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

Summary

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

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

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....

Protocol

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 ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
QIAquick PCR Purification Kit	QIAGEN	28104
QIAquick Gel Extraction Kit	QIAGEN	28704