Method Article

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

DOI:

10.3791/52814

⸱

March 25th, 2015

* These authors contributed equally

In This Article

Summary

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

Abstract

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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

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Highly efficient and versatile protein delivery strategies are critical for many basic research and therapeutic applications. The direct delivery of purified proteins into cells represents one of the safest and easiest methods for achieving this.1,2 Unlike strategies that rely on gene expression from nucleic acids,3-5 protein delivery poses no risk of insertional mutagenesis, is independent of the cellular transcription/translation machinery and allows for an immediate effect. However, the lack of simple and generalizable methods for endowing cell-penetrating activity onto proteins routinely confounds their direct entry into cells. Current method....

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Protocol

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1. Cloning

  1. Obtain alanine-substituted two-finger ZiF domains that have been sub-cloned into the pET-28 expression vector system and are available upon request (pET-2F-ZiF).34
  2. PCR amplify EmGFP from the plasmid Emerald-pBAD with the primers 5’ XmaI-EmGFP (5’-GGAAATTGCCCGGGATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3’; XmaI site in bold) and 3’ SacI-EmGFP (5’-CGGATCTGAGCTCTTACTTGTACAGCTCGTCCATGCCGAG-3’; SacI site in bold).
    1. Use 5 ng of template DNA, 10 μl of 10x polymerase buffer, 1 Units (U) of Taq DNA polymerase, 0.2 mM each dNTP and 0.2 μM ....

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Results

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Two-finger ZiF-EmGFP fusion proteins can be expressed in E. coli with >95% homogeneity and high yields (>25 mg/ml) (Figure 2). In general, one- and two-finger ZiF fusion proteins can be produced in quantities nearly identical to those of wild-type unmodified protein. However, in some contexts, five- and six-finger ZiF fusion proteins are unable to be produced in yields high enough for downstream applications.

Direct application of two-finger ZiF-EmGFP protein on.......

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Discussion

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Here, a step-by-step protocol for protein delivery using cell-permeable zinc-finger (ZiF) domains is presented. The ZiF domain does not reduce the activity of fused enzymatic cargo34; allows for the production and purification of proteins in yields nearly identical to those observed with unmodified protein; and can transport proteins and enzymes into a wide range of cell types with efficiencies that exceed traditional cell-penetrating peptide or protein transduction domain systems. Together, these findings ind.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported by the National Institutes of Health (DP1CA174426 to Carlos F. Barbas) and ShanghaiTech University, Shanghai, China (to J.L). Molecular graphics were generated using PyMol.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
XmaINew England BiolabsR0180L
SacINew England BiolabsR0156L
Expand High Fidelity PCR systemRoche11759078001
dNTPsNew England BiolabsN0446S
4%-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wellsLife TechnologiesEC6028BOX
2x Laemmli Sample BufferBioRad161-0737
T4 DNA LigaseLife Technologies15224-017
BL21 (DE3) Competent E. coliNew England BiolabsC2527I
IPTGThermo ScientificR0391
Zinc ChlorideSigma-Aldrich208086-5G
Kanamycin SulfateFisher ScientificBP906-5
GlucoseSigma-AldrichG8270-100G
Tris BaseFisher ScientificBP152-25
Sodium ChlorideSigma-AldrichS9888-25G
DTTFisher ScientificPR-V3151 
PMSFThermo Scientific36978
Ni-NTA Agarose ResinQIAGEN30210
GlycerolSigma-AldrichG5516-500ML
ImidazoleSigma-AldrichI5513-25G
Amicon Ultra-15 Centrifugal Filter UnitsEMO MilliporeUFC900324
DMEMLife Technologies11966-025
Fetal Bovine SerumLife Technologies10437-028
Antibiotic-Antimycotic Life Technologies15240-062
24-Well Flat Bottom PlateSigma-AldrichCLS3527-100EA
Poly-LysineSigma-AldrichP7280
DPBS, No Calcium, No MagnesiumLife Technologies21600010
Heparan SulfateSigma-AldrichH4777
TrypsinLife Technologies25300054
HeLa cellsATCCCCL-2
Nano Drop ND-1000 spectrophotometer Thermo Fisher Scientific
QIAquick PCR Purification KitQIAGEN28104
QIAquick Gel Extraction KitQIAGEN28704

References

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  1. Berg, A., Dowdy, S. F. Protein transduction domain delivery of therapeutic macromolecules. Curr. Opin. Biotechnol. 22, 888-893 (2011).
  2. Lindsay, M. A. Peptide-mediated cell delivery: application in protein target validation. Curr. Opin. Pharmacol....

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Tags

Zinc Finger DomainsProtein DeliveryMammalian CellsFlow CytometryProtein PurificationGene FusionBacterial ExpressionCell TransductionFluorescent ProteinCytosolic Delivery

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