der Leistung des Systems ausgelegt werden ebenfalls diskutiert.

<table><tbody><tr><td>XmaI</td><td>New England Biolabs</td><td>R0180L</td><td></td></tr><tr><td>SacI</td><td>New England Biolabs</td><td>R0156L</td><td></td></tr><tr><td>Expand High Fidelity PCR system</td><td>Roche</td><td>11759078001</td><td></td></tr><tr><td>dNTPs</td><td>New England Biolabs</td><td>N0446S</td><td></td></tr><tr><td>4%-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td><td>Life Technologies</td><td>EC6028BOX</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>BioRad</td><td>161-0737</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>Life Technologies</td><td>15224-017</td><td></td></tr><tr><td>BL21 (DE3) Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>New England Biolabs</td><td>C2527I</td><td></td></tr><tr><td>IPTG</td><td>Thermo Scientific</td><td>R0391</td><td></td></tr><tr><td>Zinc Chloride</td><td>Sigma-Aldrich</td><td>208086-5G</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>Fisher Scientific</td><td>BP906-5</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270-100G</td><td></td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-25</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S9888-25G</td><td></td></tr><tr><td>DTT</td><td>Fisher Scientific</td><td>PR-V3151&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Thermo Scientific</td><td>36978</td><td></td></tr><tr><td>Ni-NTA Agarose Resin</td><td>QIAGEN</td><td>30210</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516-500ML</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I5513-25G</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units</td><td>EMO Millipore</td><td>UFC900324</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11966-025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10437-028</td><td></td></tr><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>Life Technologies</td><td>15240-062</td><td></td></tr><tr><td>24-Well Flat Bottom Plate</td><td>Sigma-Aldrich</td><td>CLS3527-100EA</td><td></td></tr><tr><td>Poly-Lysine</td><td>Sigma-Aldrich</td><td>P7280</td><td></td></tr><tr><td>DPBS, No Calcium, No Magnesium</td><td>Life Technologies</td><td>21600010</td><td></td></tr><tr><td>Heparan Sulfate</td><td>Sigma-Aldrich</td><td>H4777</td><td></td></tr><tr><td>Trypsin</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>Nano Drop ND-1000 spectrophotometer&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>QIAGEN</td><td>28104</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704</td><td></td></tr></tbody></table>

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.

Zwei-Finger-ZiF-EmGFP Fusionsproteine ​​in E. ausgedrückt werden coli mit&gt; 95% Homogenität und hoher Ausbeute (&gt; 25 mg / ml) (Abbildung 2). Im allgemeinen Ein- und Zwei-Finger kann in Mengen nahezu identisch mit denen des Wildtyp-unmodifizierte Protein ZiF Fusionsproteine ​​hergestellt werden. Jedoch in einigen Kontexten sind fünf- und sechs Finger ZiF Fusionsproteine ​​nicht in Ausbeuten hoch genug für nachfolgende Anwendungen hergestellt werden. <p class="jo...

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

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

Zinkfingerdomänen sind eigen zellpermeablen und fähig zur Vermittlung Proteinabgabe in einem weiten Bereich von Säugetierzelltypen. Hier wird eine detaillierte Schritt-für-Schritt-Protokoll zur Durchführung Zinkfinger-Technologie für die intrazelluläre Proteinabgabe dargestellt.

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 ...

direct-protein-delivery-to-mammalian-cells-using-cell-permeable-cys2

Research

JoVE Journal

Biology

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

9.9K Aufrufe.  The Scripps Research Institute.  Zinkfingerdomänen sind eigen zellpermeablen und fähig zur Vermittlung Proteinabgabe in einem weiten Bereich von Säugetierzelltypen. Hier wird eine detaillierte Schritt-für-Schritt-Protokoll zur Durchführung Zinkfinger-Technologie für die intrazelluläre Proteinabgabe dargestellt. 

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

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

Peptid abgeleitete Methode zu schleusen Gene und Proteine ​​über zelluläre und Organell-Barrieren in Pflanzen

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Forschung

Genetik

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Biotinylierte Zelle eindringen Peptide, intrazelluläre Protein-Protein-Wechselwirkungen zu studieren

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Biochemie

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Zellzusammendrücken als Robust, Mikrofluidik Intrazelluläre Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Biotechnik

Engineering Intracellular Protein Sensors in Mammalian Cells

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect extracellular proteins have been developed, a modular framework that can be easily re-engineered to sense different intracellular proteins was missing.
Here, we describe a protocol to implement a modular genetic platform that senses intracellular proteins and activates a specific cellular response. The device operates on intracellular antibodies or small peptides to sense with high specificity the protein of interest, triggering the transcriptional activation of output genes, through a TEV protease (TEVp)-based actuation module. TEVp is a viral protease that selectively cleaves short cognate peptides and is widely used in biotechnology and synthetic biology for its high orthogonality to the cleavage site. Specifically, we engineered devices that recognize and respond to protein-biomarkers of viral infections and genetic diseases, including mutated huntingtin, NS3 serine-protease, Tat and Nef proteins to detect Huntington&#8217;s disease, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, respectively. Importantly, the system can be hand tailored for the desired input-output functional outcome, such as fluorescent readouts for biosensors, stimulation of antigen presentation for immune response, or initiation of apoptosis to eliminate unhealthy cells.

<meta charset="utf8"></head><body>Entwicklung intrazellulärer Proteinsensoren in Säugetierzellen

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

Entwicklung intrazellulärer Proteinsensoren in Säugetierzellen

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Lieferung von Proteinen, Peptiden oder Handy-undurchlässige kleine Moleküle in lebenden Zellen durch Inkubation mit dem endosomolytische Reagenz dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Engineering Cell-permeable 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 make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

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 ...

Biologie

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Eine bequeme und allgemein Expression Plattform für die Produktion von sekretierten Proteinen aus menschlichen Zellen

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

Expression rekombinanter Proteine ​​für Strukturbiologie in HEK 293F Suspensionszellen: eine neue und Barriere Ansatz

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

Eine hohe Ausbeute und kosteneffiziente Expression System der Menschen Granzyme in Säugerzellen

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Direkter Protein Lieferung Säugerzellen mit Zellpermeable Cys

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

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Biotinylierte Zelle eindringen Peptide, intrazelluläre Protein-Protein-Wechselwirkungen zu studieren

Zellzusammendrücken als Robust, Mikrofluidik Intrazelluläre Delivery Platform

Entwicklung intrazellulärer Proteinsensoren in Säugetierzellen

Entwicklung intrazellulärer Proteinsensoren in Säugetierzellen

Lieferung von Proteinen, Peptiden oder Handy-undurchlässige kleine Moleküle in lebenden Zellen durch Inkubation mit dem endosomolytische Reagenz dfTAT

Engineering zellpermeablen Protein

Eine bequeme und allgemein Expression Plattform für die Produktion von sekretierten Proteinen aus menschlichen Zellen

Expression rekombinanter Proteine für Strukturbiologie in HEK 293F Suspensionszellen: eine neue und Barriere Ansatz

Eine hohe Ausbeute und kosteneffiziente Expression System der Menschen Granzyme in Säugerzellen

Direkter Protein Lieferung Säugerzellen mit Zellpermeable Cys

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Peptid abgeleitete Methode zu schleusen Gene und Proteine ​​über zelluläre und Organell-Barrieren in Pflanzen

Biotinylierte Zelle eindringen Peptide, intrazelluläre Protein-Protein-Wechselwirkungen zu studieren

Zellzusammendrücken als Robust, Mikrofluidik Intrazelluläre Delivery Platform

Entwicklung intrazellulärer Proteinsensoren in Säugetierzellen

Entwicklung intrazellulärer Proteinsensoren in Säugetierzellen

Lieferung von Proteinen, Peptiden oder Handy-undurchlässige kleine Moleküle in lebenden Zellen durch Inkubation mit dem endosomolytische Reagenz dfTAT

Engineering zellpermeablen Protein

Eine bequeme und allgemein Expression Plattform für die Produktion von sekretierten Proteinen aus menschlichen Zellen

Expression rekombinanter Proteine ​​für Strukturbiologie in HEK 293F Suspensionszellen: eine neue und Barriere Ansatz

Eine hohe Ausbeute und kosteneffiziente Expression System der Menschen Granzyme in Säugerzellen

Peptid abgeleitete Methode zu schleusen Gene und Proteine über zelluläre und Organell-Barrieren in Pflanzen

Expression rekombinanter Proteine für Strukturbiologie in HEK 293F Suspensionszellen: eine neue und Barriere Ansatz