A peptide carrier for the delivery of biologically active proteins into mammalian cells

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Most notably, the presence of Pep-1 did not alter the enzymatic activity of beta-Gal upon delivery into cells. Finally, efficient transfection of beta-Gal was equally observed when transfection was performed at 4?C (Fig. 3C), again supporting the idea that Pep-1-mediated transfection is independent of the endosomal pathway. In the case of GFP, >80% of the cells exhibited fluorescent GFP staining in their cytoplasm. As observed for peptide delivery, the efficiency of protein delivery was not affected by the presence of 10% FBS, and no protein delivery was observed for concentrations of Pp-1 lower than 0.5 muM or greater than 50 muM (Fig. 3D), supporting the idea that the size of the particle and the number of Pep-1 molecules are important for delivery.
We next verified that Pep-1 did not affect the proper cellular localization of larger proteins. To this aim, we investigated the subcellular localization of FITC-conjugated antibodies following Pep-1-mediated delivery. We evaluated delivery of two different FITC-conjugated antibodies: anti-Lamp-1, which recognizes the lysosome-associated membrane protein 1 (LAMP-1), and monoclonal anti-beta-actin. Antibodies were used at a concentration of 0.1 muM and incubated with Pep-1 (5 muM), as described for GFP and beta-Gal delivery. As shown in Figure 3E and F, characteristic labeling of both actin and lysosomes was observed, confirming that Pep-1 is able to deliver antibodies into cells, while preserving their ability to recognize antigens within cells. Our observation that Pep-1 is able to promote delivery and proper localization of two different antibodies to their target antigens confirms that although Pep-1 alone localizes to the nucleus, it does not affect the appropriate subcellular localization of the proteins it delivers.
Peptide carriers are one of the most promising tools for delivering biologically active molecules into cells18, 19. In addition to their usefulness in the laboratory, they are promising reagents for therapeutic screens and may potentially enable direct targeting of specific events throughout the cell cycle. Protein delivery could improve the development of vaccines and therapies in a variety of viral diseases and cancers. Recently, the use of PTD-mediated transfection has proved that "protein therapy" can have a major impact on future treatments1, 2, 3. Here we describe a strategy for the delivery of full-length proteins and peptides into mammalian cells, based on a short peptide carrier, Pep-1, which allows the delivery of several distinct proteins and peptides into different cell lines without the need for crosslinking or denaturation steps. This carrier presents several advantages for protein therapy, including the rapid delivery of proteins into cells with very high efficiency, stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Taken together, the data presented in this study reveal that Pep-1 technology constitutes a useful tool for basic research, for studying the role of proteins and protein-protein interactions in living cells, as well as in a therapeutic context for the screening of potential therapeutic proteins and peptides.
Experimental protocol
Peptide synthesis and analysis.
All peptides were synthesized by solid-phase peptide synthesis using AEDI-Expansin resin with a 9050 Pep Synthesizer (Millipore, Wartford, UK) according to the Fmoc/tBoc method, and purified as already described15. Peptides (Pep-1, Pep-A, Pep-B) were acetylated at their N terminus. Pep-1 and Pep-B were synthesized with a cysteamine group at their C terminus, so as to enable coupling of fluorescein maleimide or coumarin maleimide (Molecular Probes, Eugene, OR).
Proteins and products.
beta-Gal was purchased from Novagen (Madison, WI), FITC-conjugated anti-Lamp-1 (CD107a, clone 1D4B) and FITC-conjugated anti-beta-actin (clone AC15, F 3022) were from PharMingen (San Diego, CA) and Sigma (St. Louis, MO), respectively. His-tagged GFP was subcloned into pET28a vector (Novagen), expressed in Escherichia coli, and purified to homogeneity using a nitrilotriacetic acid (NTA)-agarose column, followed by size-exclusion column.
Size-exclusion chromatography experiments.
Experiments were done in PBS buffer containing 200 mM NaCl. Pep-1/FITC-Pep-A and Pep-1/GFP complexes were formed at a molar ratio of 10:1 and 30:1 in PBS buffer, then loaded onto a HiLoad 16/60 Superdex 75 gel filtration column (Pharmacia, Uppsala, Sweden). Elution was carried out at a flow rate of 0.8 ml/min, and both protein absorption at 280 nm and fluorescence elution profiles were monitored. The column was calibrated in the same buffer with calibration markers: vitamin B12 (1.35 kDa), myoglobin (17 kDa), ovalbumin (44 kDa), albumin (66 kDa), IgG (158 kDa), and thyroglobin (670 kDa).
Cell culture, cytotoxicity assays, Pep-1-mediated delivery.
Adherent fibroblastic HS-68, NIH-3T3, 293, Jurkat T, and Cos-7 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% (vol/vol) 200 mM glutamine, 1% (vol/vol) antibiotics (streptomycin, 10,000 mug/ml; penicillin, 10,000 IU/ml), and 10% (wt/vol) FBS, at 37?C in a humidified atmosphere containing 5% CO2 as described15. The cytotoxicity of Pep-1 was investigated in the cell lines mentioned above. Cells grown in 35-mm-diameter dishes to 75% confluency (0.5 to 1 times 106 cells per dish) were incubated with 0.1-10 mM Pep-1, in 1 ml DMEM for 1 h, after which 10% (vol/vol) serum was added. Cell proliferation was measured over four days, and cytotoxicity was evaluated in a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), after removing cell culture medium and replacing it with PBS containing 5 mg/ml of MTT (ref. 20). For Pep-1-mediated delivery of proteins and peptides, Pep-1/protein or Pep-1/peptide complexes were formed in DMEM or PBS (500 mul of DMEM containing 0.25 mug of protein or peptide and a variable Pep-1:protein molecular ratio from 1:1 to 40:1) and incubated for 30 min at 37?C. Cells grown to 75% confluency were then overlaid with these preformed complexes. After 30 min incubation at 37?C, 1 ml of fresh DMEM supplemented with 10% FBS was added to the cells, without removing the overlay of Pep-1:peptide or Pep-1:protein, and cells were returned to the incubator for another 30 min. Cells were then extensively washed with PBS and examined for GFP or FITC fluorescence. For beta-Gal staining, cells were fixed with 2% formalin (Sigma), then incubated with 1 mg X-Gal in buffer containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 2 mM MgCl2. For transfection in the presence of serum, Pep-1/peptide and Pep-1/protein complexes were preincubated for 30 min in DMEM or PBS in the absence of serum, to which 10% FBS was added before addition to the cells.