Department of Bioengineering, University of Washington, Seattle, WA 98195, USA
1 To whom correspondence should be addressed. E-mail: stayton{at}u.washington.edu
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Abstract |
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Keywords: drug delivery/smart polymer/streptavidin/TAT peptide
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Introduction |
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The highly cationic 11 amino acid residue (YGRKKRRQRRR) PTD from the human immunodeficiency virus (HIV-1) TAT protein (Frankel and Pabo, 1988; Green and Loewenstein, 1988
) has been one of the most well-studied translocating peptides. In-frame fusion proteins containing the TAT sequence were shown to direct cellular uptake of proteins that retained their activity intracellularly (Nagahara et al., 1998
; Kwon et al., 2000
; Becker-Hapak et al., 2001
; Jo et al., 2001
; Xia et al., 2001
; Cao et al., 2002
; Joshi et al., 2002
; Kabouridis et al., 2002
; Peitz et al., 2002
). Subsequently, a diverse collection of over 60 full-length proteins with functional domains from 15 to 120 kDa have been engineered to date. Various studies employing TAT-fusion methodologies have demonstrated transduction in a variety of both primary and transformed mammalian and human cell types, including peripheral blood lymphocytes, diploid fibroblasts, keratinocytes, bone marrow stem cells, osteoclasts, HeLa cells and Jutkat T-cells (Fawell et al., 1994
; Nagahara et al., 1998
; Gius et al., 1999
; Vocero-Akbani et al., 1999
, 2000
, 2001
; Becker-Hapak et al., 2001
). Furthermore, in vivo intracellular delivery by injection of a TATß-gal fusion has been demonstrated (Schwarze et al., 1999
; Barka et al., 2000
).
In addition to drug delivery, there are many potential in vitro applications in areas such as drug discovery and laboratory assays that could benefit from improved intracellular delivery of biomolecules and macromolecular cargo. Here we have investigated whether the membrane-traversing capability of the TAT peptide could be conferred upon the versatile streptavidin molecular adaptor protein (Figure 1). TAT-SA was assayed for its retention of biotin binding and also for its ability to translocate through the cell membrane of human Jutkat T-cells. Cellular uptake and intracellular distribution were monitored by fluorescence activated cell sorting (FACS), biological activity assays and fluorescence microscopy of labeled TAT-SA. In addition, the ability of an endosomal-releasing polymer to enhance cytoplasmic delivery of internalized TAT-SA complexes was investigated.
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Materials and methods |
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The TAT-SA gene was constructed using core streptavidin (Chilkoti et al., 1995) in pUC18 plasmid (New England Biolabs, Beverly, MA) and overhang-primer polymerase chain reaction (PCR). The following oligonucleotide primers were used: forward overhang (5' ACG GGG AAT CAT ATG TAC GGT CGT AAA AAA CGT CGT CAG CGT CGT CGT GGT GCT GAA GCT GGT ATC ACC 3') and reverse (5' TTC GAA CCG TGA CCG GCA GC 3') from Integrated DNA Technologies (Coralville, IA), where the bold text denotes the TAT sequence. The PCR-generated cassette was digested with NdeI and HindIII restriction endonucleases (New England Biolabs) and subsequently ligated into NdeI/HindIII-linearized pUC18. The ligation products were transformed into TOP10F' competent cells (Invitrogen, Carlsbad, CA) and successful production of the TAT-SA gene was confirmed by DNA sequencing with an ABI Prism BigDye Terminator Cycle Sequencing-Ready Reaction Kit (Perkin-Elmer, Boston, MA). The TAT-SA gene construct was subsequently subcloned into the pET21a (Invitrogen) expression vector and transformed into BL21(DE3) cells (Novagen, Madison, WI) in preparation for large-scale expression. The TAT-SA construct in pET21a was expressed and purified as described previously (Klumb et al., 1998
). Briefly, TAT-SA was expressed as insoluble inclusion bodies that were isolated and then dissolved in 6 M guanidine hydrochloride, followed by refolding via dilution into Tris buffer, pH 8.0. Refolded, functional protein was purified by affinity chromatography using a 2-iminobiotin agarose column (Sigma, St. Louis, MO). Protein concentrations were determined using an extinction coefficient of 34 000 M1 cm1/subunit. Characterization of the TAT-SA fusion protein included matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, SDSPAGE and biotin off-rate determination using [3H]biotin (Amersham Biosciences, Piscataway, NJ) according to previously described methods (Klumb et al., 1998
).
Fluorescent labeling
WT-SA and TAT-SA were labeled using Alexa Fluor 488 (Molecular Probes, Eugene, OR). The fluorophore was dissolved in dimethylformamide at a concentration of 2 mg/ml and the conjugation reaction was performed with a 5-fold molar excess of the fluorophore in 0.1 M sodium carbonatesodium bicarbonate, pH 9.0. The reaction was performed for 1 h at room temperature, followed by dialysis with a 3500 MWCO Slide-A-Lyzer dialysis cassette (Pierce, Rockford, IL) in phosphate-buffered saline (PBS), pH 7.4. The protein concentration in the dialyzed sample was determined spectrophotometrically by subtracting 0.11A495 nm of the fluorophore from the A280 nm of the protein, using the tetramer WT-SA extinction coefficient of 136 000 M1 cm1. The degree of labeling was calculated by dividing the A495 nm of the fluorophore by the molar concentration of the protein and the dye extinction coefficient of 71 000 M1 cm1. WT-SA and TAT-SA were successfully labeled with Alexa Fluor 488 with a degree of labeling of 1.63 and 2.68 fluorophores per tetramer, respectively.
Preparation of TAT-SA complexes
TAT-SA was complexed with a biotinylated version of the 240 kDa fluorescent protein R-phycoerythrin (R-PE) (Molecular Probes) by incubation at varying molar ratios for 15 min at room temperature. TAT-SAalkaline phosphatase complexes were formed similarly using biotinylated calf intestinal alkaline phosphatase (AP) (Pierce) to examine the delivery of a large, active enzyme (140 kDa). For quenching studies, TAT-SAAP complexes were formed under the same conditions with Alexa-488-labeled biotinylated calf intestinal AP (US Biological, Swampscott, MA) at a 1:2 molar ratio.
Cell culture
Human Jurkat T-lymphoma cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine. Jurkat cells were grown on T75 tissue culture flasks to a density of 1 x 106 cells/ml before subculturing. Cells were incubated at 37°C in a 5% carbon dioxide atmosphere.
Flow cytometry
The internalization of fluorescently labeled TAT-SA, fluorescent TAT-SA(R-PE) complexes and fluorescent TAT-SAAP complexes were analyzed in Jurkat cells which had been plated at a density of 105 cells in 0.2 ml of RPMI medium in a 96-well microtiter plate. Triplicate protein samples were prepared in PBS, pH 7.4 and incubated at 37°C in a 5% carbon dioxide atmosphere for 3 h. The cells were then washed in 0.2 ml of PBS, pH 7.4 three times before resuspension in fresh buffer for analysis on a Coulter Epics FACS analyzer with excitation at 488 nm. TAT-SAAP-treated cells were washed three times and then resuspended in PBS, pH 7.4 containing a 2x equivalent of anti-Alexa Fluor 488 (Molecular Probes) at room temperature for 20 min, followed by FACS analysis. For each protein sample, data were collected for 10 000 events of the gated population and included forward scatter (FS), side scatter (SS), fluorescence-1 (FL-1, Alexa Fluor 488) and fluorescence-2 [FL-2, (R)-phycoerythrin] measurements. In all analyses, viable cells were chosen based on FS and SS values, where cells displaying high FS and low SS were considered intact and uncompromised because of their light diffraction profiles. The number of viable cells considered positive for protein uptake was determined by gating cells with FL-1 greater than the maximum of control wells with no labeled protein added.
Alkaline phosphatase activity assay
Jurkat cells were prepared as described above. Triplicate samples of unlabeled WT-SAAP and TAT-SAAP complexes were prepared at different molar ratios of AP, while holding the concentration of each SA species constant at 80 nM. Cells were treated and incubated at 37°C in a 5% carbon dioxide atmosphere for 4 h. The cells were then washed three times with PBS, pH 7.4 and resuspended in 0.1 ml of 1x Reporter Lysis Buffer (RLB) (Promega, Madison, WI). Cell lysates were incubated at room temperature for 15 min with intermittent shaking and then centrifuged at 300 g for 5 min to sediment cellular debris and unlysed cells. Colorimetric reactions were initiated by pipetting 50 µl of the supernatant from each well into a fresh 96-well plate and subsequently adding 50 µl of 1-Step pNPP Buffer (Pierce). After incubating at 37°C for 30 min, reactions were stopped by the addition of 150 µl of 1 M sodium carbonate and the absorbance at 405 nm was measured on a plate reader (SoftMax). Calibration curves were generated using a series of dilutions of biotinylated AP (Pierce) in PBS, pH 7.4 and 1x RLB.
Optical microscopy
Jurkat cells were plated at a density of 105 cells and incubated with 80 nM Alexa 488-labeled TAT-SA for 3 h. Jurkat cells were washed three times, resuspended in 15 µl of PBS, pH 7.4, mounted on a glass slides for imaging using a confocal Leica TCS NT/SP on a DMIRBE inverted microscope. Images were acquired using a 100x inverted oil immersion objective and the appropriate filter set for Alexa-488.
Preparation of polymer complexes
Amine-terminated poly(propylacrylic acid) (PPAA) was synthesized and end-biotinylated using sulfosuccinimidyl-6-(biotinamido)-6-hexanoate (EZ-Link Sulfo-NHS-LC-Biotin, Pierce) as described previously (Lackey et al., 1999; Murthy et al., 1999
; Lackey et al., 2002
). The weight-average molecular weight of PPAA before biotinylation was 38 kDa and the biotinylation efficiency was determined as
100% (data not shown). TAT-SAPPAA complexes were assembled by mixing together the two components at a 1:1 molar ratio at room temperature for 15 min. SDSPAGE was used to characterize the complexes.
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Results |
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MALDI mass spectrometry of refolded, purified TAT-SA yielded a molecular weight of 15 001.01 Da, which is in a good agreement with the calculated theoretical mass of 15 001.50 Da (ExPASy) per monomer (data not shown). The biotin off-rate kinetics of TAT-SA were measured and compared with WT-SA to determine possible effects of the TAT sequence on the biotin-binding properties of the mutant. Biotin-bound WT- and TAT-SA displayed monoexponential, first-order dissociation kinetics. The first-order dissociation rate constant (koff) for WT- and TAT-SA at 37°C was found to be 25 x 106 and 30 x 106 s1, respectively.
Internalization of TAT-SA and TAT-SA(R-PE) complexes
Flow cytometry was used to quantify the uptake of the Alexa-488-labeled TAT-SA and fluorescent TAT-SA(R-PE) complexes. Test samples were compared with untreated cells and included cells incubated with WT-SA alone, WT-SA(R-PE) complexes, TAT-SA alone or TAT-SA(R-PE) complexes. Jurkat cells were incubated with a fixed 80 nM concentration of WT-SA or TAT-SA, each complexed with a varying molar ratio of R-PE as indicated. FACS analysis of washed cells 3 h after treatment with TAT-SA revealed a strong Alexa-488 fluorescence signal (Figure 2A) compared with gated WT-SA-treated cells, indicating that internalization was mediated by the NH2-terminal TAT peptide. Similar FACS results were obtained when transfections were performed at 37 or 4°C. Samples treated with TAT-SA(R-PE) complexes at a 1:1 molar ratio displayed strong phycoerythrin fluorescence intensity, whereas those treated with WT-SA(R-PE) did not (Figure 2B).
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TAT-SA complexes were formed with biotinylated calf intestinal AP which had been labeled with Alexa-488. Nearly 85% of cells treated with TAT-SAAP complexes were positive for Alexa-488 fluorescence as compared with 8% of cells treated with WT-SAAP complexes (Figure 3B). Post-treatment of parallel cell populations with the impermeable quenching antibody resulted in a slight, statistically insignificant (P > 0.05) decrease in the fluorescence intensity of gated cells, indicating again that a majority of the detected fluorescence signal was due to internalized TAT-SAAP complexes. AP activity was measured in the cellular lysates of TAT-SAAP-treated cells 4 h after treatment. Enzymatic activity increased with increasing molar ratio of AP, illustrating the ability for one TAT-SA molecule to bind and internalize multiple AP molecules with no apparent loss of enzymatic function (data not shown) (Figure 4). WT-SAAP conjugates were not internalized and the treated samples displayed an amount of activity consistent with the negative controls. These data confirmed that TAT-SA is capable of delivering a large biotinylated enzyme with retention of biological activity.
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The intracellular localization of internalized TAT-SA was investigated through the use of confocal microscopy and fluorescently labeled TAT-SA and TAT-SApolymer complexes. Representative images of live Jurkat cells 3 h after treatment with Alexa-488-labeled TAT-SA are shown in Figure 5a. WT-SA-treated cells showed low or no fluorescence (data not shown), consistent with low-level, non-specific uptake, whereas cells treated with TAT-SA displayed punctate patches of intracellular fluorescence. These results reveal that the TAT sequence does not direct cytoplasmic delivery in this streptavidin fusion protein. The punctate fluorescence distribution is strongly suggestive of endosomal/lysosomal compartmentalization. The strongly positive TAT sequence was likely directing pinocytosis of electrostatically bound TAT-SA, which would result in endosomal/lysosomal localization.
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Discussion |
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The data presented here demonstrate that the TAT-SA fusion does not function/act through a truly transductive process, but instead directs internalization through an electrostatically mediated cell surface adherence. As confirmed through live cell microscopy, the TAT-SA fusion protein is internalized with high efficiency but sequestered in small vesicular compartments, suggestive of an endocytic mode of entry. The internalization of TAT-SA complexed to a range of biotinylated cargo proteins gave similar results. In order to achieve cytoplasmic delivery, the biomimetic, pH-sensitive polymer PPAA was used to direct endosomal release. The addition of the biotinylated PPAA to TAT-SA significantly altered the intracellular distribution of the internalized fusion protein and cargo, resulting in diffuse cytoplasmic localization. This work therefore demonstrates that TAT-SA and PPAA can be used jointly as a biomolecular vehicle for delivering heterogeneous cargo proteins of large size in an apparent stepwise uptake and endosomal release mechanism.
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Acknowledgements |
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Received January 24, 2005; accepted February 22, 2005.
Edited by Ashutosh Chilkoti
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