Name: direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains
Uploaded: 2015-03-25T15:00:00.000+00:00
Duration: 11 min 24 s
Description: Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Questo lavoro è stato sostenuto dal National Institutes of Health (DP1CA174426 di Carlos F. Barbas) e ShanghaiTech University di Shanghai, in Cina (a JL). Grafici molecolari sono stati generati utilizzando PyMol.

1. Clonazione <ol><li> Ottenere domini Zif due dita alanina-sostituiti che sono stati sub-clonati nel sistema vettore di espressione pET-28 e sono disponibili su richiesta (PET-2F-ZIF). 34 </li><li> PCR amplifica EmGFP dal plasmide Emerald-pBAD con primer 5 &#39;XmaI-EmGFP (5&#39;-GGAAATTG CCCGGG ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#39;; Xma sito ho in grassetto) e 3 &#39;SacI-EmGFP (5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3&#39; ; Sac sito che ho in grassetto). <ol><li> Utilizzare 5 ng di DNA stampo, 10 microlitri di tampone 10x polimerasi, 1 unità (U) di DNA polimerasi Taq, 0,2 mM ciascun dNTP e 0,2 mM di ciascun primer in una soluzione di 100 microlitri con il volume rimanente costituiti di acqua distillata / deionizzata . Utilizzare le condizioni di ciclismo: 95 ° C per 5 minuti; 30 cicli di 95 ° C per 30 sec, 55 ° C per 30 sec e 72 ° C per 1 min; 72 ° C per 10 min. Purificare il prodotto PCR gel extraction e determinare la concentrazione di DNA utilizzando uno spettrofotometro misura Abs 260 x 50 ug / ml. </li></ol></li><li> Digest pET-2F-ZIF e l&#39;inserto codifica EmGFP con gli enzimi di restrizione Xma I e Sac I in tampone raccomandato per 3 ore a 37 ° C utilizzando 10 U di enzima per 1 mg di DNA. Visualizza DNA mediante elettroforesi su gel di agarosio utilizzando un colorante fluorescente intercalante, come bromuro di etidio. </li><li> Purificare il DNA digerito dalla kit estrazione gel e determinare la concentrazione di DNA da uno spettrofotometro misurando Abs 260 x 50 mg / ml. </li><li> Legare il DNA EmGFP-codifica purificato in 50-100 ng di PET-2F-ZIF con 1 U di T4 DNA Ligase per almeno 1 ora a temperatura ambiente. Per ottenere i migliori risultati, effettuare la reazione di ligazione utilizzando un 6: rapporto molare 1 inserto a vettoriale. </li><li> Scongelare 50 ml di eventuali chimicamente competenti XL-1 blu Escherichia coli su ghiaccio e mescolare delicatamente con 10-20 ng di legatura PET-2F-ZIF-EmGFP. </li><li> Tenere in ghiaccio per 30 min. Urti Riscaldare la miscela a 42 ° C per 90 sec e recuperare le cellule in 2 ml di brodo con Super Optimal Cataboliti rimozione (SOC) per 1 ora a 37 ° C con agitazione. </li><li> Stendere 100 ml di cultura recupero su un brodo lisogenia (LB) piastra di agar con 50 mg / ml kanamicina e incubare O / N a 37 ° C. </li><li> Il giorno seguente, inoculare 6 ml di Super Broth (SB) o di coltura LB contenente 50 ug / ml kanamicina con una colonia dalla piastra di agar LB e cultura O / N a 37 ° C. Nota: Colony PCR utilizzando i primer 5 &#39;XmaI-EmGFP e 3&#39; SacI-EmGFP potrebbe essere utilizzata per identificare cloni positivi prima miniprep. </li><li> Purificare pET-2F-ZIF-EmGFP da miniprep e confermare plasmide dal sequenziamento del DNA utilizzando promotore T7 (5&#39;-TAATACGACTCACTATAGGG-3 &#39;). </li></ol> 2. Espressione e purificazione <ol><li> Scongelare 50 ml di chimicamente competente BL21 E. coli su ghiaccio e mescolare delicatamente con 100 ng di PET-2F-ZIF-EmGFP plasmid. Trasforma come descritto nei passaggi 1,7-1,8. </li><li> Il giorno seguente, inoculare 10 ml di terreno LB contenente 50 mg / ml kanamicina con una singola colonia e crescere O / N a 37 ° C con agitazione. </li><li> Il giorno seguente, diluire i 10 ml di O / N cultura in 1 l di terreno LB integrata con 50 mg / ml kanamicina, 0,2% di glucosio e 100 micron ZnCl 2. Crescere la coltura a 37 ° C con agitazione ad una densità ottica a 600 nm (OD 600) di 0,8 e indurre l&#39;espressione di proteine ​​con 2 mM isopropil-β-D-1-thiogalactopyranoside (IPTG). Dopo 6 ore di crescita a 37 ° C, le cellule raccolto per centrifugazione a 5.000 xg per 10 min a 4 ° C. Nota: Le condizioni induzione sono molto variabili e dipendono dalla stabilità delle proteine ​​espressi. Monitorare l&#39;OD 600 ogni 30 minuti fino a quando un OD 600 di 0,8 è raggiunto. </li><li> Risospendere il pellet di cellule in 5 ml di tampone di lisi (50 mM Tris-HCl, NaCl 500 mM, 100 mM ZnCl 2, 1 mM ditiotreitolo (DTT), 1 mM MgCl 2, 1 mM phenylmethylsulfonyl fluoruro (PMSF) e imidazolo 10 mM, pH 8,0). Lisare le cellule sul ghiaccio di ultrasuoni con la seguente impostazione: uscita di potenza del 50%, 4 min di tempo di processo con 5 sec su / 10 sec off intervalli. Evitare il surriscaldamento della soluzione. </li><li> Centrifugare il lisato cellulare a 25.000 xg per 30 min a 4 ° C e trasferire il surnatante in una provetta di raccolta fresco. Per ottenere i migliori risultati, eseguire le seguenti operazioni a 4 ° C. </li><li> Eseguire il surnatante con una colonna pre-compresso con 1 ml di equilibrata liquami Ni-NTA. Lavare la resina con 20 ml di tampone di lavaggio (50 mM Tris-HCl, NaCl 500 mM, 100 mM ZnCl 2, 1 mM DTT, 1 mM MgCl 2 e imidazolo 30 mM, pH 8,0). </li><li> Eluire la proteina con 5 ml di tampone di eluizione (50 mM Tris-HCl, NaCl 500 mM, 100 mM ZnCl 2, 1 mM DTT, 1 mM MgCl 2, e imidazolo 300 mM, pH 8,0). </li><li> Buffer scambiare la proteina eluita con tampone di stoccaggio (50 mMTris-HCl, NaCl 500 mM, 100 mM ZnCl 2, 1 mM DTT, 1 mM MgCl 2 e 10% glicerolo, pH 8,0) e concentrare la proteina di almeno 40 mM usando un concentratore rotazione mediante centrifugazione secondo le istruzioni del produttore. </li><li> Determinare la concentrazione di proteine ​​da BCA o saggio Bradford. Mescolare 2 ml di proteine ​​purificate con 2 ml 2 × SDS-PAGE loading dye, ebollizione a 95 ° C per 10 minuti e poi risolvere il 4% -20% Tris-Glycine SDS-PAGE per valutare la proteina purezza (Figura 2). </li></ol> 3. Protein bagagli <ol><li> Aliquota la proteina concentrata, flash congelare in azoto liquido e conservare a -80 ° C. Per proteine ​​di fusione EmGFP, coprire il tubo con un foglio di alluminio per proteggere la proteina dalla luce. </li><li> Evitare congelamenti e scongelamenti ripetuti della soluzione proteica. Nota: le proteine ​​Zif-fuse sono stabili in queste condizioni per almeno 1 mese. O&gt; 4 stoccaggio inappropriato mese può portarealle proteine ​​precipitazione o photobleaching di EmGFP. </li></ol> 4. proteina di trasduzione <ol><li> Mantenere cellule HeLa in terreno Eagle modificato di Dulbecco (DMEM) contenente 10% (v / v) di siero fetale bovino (FBS) e 1% di antibiotico-antimicotico a 37 ° C in un ambiente completamente umidificata con 5% di CO 2. Nota: Le celle diversi passaggi più di 30 volte non sono raccomandati per la proteina di trasduzione. </li><li> Pre-coat una piastra a 24 pozzetti con 500 ml di 50 mg / ml di poli-lisina per 30 a 60 minuti a 25 ° C. Cellule Seed su una piastra da 24 pozzetti ad una densità di 2 x 10 5 cellule per pozzetto. A 24 ore dopo la semina, o una volta cellule sono tra l&#39;80% -90% confluenti, rimuovere i supporti da ciascun pozzetto e lavare con 500 ml di pre-riscaldato DMEM senza siero (SFM). </li><li> Per ogni bene, aggiungere 250 ml di SFM contenente 2 mM di proteine ​​Zif-EmGFP e 100 micron ZnCl 2. Incubare le cellule a 37 ° C per 1 ora. Nota: i domini ZIF entrano nelle cellule primarily attraverso macropinocitosi, 34 che è un meccanismo di energia-dipendente. Pertanto, le cellule devono essere incubate a 37 ° C per un efficiente internalizzazione proteine. </li><li> Rimuovere supporti dalle cellule e lavare tre volte con 500 ml di calcio-magnesio e senza tampone fosfato salino di Dulbecco (DPBS) supplementato con 0,5 mg / ml di eparina. Nota: L&#39;eparina è necessario rimuovere legato alle proteine ​​di superficie che potrebbe complicare le analisi a valle. </li><li> (Opzionale) Ripetere i trattamenti fino a tre volte per la consegna maggiore. </li><li> Isolare cellule trattate immediatamente dopo eparina lavaggio mediante digestione con 0,05% tripsina-EDTA a 37 ° C per 2 min. Risospendere le cellule staccate in 0,5 ml di DPBS addizionato con 1% FBS. </li><li> Misurare l&#39;intensità di fluorescenza di ciascun campione mediante citometria di flusso 35 utilizzando il canale di isotiocianato di fluoresceina (FITC) (Figura 3). Regolare il forward scatter (FSC) e side scatter (SSC) per posizionare la popolazioneinteressi su scala, assicurando che le popolazioni con proprietà differenti vengono risolte tra loro. </li><li> Raccogliere 10.000 eventi live per ogni campione e analizzare i dati in citometria a flusso di analisi dei dati del software. 36 Normalizzare fluorescenza da cellule HeLa trattate con ZIF EmGFP a quelle cellule trattate solo con SFM. </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>

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Qui, un protocollo di step-by-step per la consegna delle proteine ​​usando zinc-finger (ZIF) domini cellulari permeabili è presentato. Il dominio ZIF non riduce l&#39;attività del fuso carico enzimatica 34; consente la produzione e purificazione di proteine ​​dei rendimenti quasi identici a quelli osservati con proteine ​​non modificato; e può trasportare proteine ​​ed enzimi in una vasta gamma di tipi di cellule con efficienze superiori peptide o proteina sistemi tradizionali dominio trasd...

Altamente strategie consegna proteine ​​efficienti e versatili sono fondamentali per molti la ricerca di base e applicazioni terapeutiche. La consegna diretta di proteine ​​purificate in cellule rappresenta uno dei metodi più sicuri e più semplici per raggiungere questo. 1,2 differenza strategie che si basano su espressione genica da acidi nucleici, proteine ​​3-5 consegna non comporta alcun rischio di mutagenesi inserzionale, è indipendente dal cellular trascrizione macchinari e / definizione permette un effetto immediato. Tuttavia, la mancanza di metodi semplici e generalizzabili per dotare l&#39;attività delle cellule-penetrante su proteine ​​confonde regolarmente il loro ingresso diretto nelle cellule. Gli attuali metodi per facilitare la consegna proteina intracellulare si basano sull&#39;uso di naturalmente 6-8 o progettati peptidi cellula-penetranti, 9-12 domini di trasduzione sovralimentati, 13,14 nanoparticelle 15 e liposomi, 16 particelle simili al virus 17,18 </sup&gt; materiali polimerici microsfere. 19 Purtroppo, molti di questi approcci sono ostacolati da bassi tassi di assorbimento cellulare, 20,21 scarsa stabilità, 22 involontaria tipo cellulare specificità, 23 basso endosomali proprietà di fuga di 24 e la tossicità. 25 Inoltre, molti proteine ​​e tecnologie di trasduzione riducono la bioattività delle proteine ​​consegnati. 14 Il nostro laboratorio in precedenza dimostrato che zinc-finger nucleasi (ZFN) proteine ​​- restrizione endonucleasi chimerico costituito da una Cys 2 -Il suo 2 zinc-finger proteina-DNA-binding programmabile e il dominio scissione del FokI endonucleasi di restrizione 26-28 - sono intrinsecamente cell- permeabile. 29 L&#39;attività delle cellule-penetrante sorprendente ha dimostrato di essere una proprietà intrinseca del dominio zinc-finger design personalizzato, una piattaforma di legame al DNA che è emersa come un potente strumento per la mirata del genoma enEngineering, 30-32 e considerato il risultato della costellazione di sei residui carichi positivamente sulla superficie della proteina. Infatti, diverse proteine ​​che legano il DNA, tra cui c-Jun e N-DEK hanno dimostrato di possedere una capacità innata di attraversare le membrane cellulari. 33 Più recentemente, il nostro laboratorio espanso su questi risultati e dimostrato che l&#39;attività delle cellule-penetrante di zinco finger (ZIF) domini potrebbero essere sfruttate per la consegna delle proteine ​​intracellulari. La fusione genetica di entrambi i domini ZIF uno o due dita per uno specifico carico di proteine ​​portato ad assorbimento di efficienza che hanno superato molti sistemi di consegna di peptidi cellula-penetranti convenzionali. 34 In particolare, la consegna Zif-mediata non ha compromesso l&#39;attività di carico enzimatica fuso e facilitato alti livelli di consegna citosolico. Collettivamente, questi risultati dimostrano il potenziale del dominio ZIF per facilitare la consegna efficiente e facile delle proteine, e potenzialmente più diversi tipi di macromolecole, nelle cellule. Qui, un protocollo dettagliato passo-passo su come implementare la tecnologia ZIF per la consegna delle proteine ​​in cellule di mammifero è presentato. Abbiamo già costruito una suite di uno, due, tre, quattro, cinque e sei dita domini ZIF che non hanno la capacità di legare il DNA, a causa della sostituzione di ciascuno dei DNA-binding residui α-elica, ma sono in grado di fornire proteine ​​in cellule 34 (Figura 1). La produzione e la trasduzione di Emerald GFP (EmGFP) in cellule HeLa utilizzando una Zif dominio a due dita è descritto. Questo protocollo è estensibile a quasi qualsiasi proteina in grado di espressione solubile in Escherichia coli e quasi ogni tipo di cellula di mammifero. I risultati attesi sono forniti e strategie per massimizzare le prestazioni di questo sistema vengono anche discussi.

<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.

Due dita proteine ​​di fusione Zif-EmGFP possono essere espressi in E. coli con&gt; 95% omogeneità e rese elevate (&gt; 25 mg / ml) (Figura 2). In generale, a uno o due dita proteine ​​di fusione ZIF possono essere prodotti in quantità quasi identiche a quelle del wild-type di proteine ​​non modificato. Tuttavia, in alcuni contesti, proteine ​​di fusione ZIF cinque e sei dita sono in grado di essere prodotti in rendimenti abbastanza elevati per applicazioni a valle. <p cla...

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

Domini zinco-dita sono intrinsecamente cellula-permeabile e capace di mediare consegna proteine ​​in una vasta gamma di tipi di cellule di mammifero. Qui, un protocollo dettagliato passo-passo per l&#39;attuazione tecnologia zinc finger-consegna proteina intracellulare è presentato.

Diretto Protein Consegna a cellule di mammifero Utilizzando Cell-permeabile Cys<sub&gt; 2</sub&gt; -Il suo<sub&gt; 2</sub&gt; domini zinc-finger

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

9.9K Views.  The Scripps Research Institute.  Domini zinco-dita sono intrinsecamente cellula-permeabile e capace di mediare consegna proteine ​​in una vasta gamma di tipi di cellule di mammifero. Qui, un protocollo dettagliato passo-passo per l'attuazione tecnologia zinc finger-consegna proteina intracellulare è presentato. 

Video: 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.

Peptide di derivazione Metodo di Trasporto geni e proteine ​​Attraverso cellulari e barriere organelli in Piante

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

Ricerca

Genetica

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.

Biotinylated Cell-penetrating peptidi per studiare le interazioni proteina-proteina intracellulare

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

Biochimica

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.

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

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

Bioingegneria

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.

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.

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.

Consegna di proteine, peptidi o cella-impermeabile piccole molecole in cellule vive mediante incubazione con il reagente Endosomolytic 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.

Ingegneria Cell-permeabile proteine

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

Biologia

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.

Una piattaforma espressione conveniente e generale per la produzione di proteine ​​secrete da cellule umane

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.

Espressione della proteina ricombinante di Biologia Strutturale in HEK 293F cellule in sospensione: un approccio nuovo e accessibile

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.

A High Yield e Cost-efficient Sistema Espressione di granzimi umane in cellule di mammifero

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

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

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

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

Efficiente Espressione cellulari di mammifero e Passo singolo Purificazione di extracellulare glicoproteine ​​per cristallizzazione

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

Diretto Protein Consegna a cellule di mammifero Utilizzando Cell-permeabile Cys

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Capitoli in questo video

Peptide di derivazione Metodo di Trasporto geni e proteine Attraverso cellulari e barriere organelli in Piante

Biotinylated Cell-penetrating peptidi per studiare le interazioni proteina-proteina intracellulare

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

Engineering Intracellular Protein Sensors in Mammalian Cells

Consegna di proteine, peptidi o cella-impermeabile piccole molecole in cellule vive mediante incubazione con il reagente Endosomolytic dfTAT

Ingegneria Cell-permeabile proteine

Una piattaforma espressione conveniente e generale per la produzione di proteine secrete da cellule umane

Espressione della proteina ricombinante di Biologia Strutturale in HEK 293F cellule in sospensione: un approccio nuovo e accessibile

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