Potent delivery of functional proteins into Mammalian cells in vitro and in vivo using a supercharged protein

Abstract

The inability of proteins to potently penetrate mammalian cells limits their usefulness as tools and therapeutics. When fused to superpositively charged GFP, proteins rapidly (within minutes) entered five different types of mammalian cells with potency up to approximately 100-fold greater than that of corresponding fusions with known protein transduction domains (PTDs) including Tat, oligoarginine, and penetratin. Ubiquitin-fused supercharged GFP when incubated with human cells was partially deubiquitinated, suggesting that proteins delivered with supercharged GFP can access the cytosol. Likewise, supercharged GFP delivered functional, nonendosomal recombinase enzyme with greater efficiencies than PTDs in vitro and also delivered functional recombinase enzyme to the retinae of mice when injected in vivo.

Figure 1

Comparison of mCherry delivery by +36 GFP, Tat, Arg₁₀, and penetratin. a) Flow cytometry of HeLa, BSR, 3T3, PC12, and IMCD cells incubated in the presence of the specified concentrations of +36 GFP-mCherry, Tat-mCherry, Arg₁₀-mCherry, penetratin-mCherry, or wild-type mCherry alone for 4 h at 37 °C. Cells were washed three times with 20 U mL⁻¹ heparin in PBS to remove membrane-bound protein before analysis. Error bars represent the standard error of three independent biological replicates. b) Confocal fluorescence microscopy of live cells incubated with 100 nM +36 GFP-mCherry for 4 h at 37 °C. Red color represents mCherry signal; green color represents +36 GFP signal. The scale bar is 15 μm.

Figure 2

Deubiquitination suggests cytosolic exposure of a ubiquitin-+36 GFP fusion protein. a) Western blots using anti-GFP antibodies. Lanes 1−3: purified protein samples of +36 GFP, wild-type ubiquitin-+36 GFP fusion (wt), or G76V mutant ubiquitin-+36 GFP fusion (mut). Lanes 4 and 5: purified protein spiked into HeLa cell lysate to confirm that lysis conditions do not affect fusion protein integrity. Lanes 6−11: the indicated cells were treated with 100 nM of either the wt or mutant ubiquitin-+36 GFP for 1 h and then lysed. b) Mean extent of deubiquitination of wt ubiquitin-+36 GFP fusion protein in HeLa, 3T3, and BSR cells. Error bars reflect the standard deviation of three independent biological replicates. c) In vitro deubiquitination control experiment. Ubiquitin-+36 GFP fusion proteins were incubated in either HeLa cytosolic extract or in HeLa cytosolic extract containing one of two DUB inhibitors, 10 mM N-ethylmaleimide (NEM) or 20 μg mL⁻¹ ubiquitin-aldehyde (Ub-Al), for 1 h at 37 °C.

Figure 3

Delivery of active Cre recombinase into mammalian cells in vitro and in vivo. a) Cre-mediated recombination in HeLa cells transiently transfected with pCALNL-DsRed2 and treated with +36 GFP-Cre, Tat-Cre, Arg₁₀-Cre, or penetratin-Cre for 4 h at 37 °C. The image is an overlay of DsRed2 signal and brightfield images of HeLa cells transfected with pCALNL-DsRed2 and treated with 100 nM +36 GFP-Cre. b) Cre-mediated recombination in 3T3.LNL.LacZ cells treated with +36 GFP-Cre, Tat-Cre, Arg₁₀-Cre, or penetratin-Cre for 4 h at 37 °C. The image is of 3T3.loxP.lacZ cells treated with 500 nM +36 GFP-Cre and stained with X-Gal. c) Cre-mediated recombination in BSR.LNL.tdTomato cells treated with +36 GFP-Cre, Tat-Cre, Arg₁₀-Cre, or penetratin-Cre for 4 h at 37 °C. The image is an overlay of tdTomato signal and brightfield images of BSR.LNL.tdTomato cells treated with 100 nM +36 GFP-Cre. d) Identical to panel c but with the addition of 100 μM chloroquine during and after protein treatment. In panels a−d, error bars reflect the standard error of three independent biological replicates. e) Fluorescence microscopy of a retinal section of a CD1 adult mouse injected with 0.5 μL of 100 μM +36 GFP. The retina was harvested and analyzed 6 h after injection. GFP fluorescence is shown in green, and DAPI nuclear stain is shown in blue. f) Retinal sections of neonatal RC::PFwe mouse pups harboring a nuclear LacZ reporter of Cre activity. Three days after injection of 0.5 μL of 40 μM wild-type Cre, Tat-Cre, or +36 GFP-Cre, retinae were harvested, fixed, and stained with X-gal. Dots on the graph represent the total number of recombined cells counted in each retina. The horizontal bar represents the average number of recombined cells per retina for each protein injected (n = 4 for wild-type Cre, n = 6 for Tat-Cre, n = 6 for +36 GFP-Cre).