Direkte Protein Delivery for pattedyrsceller Brug Cell-permeable Cys<sub> 2</sub> -His<sub> 2</sub> Zink-finger Domæner
Summary
Zinkfinger-domæner er uløseligt celle-permeable og i stand til at mediere protein levering i en bred række af mammale celletyper. Her er en detaljeret trin-for-trin-protokollen til gennemførelse zink-finger-teknologi til intracellulær protein levering fremlagt.
Abstract
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.
Introduction
Meget effektive og alsidige strategier protein levering er afgørende for mange grundforskning og terapeutiske anvendelser. Den direkte levering af oprensede proteiner i celler er et af de sikreste og letteste metoder til at opnå dette. 1,2 modsætning strategier, der er afhængige af genekspression fra nukleinsyrer, 3-5 protein levering udgør nogen risiko for insertionsmutagenese, er uafhængig af den cellulær transskription / translation maskiner og giver mulighed for en øjeblikkelig virkning. Men manglen på enkle og generalisere metoder til tilføre celle-gennemtrængende aktivitet på proteiner rutinemæssigt forvirrer deres direkte indrejse i celler. Nuværende fremgangsmåder til at lette intracellulær protein levering er baseret på anvendelsen af naturligt forekommende 6-8 eller konstrueret cellepenetrerende peptider, 9-12 trykladning transduktion domæner, 13,14 nanopartikler 15 og liposomer, 16 viruslignende partikler 17,18 </sup> og polymere mikrosfære materialer. 19 Desværre har mange af disse metoder er hæmmet af lave celleoptagelse rater, 20,21 dårlig stabilitet, 22 utilsigtet celletype specificitet, 23 lav endosomale escape egenskaber 24 og toksicitet. 25 Endvidere er mange protein transduktion teknologier reducere bioaktiviteten af de leverede proteiner. 14
Vores laboratorium tidligere vist, at zink-finger nuclease (ZFN) proteiner – kimære restriktionsendonukleaser bestående af en programmerbar Cys 2 -His 2 zink-finger-DNA-bindende protein og spaltning domæne af Fokl restriktionsendonuklease 26-28 – er i sagens natur celle- gennemtrængelig. 29 Denne overraskende celle-gennemtrængende aktivitet viste sig at være en iboende egenskab ved specialdesignede zink-finger-domæne, et DNA-bindende platform, der er opstået som et effektivt redskab til målrettet genom enEngineering, 30-32 og anses for at være resultatet af konstellation af seks positivt ladede rester på proteinet overflade. Faktisk adskillige DNA-bindende proteiner, herunder c-Jun og N-DEK er blevet vist, at besidde en medfødt evne til at krydse cellemembraner 33 nylig vores laboratorium udvidet på disse resultater og demonstreret. At cellepenetrerende aktivitet zink- finger (ZIF) domæner kunne udnyttes til intracellulært protein levering. Genetisk fusion af enten et eller to finger ZIF domæner til specifikt protein last ført til optagelse effektivitetsgevinster, der oversteg mange konventionelle celle-gennemtrængende peptid leveringssystemer. 34 Mest bemærkelsesværdigt var ZIF-medieret levering ikke kompromittere aktiviteten af smeltet enzymatisk fragt og lettet høje niveauer af cytosolisk levering. Kollektivt, disse resultater demonstrere potentialet i ZIF-domænet for at lette en effektiv og let levering af proteiner, og potentielt mere forskelligartede former for makromolekyler i celler.
Her er en detaljeret trin-for-trin-protokollen om, hvordan man gennemfører ZIF teknologi til protein levering i pattedyrceller fremlagt. Vi har tidligere fremstillet en suite af en-, to-, tre-, fire-, fem- og seks-finger ZIF domæner, der mangler evnen til at binde DNA, som følge af substitution af hver af de α-helix DNA-bindende rester, men er i stand til at levere proteiner i celler 34 (figur 1). Produktion og transduktion af Emerald GFP (EmGFP) ind i HeLa-celler ved hjælp af en to-finger ZIF domænet er beskrevet. Denne protokol kan udvides til næsten ethvert protein i stand til opløselig ekspression i Escherichia coli og næsten enhver type pattedyrcelle. Forventede resultater leveres og strategier for at maksimere effektiviteten af dette system er også drøftet.
Protocol
Representative Results
Discussion
Her er en trin-for-trin-protokol for protein levering ved hjælp af celle-permeable zink-finger (ZIF) domæner præsenteres. ZIF-domænet ikke reducere aktiviteten af smeltet enzymatisk fragt 34; giver mulighed for produktion og oprensning af proteiner i udbytter næsten identiske med dem observeret med umodificeret protein; og kan transportere proteiner og enzymer i en lang række celletyper med en virkningsgrad, der overskrider traditionelle cellepenetrerende peptid- eller proteintransduktionsdomæne system…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af National Institutes of Health (DP1CA174426 til Carlos F. Barbas) og ShanghaiTech University, Shanghai, Kina (til JL). Molekylære grafik blev dannet ved anvendelse PyMOL.
Materials
| XmaI | New England Biolabs | R0180L | |
| SacI | New England Biolabs | R0156L | |
| Expand High Fidelity PCR system | Roche | 11759078001 | |
| dNTPs | New England Biolabs | N0446S | |
| 4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells | Life Technologies | EC6028BOX | |
| 2x Laemmli Sample Buffer | BioRad | 161-0737 | |
| T4 DNA Ligase | Life Technologies | 15224-017 | |
| BL21 (DE3) Competent E. coli | New England Biolabs | C2527I | |
| IPTG | Thermo Scientific | R0391 | |
| Zinc Chloride | Sigma-Aldrich | 208086-5G | |
| Kanamycin Sulfate | Fisher Scientific | BP906-5 | |
| Glucose | Sigma-Aldrich | G8270-100G | |
| Tris Base | Fisher Scientific | BP152-25 | |
| Sodium Chloride | Sigma-Aldrich | S9888-25G | |
| DTT | Fisher Scientific | PR-V3151 | |
| PMSF | Thermo Scientific | 36978 | |
| Ni-NTA Agarose Resin | QIAGEN | 30210 | |
| Glycerol | Sigma-Aldrich | G5516-500ML | |
| Imidazole | Sigma-Aldrich | I5513-25G | |
| Amicon Ultra-15 Centrifugal Filter Units | EMO Millipore | UFC900324 | |
| DMEM | Life Technologies | 11966-025 | |
| Fetal Bovine Serum | Life Technologies | 10437-028 | |
| Antibiotic-Antimycotic | Life Technologies | 15240-062 | |
| 24-Well Flat Bottom Plate | Sigma-Aldrich | CLS3527-100EA | |
| Poly-Lysine | Sigma-Aldrich | P7280 | |
| DPBS, No Calcium, No Magnesium | Life Technologies | 21600010 | |
| Heparan Sulfate | Sigma-Aldrich | H4777 | |
| Trypsin | Life Technologies | 25300054 | |
| Hela cells | ATCC | CCL-2 | |
| Nano Drop ND-1000 spectrophotometer | Thermo Fisher Scientific | N/A | |
| QIAquick PCR Purification Kit | QIAGEN | 28104 | |
| QIAquick Gel Extraction Kit | QIAGEN | 28704 |
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