Protein transduction in human cells is enhanced by cell-penetrating peptides fused with an endosomolytic HA2 sequence.

2.1. Plasmid construction The mCherry plasmid (kindly provided by Dr. Roger Y. Tsien, University of California, San Diego, CA, USA) is a prokaryotic expression vector that encodes a hexa-histidine (6His)-tagged monomeric RFP sequence ( Table 1 ) [ 44 ]. The pR9-mCherry plasmid containing a nona-arginine (R9, a CPP) and HA tag (YPYDVPDYA) fused RFP coding region under the control of the T7 promoter was described previously [ 54 ]. This HA tag does not appear to interfere with the bioactivity of recombinant fusion proteins and facilitates the detection, isolation and purification of many protein fusions [ 47 ]. The pR9-HA2-mCherry plasmid consisting of an R9 and HA2 (INF7) fused RFP coding sequence was constructed by the annealing and digestion of two overlapping primers (HA2-U [5′-TT AGATCT AGGCCTATTCGAGGCAATAGAAGGTTTCATAGAAAATGGTTGGGA-3′, with the Bgl II site underlined] and HA2-D [5′-TT GGATCC CCGTACCAACCGTCTATCATTCCCTCCCAACCATTTTCTATGAAA-3′, with the Bam HI site underlined]) cloned into the Bam HI site of the pR9-mCherry plasmid. The pR9-NLS-mCherry plasmid containing an R9 and NLS fused RFP was generated using two overlapping 5′ phosphorylated primers (NLS-U [5′-GATCCCAAGAAGAAGAGGAAAGTC-3′] and NLS-D [5′-GATCGACTTTCCTCTTCTTCTTGG-3′] cloned into the Bam HI site of the pR9-mCherry plasmid. The pR9-HA2-NLS-mCherry plasmid consisting of an R9, HA2 and NLS fused RFP was constructed by cloning the NLS-U/D fragment at the Bam HI site of the pR9-HA2-mCherry plasmid. All constructs were confirmed by DNA sequencing. 2.2.

Protein purification and characterization

Protein expression and purification were as described previously with modifications [ 3 , 27 ]. All plasmid constructs were transformed into Escherichia coli KRX strain (Promega, Madison, WI, USA). Bacteria were grown to OD 600 = 0.4 and then induced with 0.1% (w/v) rhamnose (Sigma-Aldrich, St. Louis, MO, USA) overnight at 37 °C. Expressed proteins were purified by metal chelation chromatography. The binding (5 mM imidazole, 0.5 M NaCl, 20 mM Tris–HCl, pH 7.9) and wash (80 mM imidazole, 0.5 M NaCl, 20 mM Tris–HCl, pH 7.9) buffers were used during purification. Purified proteins were concentrated as well as dialyzed using Amicon Ultra-4 centrifugal filter devices (Millipore, Billerica, MA, USA) and quantified by a protein assay kit (Bio-Rad, Hercule, CA, USA). Luminescent images of proteins were captured using the Typhoon FLA 9000 Biomolecular Imager (GE Healthcare, Piscataway, NJ, USA) with the excitation wavelength at 532 nm for RFPs [ 32 ]. Emission spectra of RFPs were evaluated using an EnSpire 2300 Multilabel Reader (PerkinElmer, Waltham, MA, USA) with excitation at 488 nm and emission varying from 510 to 700 nm. 2.3. Protein transduction, subcellular colocalization and mechanistic assays Human lung carcinoma A549 cells (American Type Culture Collection, Manassas, VA, USA; CCL-185) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Invitrogen, Carlsbad, CA, USA) containing phenol red supplemented with 10% (v/v) bovine serum (Gibco), as previously described [ 54 ]. To determine the optimal concentration for protein transduction, cells were seeded at a density of 1 × 10 5 cells per 35-mm petri dish and then incubated overnight in 1 ml of growth medium. Cells were washed twice with 1 ml of phosphate buffered saline (PBS) and then treated with various proteins at the final concentrations of 1, 5, 10, 30 and 60 μM in RPMI 1640 medium with neither phenol red nor serum at 37 °C for 1 h. Cells were washed five times with PBS to remove free proteins. To determine subcellular colocalization, cells were treated with 30 μM of various proteins for different durations (30 min, 1, 3, 6, 12, 18 and 24 h) in RPMI medium with neither phenol red nor serum at 37 °C. Cells were washed with PBS for five times to remove free proteins followed by staining with fluorescent organelle-specific trackers [ 27 ]. Hoechst 33342, LysoSensor Green DND-153 (Invitrogen) and fluorescein isothiocyanate (FITC) mouse anti-human early endosome antigen 1 protein (EEA1) antibody (BD Biosciences, Franklin Lakes, NJ, USA) were utilized to visualize nuclei, lysosomes and early endosomes, respectively. Fluorescent images were detected using a BD Pathway 435 System (BD Biosciences). To conduct energy-dependent uptake experiments, cells were incubated for 30 min at 4 °C at which temperature energy-dependent molecular movement in the cell membrane is essentially arrested [ 49 ]. Cells were then treated with 30 μM of R9-HA2-mCherry at 4 °C for 6 h followed by staining with Hoechst 33342 for 40 min and LysoSensor trackers for 30 min. The influence of modulators that inhibit or enhance uptake processes was studied in cells treated without or with cytochalasin D (CytD), nocodazole [ 33 ] or chloroquine (Sigma–Aldrich) [ 60 ]. Cells were pretreated in the absence or presence of 10 μM of CytD, 10 μM of nocodazole or 25 μM of chloroquine at 37 °C for 30 min. These cells were then treated with 30 μM of R9-HA2-mCherry in the absence or presence of CytD or nocodazole at 37 °C for 6 h, or chloroquine for 12 h followed by staining with Hoechst 33342 and LysoSensor trackers. To test any synergistic effect of the HA2 and chloroquine combination [ 57 ], cells were pretreated in the absence or presence of chloroquine at 37 °C for 30 min. These cells were then treated with 30 μM of R9-mCherry or R9-HA2-mCherry in the absence or presence of chloroquine for 12 h. To assess the CPP-HA2-mediated cargo delivery, cells were treated with collagen-fluorescein (Sigma–Aldrich) [ 23 ], R9-HA2-mCherry or R9-HA2-mCherry/collagen-fluorescein complexes at various ratios. Fluorescent images were detected using a BD Pathway 435 System (BD Biosciences). 2.4.

Confocal and fluorescent microscopy

Fluorescent and bright-field live cell images were recorded using a BD Pathway 435 System (BD Biosciences) equipped with the Olympus 20× and 60× oil objectives (Olympus, Tokyo, Japan) [ 27 ]. This system includes both confocal and fluorescent microscopy sets. Excitation filters were set at 377/50 nm, 482/35 nm and 543/22 nm for blue, green and red fluorescence, respectively. Emission filters were set at 435LP (long-pass), 536/40 nm and 593/40 nm for blue (BFP), green fluorescent protein (GFP) and RFP channels, respectively. Transmitted light with the 536/40 nm emission filter was used to observe cell morphology as bright-field images. 2.5.

Flow cytometric analysis

A549 cells were seeded at a density of 1 × 10 5 cells per well in 24-well plates and then incubated overnight in 500 μl/well of culture medium. Cells were treated with five proteins for designated durations (1, 5, 10, 30, 60 min, 3, 6, 12, 18 and 24 h) in RPMI medium with neither phenol red nor serum at 37 °C and then washed five times with PBS. Cells were analyzed using a Cytomics FC500 Flow Cytometer (Beckman Coulter, Fullerton, CA, USA), as previously described [ 24 ]. For RFP detection, excitation was set at 488 nm and emission at 615 nm with a FL3 filter. Results are reported as the percentage of the total cell population. 2.6.

Cytotoxicity assay

A549 cells were plated at a density of 1 × 10 4 cells in 96-well plates and incubated overnight in 200 μl/well of growth medium. Cells were treated with 1, 5, 10, 30 and 60 μM of five proteins in RPMI medium without phenol red nor serum, washed with PBS, and cultured in RPMI medium without serum at 37 °C for 24 h. Cells were treated with PBS as a negative control and 70% alcohol as a positive control. Cytotoxicity was assessed by the ability of the cells to reduce 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) [ 23 , 54 ]. MTT absorbance was measured at 570 nm wavelength using a Model 680 Microplate Reader (Bio-Rad). 2.7.

Statistical analysis

Data are expressed as mean ± standard deviation. Mean values and standard deviations were calculated from at least three independent experiments carried out in triplicates in each group. Statistical comparisons between the control and treated groups were performed by the Student t-test, using levels of statistical significance of P < 0.05 (*, †, ‡) and 0.01 (**, ††, ‡‡), as indicated.