1
A gene delivery system based on the N-terminal domain of human toposiomerase I
1
Yi-An Chen1,5,§, Hsiao-Che Kuo1,3,4,§, Young-Mao Chen1,3,4, Shin-Yi Huang1, Yu-Ru Liu1, 2
Su-Ching Lin1,2, Huey-Lang Yang2,3,4 and Tzong-Yueh Chen1,3,4,* 3
1Laboratory of Molecular Genetics and 2Laboratory of Aquatic Animal Disease, Institute of 4
Biotechnology, College of Bioscience and Biotechnology; 3Research Center of Ocean 5
Environment and Technology; 4Agriculture Biotechnology Research Center, National Cheng 6
Kung University, Tainan 70101, Taiwan. 5Department of Food Nutrition, Chung Hwa 7
University of Medical Technology, Tainan 71703, Taiwan. 8
§these authors contributed equally to this work 9
Running title: DNA delivery based on human topoisomerase I 10
*Corresponding author: Dr. Tzong-Yueh Chen 11
Laboratory of Molecular Genetics, Institute of Biotechnology, College of Bioscience and 12
Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan 13
Telephone: 886-6-2757575 ext. 65622 ext. 610 14 Fax: 886-6-2766505 15 E-mail: [email protected] 16 17
Source:Biomaterials, Vol. 32, No. 17, pp. 4174-4184 Year of Publication: 2011
ISSN:0142-9612 Publisher:Elsevier
DOI :10.1016/j.biomaterials.2011.02.041 © 2011 Elsevier Ltd. All rights reserved
2 Abstract
18
The N-terminal 200 amino acid residues of topoisomerase I (TopoN) is highly positive in 19
charge and has DNA binding activity, without DNA sequence and topological specificity. 20
Here, a fusion protein (6×His-PTD-TopoN) containing a hexahistidine (6×His) tag, a 21
membrane penetration domain and TopoN (amino acid 3–200) was designed and developed. 22
The protein can bind to different sizes (3.0–8.0 kb) and forms (circular and linear) of DNA 23
and translocates the bound DNA to the nucleus. The protein also showed low cytotoxicity to 24
GF-1 grouper fish fin cells that were previously very sensitive and difficult to transfect in 25
vitro. Maintaining the hexahistidine tag increased the protein’s transfection efficiency in 26
COS7 African green monkey kidney cells and simplified the purification process. The 27
plasmid pEGFP-N1 was delivered into COS7 cells by the protein in ATP- and 28
temperature-dependent manners. The results indicate that the binding ability of TopoN is very 29
useful for DNA delivery and the carrier protein can be expressed in Escherichia coli without 30
removal of the hexahistidine tag. 31
Keywords: gene delivery system; protein transduction domain; topoisomerase I
32 33
3 1. Introduction
34
Efficient delivery of genetic materials through the cell membrane is the key step for success 35
in gene therapy, DNA vaccines and genetically modified organisms. The cell membrane is 36
generally impermeable to exogenous bioactivity molecules such as proteins and peptides. 37
Techniques used for delivering genetic materials including calcium phosphate precipitation 38
[1], DEAE-dextran-mediated cell fusion [2] and electroporation [3] have been used in vitro 39
and in vivo, virus infection [4], microinjection [5] and liposome mediated transfer[6] have 40
been used in vivo. 41
Among those methods, the most common is virus infection, which can deliver the 42
exogenous gene into cells and even to insert the gene into a host genome. The gene can 43
maintain and function in the host cell, but is impossible to control and identify the insertion 44
location of the host genome [7–9]. Using the virus as a carrier can have higher transfection 45
efficiency, but also with higher potential risk of inflammatory response and virulence. The 46
application limitations of viral vectors are toxicity, restricted targeting of specific cell types 47
[10], limited DNA carrying capacity, production and packaging problems, recombination and 48
expense [11]. The most wildly used of non-viral vectors for delivering DNA into cells are 49
cationic lipids or polymers [12]. Other methods/vectors such as electroporation, 50
microinjection, cell fusion organic or inorganic nanoparticles [13] are also used for 51
transfection. Cationic liposomes and polymers can form electrostatic complexes with DNA 52
4
and protect these complexes from nuclease degradation by condensing DNA [14]. While 53
non-viral gene delivery systems are safer than viral gene delivery systems [15, 16] they suffer 54
from complicated operation procedures, low transfection efficiency [9] and may cause 55
damage to the cell. 56
Proteins can be taken up into cells through endocytosis. Some proteins, such as bacterial 57
toxins, growth factors, homeoproteins and viral proteins are able to pass through the cell 58
membrane when added exogenously [17–19]. Recently, membrane-permeable peptide 59
delivery systems that include short peptide segments derived from human immunodeficiency 60
virus type 1 (HIV-1) Tat [20, 21], Drosophila antennapedia (Antp) homeotic transcription 61
factor [22] and VP22 protein from herpes-simplex-virus-1 [17, 23] – which are termed 62
cell-penetrating peptide (CPP), protein transduction domain (PTD) or Trojan horse peptides – 63
have been used to deliver various molecules including proteins, small molecular weight 64
compounds, oligonucleotides and liposomes into cells [24, 25]. Most of the peptide delivery 65
vehicles contain fewer than 20 amino acids and are rich in basic or hydrophobic amino acids: 66
an example is the tryptophan-rich peptide pep-1 (KETWWETWWTEWSQPKKRKV) that, 67
when applied at a certain range of concentrations, can carry proteins and peptides into 68
mammalian cells without receptor-mediation, input of energy and harm to the cells [26]. 69
In this study, a protein containing DNA-binding and membrane transduction domains 70
was used to deliver DNA into variety of cell types. The DNA binding and nuclear 71
5
localization capabilities of the protein were bestowed by the N-terminal domain of human 72
topoisomerase I (TopoN), a 765-amino acid protein comprised of an unstructured N-terminal 73
domain of 200 amino acids, a core domain, a linker domain, and a C-terminal domain [27, 74
28]. TopoN regulates DNA topology by making single-strand breaks, allowing strand passage, 75
and then resealing the breaks independent of ATP hydrolysis [29]. It can bind positively and 76
negatively supercoiled DNA [30], and plays an important role in different aspects of DNA 77
metabolism such as DNA replication, DNA recombination and transcription [29, 31, 32]. In 78
addition to its catalytic activity on DNA, TopoN functions as a kinase to phosphorylate RNA 79
splicing factors [33]. TopoN is poorly conserved, highly positively charged [34], unstructured, 80
protease sensitive and contains nuclear localization sequences (NLSs) [27, 28, 35–37]. 81
Although the NLSs do not contribute to the catalytic activity, they are essential for the 82
nuclear translocation of the enzyme [36, 38]. TopoN also binds to DNA [10, 39] without 83
DNA sequence and topological specificity [10]. 84
The present study was undertaken to develop a new DNA delivery system based on a 85
short amphipathic peptide carrier, pep-1 [26]. The pep-1 NLS (KKRKV) and spacer domain 86
(SQP) were removed and the altered peptide was fused to TopoN. A hexahistidine (6×His) 87
tag was added in the N-terminus of the designed protein to enable purification. A spacer 88
(SQPGR) between pep-1 and TopoN harbored a proline residue to improve the flexibility and 89
integrity of the linked peptides. 90
6 Materials and methods
91
2.1. Modification of genes and construction of plasmids 92
6×His-pep1-TopoN (Fig. 1A) and 6×His-pep2-TopoN (Fig. 1B) were modified from pep-1 93
[26] and pep-2 [40], in which the spacer domain (SQP) and NLS (KKRKV) were replaced by 94
a spacer domain (SQPGR) and 198 amino acids of TopoN (3–200). We have tried different 95
sequences and mainly on the TopoN (N-terminus 198 a.a. and 98 a.a.) which is important on 96
DNA binding and nuclear localization. The result showed that 198 a.a. of human 97
topoisomerase I was better than the shorter one. In addition, use SQPGR as a spacer between 98
pep-1/2 and TopoN was more suitable in this study than SQP. For construction of the plasmid 99
expressing 6×His-pep1-TopoN and 6×His-pep2-TopoN, the oligonucleotides encoding 100
tryptophan-rich peptide (pep1, MGKETWWETWWTEW and pep2, MGKETWFETWFTEW) 101
with spacer domain (SQPGR) were synthesized (Mission Biotech, Taipei, Taiwan), cloned 102
and inserted into the pET15b vector (Novagen, Darmstadt, Germany). The vector contains 103
TopoN (Fig. 1, Suppl. Table 1) and encodes the 6×His tag at the N terminus (Figs. 1A, 1B 104
and 1D; Suppl. Table 1). TopoN was constructed with vector pET15b by use of BamHI and 105
EcoRI. Proteins without the hexahistidine tag (pep1-TopoN; Fig. 1C) and without PTD 106
domain (6×His-TopoN; Fig. 1D) were used as the controls. Gene expression was driven by 107
the T7 promoter and all constructs were sequenced and determined to be error-free. 108
2.2. Protein expression, protein purification and immunoblotting 109
7
6×His-pep1-TopoN and 6×His-pep2-TopoN were expressed in Escherichia coli BL21(DE3) 110
by isopropyl-1-thio-D-galactopyranoside (IPTG) induction. The expressed protein was 111
collected by centrifugation and resuspended in 40 ml of 1 × binding buffer (5 mM imidazole, 112
0.5 M NaCl, 20 mM Tris-Cl, pH 7.9). Cells were broken by sonication. The lysate was 113
centrifuged at 13,000 g for 30 min at 4 ºC, and the supernatant was applied to a Ni+-HiTrap 114
affinity column (Pharmacia Biotech, Upsalla, Sweden) and was concentrated by Centricon 115
plus-20 (Millipore Asia, , Taipei, Taiwan). The fractions were eluted with 250 mM and 300 116
mM imidazole containing 0.5 M NaCl and 20 mM Tris-Cl, pH 7.9. The sizes of the proteins 117
were determined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis 118
(SDS-PAGE) and Coomassie Brilliant Blue staining (Fig. 2A). Pre-stained protein marker 119
standards (Invitrogen, Carlsbad, CA) were included on each gel for molecular weight 120
estimation. Monoclonal anti-His-tag (diluted 1:2000) and monoclonal rabbit anti-PTD 121
antibodies (diluted 1:3000) were used for the detection of 6×His-PTD-TopoN and 122
PTD-TopoN, respectively. Alkaline phosphatase-conjugated goat anti-rabbit antibody (Santa 123
Cruz Biotechnology, Santa Cruz, CA.) diluted 1:2000 was used as the secondary antibody. 124
2.3. Cells and cell culture conditions 125
To exam the transfection efficiency of 6×His-pep1-TopoN and 6×His-pep2-TopoN in 126
different cells, COS-7 African green monkey kidney cells (kindly supplied by Dr. Shyh-Yu 127
Shaw, National Cheng Kung University), 3T3 mouse embryo fibroblast cells (Bioresources 128
8
Collection and Research Center (BCRC), Taipei, Taiwan; BCRC 60159) and GF-1 grouper 129
fin cell (BCRC 960094) were used. Cells were maintained in Dulbecco’s Modified Eagle’s 130
Medium (DMEM, Gibco, Grand Island, NY) and 10% (w/v) fetal bovine serum (FBS). 131
Except for GF-1, the cells were cultured as a monolayer in a humidified atmosphere 132
containing 5% CO2 at 37 ºC. GF-1 cells were grown in humidified incubator at 28°C in 133
antibiotic-free L15 medium (Life Technologies, Gaithersburg, MD) supplemented with 5% 134
v/v heat-inactivated FBS [41]. The cells were imaged by fluorescence microscopy (Olympus 135
IX70; Olympus, Tokyo, Japan) using a 488 nm excitation wavelength. 136
2.4. Lipofectamine transfections 137
Lipofectamine™ transfection was performed following the manufacturer’s instructions 138
(Invitrogen, Carlsbad, CA). Briefly, serum-free DMEM (Gibco) was used to replace all 139
existing medium on cells. For each well of 24-well plate, 1 μg DNA was added to 50 μl 140
DMEM in one tube and lipofectamineTM transfection reagent (Invitrogen) (1 μl was used in 141
Figs. 5C and 5F and 0.5, 1 and 2 μl were used in Fig. 6) with 50 μl DMEM in another tube. 142
The contents of both tubes were mixed and incubated for 20 min then added to cells. After 24 143
h incubation, the serum-free DMEM was replaced with RPMI-1640. Observation and 144
imaging of green fluorescent protein (GFP) expression in the cells was performed using 145
fluorescence microscopy (Olympus IX70; Olympus). 146
2.5. Electrophoretic mobility shift assay (EMSA) 147
9
Reaction mixture totaling 15 μl contained the 6×His-pep1-TopoN and 6×His-pep2-TopoN 148
plasmid and 1× gel retardation assay buffer (20 mM HEPES, pH 7.6, 100 mM KCl, 5 mM 149
MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol and 10% glycerol). The plasmids used in this 150
study were pBluescript SK+ (pBSK+; Stratagene, LaJolla, CA), pCMV-Mx-egfp [37], 151
pEGFP-N1 (Clontech, Mountain View, CA) and pF4X1.4hyg (Jena Bioscience GmbH, Jena, 152
Germany). Within these plasmids, pBluescript SK+ and pF4X1.4hyg were cut by BamHI and 153
XhoI, respectively, to obtain linear form DNA. The amount of DNA was fixed to 1 μg and the 154
amounts of protein were very according to the molar ration. The molecular weight (MW) of 155
pep1-TopoN and pep2-TopoN is about 36.5 KDa. The MW of BSA is 66.776 KDa and 156
protease K is 28.9 KDa. 1 base pair MW of dsDNA is 0.66 KDa. The reaction mixture was 157
incubated at 37 ºC for 30 min. Bovine serum albumin (BSA, molar ratio of 40:1 or 100:1) 158
was used as a control protein and loss-of-function was demonstrated by adding protease K 159
(ProK, Sigma-Aldrich, St. Louis, MO) to 6×His-pep1-TopoN. The concentration of protease 160
K followed the manufacture’s recommendation. Each reaction was run on a 0.7% agarose gel 161
during electrophoresis in 1 × TBE buffer, and the DNA was visualized by staining with 162
ethidium bromide. 163
2.6. Viability and proliferation assay 164
Cell viability was determined using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 165
bromide (MTT) as detailed by the manufacturer (Promega, Madison, WI) in a modification of 166
10
a previously described protocol [42]. Cells were washed with warm RPMI-1640 without 167
phenol red and a MTT working solution (0.5 mg ml-1 MTT in RPMI-1640 without phenol red) 168
was added (10 μl for each well) into wells of a 96-well plate. Except for GF-1, cells were 169
incubated in a 5% CO2 incubator at 37 ºC for 4 hours. GF-1 cells were incubated at 28 ºC. 170
The converted dye was solubilized with 1 ml acidic isopropanol (0.04 M HCl in absolute 171
isopropanol) and the absorbance was measured at 570 nm with background subtraction at 650 172
nm using a Shimadzu UV-1201 spectrophotometer and disposable plastic cuvettes. Relative 173
cell viability at 4 h was compared to control cells containing cell culture medium without 174
copolymer using the following equation: 175
Relative cell viability (%) = ([OD]test ÷ [OD]total cells )× 100% 176
[OD]test = [OD]sample – [OD] medium 177
2.7. Luciferase activity assay 178
Cells were seeded in wells of 12-well plates and grown to 70% confluence. The control 179
experiment was without BSA in the culture medium. 6×His-PTD-TopoN was mixed with 180
pGL3-Promoter vector (1 μg) (molar ratio of 15:1) and added to each well. After 2 h, the 181
culture medium with BSA was added into each well and cultured for 48 h. Cells were 182
collected and lysed directly in cell lysis buffer (100 mM potassium phosphate pH 7.8, 1 mM 183
EDTA, 10% glycerol, 1% Triton X-100, 7 mM ß-mercaptoethnaol). Cell lysates were mixed 184
with luciferase substrate (luciferase activity reagent, 25 mM Tricine pH 7.8, 15 mM 185
11
postassium phosphate pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1 mM ATP, 0.1 mM 186
dithiothreitol) and measured immediately with a Lumat LB9501 luminometer (Berthold 187
Technologies, Bad Wildbad, Germany,). All transfection experiments were performed in 188
triplicate. 189
2.8 Statistical analysis 190
The data were obtained from triplicate measurements and summarized as means ± standard 191
deviation (S.D.). Statistical differences (p < 0.05) were performed by Student’s t-test. 192
12 3. Results
194
3.1. PTD-TopoNs expression and purification 195
The constructed plasmids were transfected and expressed in E. coli BL21(DE3), and 196
production of the protein was induced by IPTG and purified (Fig. 2A). Western blotting 197
confirmed that the 6×His-pep1-TopoN and 6×His-pep2-TopoN proteins were recognized by 198
anti-PTD (data not shown) and anti-hexahistidine-tag monoclonal antibodies (Fig. 2B). 199
3.2. pep1-TopoN stability 200
The 6×His-pep1-TopoN and 6×His-pep2-TopoN proteins were transfected with similar 201
efficiency. Hereafter we only focus on 6×His-pep1-TopoN. 6×His-pep1-TopoN was purified 202
and stored at 4 ºC, -20 ºC and -80 ºC (Figs. 2C and 2D). When the proteins were analyzed by 203
12% SDS-PAGE and Coomassie Brilliant Blue staining, its stability was confirmed, with 204
only a single major band was evident after storage for 3 weeks (Fig. 2C) and 8 months (Fig. 205
2D). Freshly purified 6×His-pep1-TopoN did not display evident of appreciable degradation 206
when stored at 37ºC for 30 min, 1 h and 2 h (Fig. 2E). Experiments in which 207
6×His-pep1-TopoN was left at room temperature for at least 2 h confirmed the undiminished 208
function of the protein upon translocation. 209
3.3. DNA binding assay of PTD-TopoNs 210
6×His-pep1-TopoN and 6×His-pep2-TopoN were mixed with plasmid pBSK+ (3.0 kb) (Fig. 211
3A), pEGFP-N1 (4.7 kb) (Fig. 3B) or pCMV-Mx-egfp (8.0 kb) (Fig. 3C) in different molar 212
13
ratios (5:1, 10:1, 20:1 and 40:1). Both the 6×His-pep1-TopoN and 6×His-pep2-TopoN 213
exhibited binding to plasmids ranging in size from 3.0–8.9 kb (Fig. 3). The control (bovine 214
serum albumin) displayed no binding to DNA observed on the same agarose gels. The gel 215
mobility shift assay revealed decreasing plasmid mobility with increasing molar ratio of 216
6×His-pep1-TopoN and 6×His-pep2-TopoN (Fig. 4), consistent with the increased binding of 217
DNA to either protein. Similar results were obtained with the circular form of DNA, in which 218
more DNA was bound to 6×His-PTD-TopoN (Fig. 4). When proteinase K was added to digest 219
6×His-pep1-TopoN, the shift in DNA mobility disappeared. We had done the time course of 220
0.5, 1, 2 and 4 h. The results showed that after 0.5 h the peptide already bind to DNA and 221
continue to increase the bind amount of DNA until 2 h. No significant difference between 2h 222
and 4h. We then choose the 2h though the whole paper as the reaction time. 223
3.4. in vivo DNA delivery of PTD-TopoNs 224
COS-7 cells were incubated for 2 h in the presence of 6×His-pep1-topoN and 225
6×His-pep2-TopoN with 1 μg pGL3-Promoter plasmid encoding the reporter gene luciferase. 226
Use of different molar ratios of 6×His-PTD-TopoN to pGL3-Promoter plasmid (5:1, 10:1, 227
15:1, 20:1, 25:1 and 30:1) determined that ratios of 15:1 for pep1-TopoN to DNA and 20:1 228
for pep2-TopoN to DNA produced maximum luciferase activity in COS7 cells (Fig. 5A). The 229
transfection efficiency of 6×His-pep1-TopoN was significant (p < 0.005) higher than 230
6×His-pep2-TopoN in the molar ratio of 15:1. But opposite result was obtain in the molar 231
14
ratio of 20:1. 6×His-PTD-TopoN (6×His-pep1-TopoN) was capable of transfecting 232
pEGFP-N1 into COS-7 cells, allowing the expression of GFP. The 7.2% transfection 233
efficiency (Figs. 5D and 5G) was better than the rate achieved using lipofectamine (5.2%) 234
(Figs. 5C and 5F). In the absence of 6×His-pep1-TopoN and lipofectamine, pEGFP-N1 was 235
unable to pass through the cell membrane and no fluorescence was evident in COS7 cells 236
(Figs. 5B and 5E). 237
3.5. Cytotoxicity test of PTD-TopoNs 238
COS-7, 3T3 and GF-1 cells were used to test the cytotoxicity of 6×His-pep1-TopoN, 239
6×His-pep2-TopoN, 6×His-TopoN and lipofectamine. GF-1 cells were very sensitive to 240
commonly-used amount of lipofectamine (0.5, 1 and 2 μl for each well), while cell 241
proliferation was completely unaffected by 6×His-pep1-TopoN and 6×His-pep2-TopoN (p < 242
0.005, Fig. 6A). 3T3 cells were most resistance to exogenous proteins or lipid, with over 80% 243
of the examined populations remaining capable of proliferation (Fig. 6B). COS7 cells were 244
the most sensitive cell type to lipofectamine, 6×His-pep1-TopoN and 6×His-pep2-TopoN 245
(Fig. 6C). The results highlighted the differing responses to different cell types to exogenous 246
proteins or lipid. Even a low amount of lipofectamine (2 μl per well) could similarly affect 247
cells in the presence of higher concentrations of proteins (1,000 μM of 6×His-pep1-TopoN 248
and 6×His-pep2-TopoN). The amount (12, 24 and 48 μl) of lipofectamine indicated in Fig.6 249
were the total amount been used per 24-well plate. 250
15
3.6. Cytotoxicity test of PTD-TopoNs/DNA complex 251
3T3 and COS-7 cell populations received were used to test different concentrations of 252
6×His-PTD-TopoNs or 6×His-PTD-TopoNs/DNA complex. Increasing concentration of 253
either preparation produced increased cytotoxicity (p < 0.005, Figs. 7A and 7B). 254
Approximately 40% of the 3T3 populations survive after a 3 h exposure to 10 mM 255
6×His-PTD-TopoNs/DNA at 37 ºC in DMEM supplemented with 10% FCS (Fig. 7B). 256
Treatment with 6×His-PTD-TopoNs under the same conditions resulted in increased 257
cytotoxicity (p < 0.005,), with only about 27% of the cell populations surviving (Fig. 7A). 258
Lipofectamine produced even higher cytotoxicity at lower concentration (Fig. 6). 259
3.7. Energy requirement of PTD-TopoNs 260
Cells were pre-incubated for 1 h at 4oC or 37 ºC, or with 10 mM sodium azide and 6 mM 261
2-deoxy-D-glucose to deplete cellular ATP. Gene delivery via the 6×His-PTD-TopoNs was 262
inhibited at 4ºC (p < 0.005) and by depletion of cellular ATP (p < 0.01, Fig. 8). 263
3.8. Effect of His6-tag on pep1-TopoN transfection efficiency 264
COS7 cells were incubated for 2 h in the presence of 6×His-pep1-TopoN/DNA complexes 265
(15:1) with 1 μg pGL3-Promoter plasmid encoding the luciferase reporter gene. The 266
transfection efficiency of 6×His-pep1-TopoN was higher (p < 0.01) than pep1-TopoN (Fig. 267
9). 268
3.9. Effect of structural constraints on transfection activity 269
16
The 6×His-pep1-TopoN fusion protein was denatured with 6M urea and then tested for DNA 270
binding activity using an agarose gel mobility shift assay. For 1 μg of pCMV-Mx-EGFP-N1 271
(~8.0kb) plasmid DNA, a mobility shift of the DNA bands was first detected when denatured 272
or native forms of fusion protein were added to plasmid at a 10:1 molar ratio (Fig. 10A). 273
Denatured and native forms of the peptides on transfection to COS-7 cells showed similar 274
results (Fig. 10B). 275
17 4. Discussion
277
The results demonstrate that a fusion protein containing a PTD domain and TopoN can be 278
used to deliver functional exogenous DNA into three different cell types. The fusion protein 279
meets the criteria of a successful delivery system: in order, penetration of the cell membrane, 280
nuclear localization and binding to DNA; as well as cellular/tissue specificity [43] and lack of 281
cell and tissue toxicity. 282
The most important feature PTD is the ability to transport genes of interest into the cell 283
lines or primary cells; this effectiveness has been confirmed [26]. The designed protein, 284
6×His-PTD-TopoNs, can bind DNA and transport DNA through the cell membrane by virtue 285
of the TopoN and PTD domains, respectively, and spontaneously locates to cell nuclei due to 286
the NLSs present in TopoN. NLSs function in the active transport of exogenous proteins and 287
probes into the nucleus [44, 45]. This function of the TopoN NLSs was confirmed in the 288
TopoN of 6×His-pep1-TopoN (Figs. 5D and 5G). Unstructured and non-enzymatic 289
functioning TopoN possess DNA binding ability [10], although the details have been unclear. 290
To clarify the binding ability of TopoN, the binding of 6×His-PTD-TopoNs to different sizes 291
and forms of DNA was assessed. 6×His-PTD-TopoNs bound to various sizes of DNA (Fig. 3) 292
and to both circular and linear forms of plasmids (Figs. 3 and 4), which was indicative of a 293
broad application on DNA binding. 294
Compared with previous transfection methods, the advantages of using PTD to carry 295
18
exogenous genetic material into cells is that the PTD domain can significantly increase the 296
transfection efficiency (e.g., in primary lymphocytes) [46, 47]. In in vivo experiments, PTD 297
has broad applications, e.g., PTD can even penetrate the blood-brain barrier [48], while viral 298
vectors can only carry foreign genes to the brain artery adventitia. Not only mammalian cell 299
but also the plant cell wall can be penetrated by PTD [49, 50]. Here, we also showed that the 300
COS-7 cell transfection efficiency of the DNA delivery system (7.2 %, Figs. 5D and 5G) is 301
superior than that provided by lipofectamine (5.2 %, Figs. 5C and 5F). The transfection 302
efficiency (7.2 %) of 6×His-PTD-TopoNs was lower than poly-L-lysine-palmitic acid is (~22 303
%) and Lipofectamine TM 2000 (~11%) [51]. However, compared to other peptide carrier 304
system, 0.165-0.22 μg of 6×His-PTD-TopoNs was required to delivery 1 μg DNA which was 305
much lower than K-Antp (12 μg peptides to deliver 1 μg DNA) [52] 306
Toxicity is always the major criterion and needs to be considered when designing a gene 307
delivery system. The cytotoxicity of a gene delivery system is cell type-dependent [53] and 308
different cell types were evaluated in this study. A possible concern is that cytotoxicity could 309
be caused by elevated levels of hTopoI. But this topological poison to human cells is from the 310
3’-terminus of hTopoI [54]. in vitro applications of 6×His-PTD-TopoNs showed that different 311
cell types have different responses to the carrier protein (Figs. 6 and 7), and 3T3 and GF-1 312
cells are resistant (>80% proliferation at different concentrations of exogenous peptides and 313
lipids) to exogenously peptides or lipids. COS7 is more sensitive to 6×His-PTD-TopoNs, 314
19
especially 10 μM 6×His-pep1-TopoN (50% proliferation), which had the same cytotoxicity as 315
12 μl lipofectamine (55% proliferation) at low concentration. The elevated sensitivity of 316
COS7 cells to 6×His-PTD-TopoNs might be due to the TopoN domain. COS7 and TopoN 317
both originate from primates and might influence transfection. This might also explain the 318
superior transfection efficiency in COS7 cells (Fig. 5) as compared to 3T3 and GF-1 cells 319
(data not shown). 320
The mechanism of cellular uptake of PTD is controversial. One hypothesis posits that 321
the cell-penetrating peptide that delivers molecules into cells is independent of temperature 322
and does not require energy or receptors. In this scenario, PTD-mediated gene delivery is 323
probably through the non-endosomal pathway [21, 55–58]. More recent hypotheses include 324
the artifactual uptake of peptides upon even mild cell fixation [59], improved endosomal 325
escape as the result of photochemical reactions initiated by photosensitization of compounds 326
localized in endocytic vesicles, which induces rupture of these vesicles upon light exposure 327
[60], ATP- and temperature-dependent involvement of endocytosis [59, 61], which was 328
subsequently identified as macropinocytosis [24, 25, 62], clathrin-dependent endocytosis [61] 329
and endosomal acidification [63]. Here, experiments conducted with cells that were 330
pre-incubated for 1 h at 4 ºC [43] or with 10 mM sodium azide and 6 mM 2-deoxy-D-glucose 331
to deplete cellular ATP [59] revealed that the cellular uptake of 6×His-PTD-TopoNs/DNA 332
complexes was energy- and temperature-dependent (Fig. 8), implicating 333
20
6×His-PTD-TopoNs-mediated gene delivery through the endosomal pathway. 334
The 6×His-PTD-TopoNs developed in this study not only provide a DNA delivery 335
system but also allow the easy and inexpensive preparation of protein. Re-folding is not 336
necessary for purification of 6×His-PTD-TopoNs because there was no different in DNA 337
binding and transfection efficiency between soluble and 8M urea-denatured forms (Fig. 10). 338
6×His-PTD-TopoNs were expressed in E. coli, which lacks posttranslational modification 339
machinery, and were functionally active, negating the necessity of posttranslational 340
modifications for biological functions of the proteins. Moreover, the absence or presence of a 341
hexahistidine tag did not influence the DNA binding ability of PTD-TopoNs (Fig. 9), which 342
simplifies PTD-TopoNs production, since removal of the hexahistidine tag is unnecessary. 343
Presence of a hexahistidine tag at the N-terminus might not affect the functional C-terminal 344
peptide sequences, and even can increase the transmembrane ability of PTD-TopoNs (Fig. 9). 345
Aa arginine-rich basic PTD domain is the common use for DNA delivery. However, a 346
histidine-rich PTD also has the same cell membrane penetrating ability [66]. This may 347
answer the question why PTD-TopoNs with N-terminal poly-histidine can have a higher 348
transfection efficiency than PTD-TopoNs. 349
21 5. Conclusion
351
This study demonstrates a DNA delivery system by a fusion protein containing a PTD and 352
TopoN. PTD can cross biological membranes independent of transporters or specific 353
receptors. TopoN can bind to DNA regardless of DNA size and topology, and contains five 354
NLSs that lead the protein to the nucleus. In addition, this protein can deliver biological 355
active DNA into different cells (3T3, GF-1 and COS7). 356
22 Acknowledgements
358
The authors would like to thank Dr. Shyh-Yu Shaw for providing COS-7 cells. This research 359
was supported by the National Science Council and Blossom Biotechnologies, Taiwan 360
(NSC91-2313-B-006-006, 93-2622-B-006-001-CC3, 94-2622-B-006-001-CC3). 361
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32 Figure captions
525
Fig. 1. Schematic structure of plasmids used in protein transduction. The constructions were
526
inserted into the pET15b system and the expression was driven by T7 promoter. A. 527
6×His-pep1-TopoN with hexahistidine tag at N-terminus; B. 6×His-pep2-TopoN with 528
hexahistidine tag at N-terminus; C. pep1-TopoN without hexahistidine tag; D. 6×His-TopoN 529
with hexahistidine tag at N-terminus. T7, T7 promoter; 6xHis, hexahistidine tag; pep1, PTD 530
domain pep1 (KETWWETWWTEW); pep2, PTD domain pep2 (KETWFETWFTEW); S, a 531
spacer domain (SQPGR); TopoN, N terminal human topoisomerase I (3–200 amino acids.). 532
The NLSs are located in positions 59–65, 117–146, 150–156, 174–180 and 192–198 [32, 55]. 533
Fig. 2. Protein expression by pET system and storage. A. IPTG induction of
534
6×His-PTD-TopoN for 3 hours in E. coli BL21(DE3) and separation by 12% SDS-PAGE 535
with Coomassie Brilliant Blue staining. B. Western blotting assay. Rabbit anti-PTD antibody 536
was used to identify 6×His-PTD-TopoN (6×His-pep1-TopoN and 6×His-pep2-TopoN). M, 537
protein marker; P, purified protein. Stored 6×His-pep1-TopoN was separated by 12% 538
SDS-PAGE and stained with Coomassie Brilliant Blue. Lanes 1–3, stored at 4 ºC; lanes 4–6, 539
stored at -20 ºC; lanes 7–9, stored at -80 ºC for 3 weeks (C) and 8 months (D). E. 540
6×His-pep-1-TopoN was stored at 37 ºC for 0.5 h (lanes 1–3), 1 h (lanes 4–6) and 2 h (lanes 541
7–9). 542
Fig. 3. PTD-TopoN binding to the circular form of DNA. Gel retardation electrophoresis
33
assay on the binding ability of 6×His-PTD-TopoNs (6×His-pep1-TopoN and 544
6×His-pep2-TopoN) to different sized plasmids. A. pBSK+ (~3.0kb); in molar ratio of 5:1, 545
10:1, 20:1 and 40:1 (protein: DNA), the amount of protein is 9.125×10-2μg, 1.825×10-1μg, 546
3.65×10-1μg and 7.3×10-1μg, respectively. B. pCMV-Mx-egfp-N1 (~8.0kb); in molar ratio of 547
5:1, 10:1, 20:1 and 40:1 (protein: DNA), the amount of protein is 3.5×10-2μg, 7.0×10-2μg, 548
1.4×10-1μg and 2.8×10-1μg, respectively.C. pEGFP-N1 (4.7kb); in molar ratio of 5:1, 10:1, 549
20:1 and 40:1 (protein: DNA), the amount of protein is 5.8×10-2μg, 1.16×10-1μg, 2.32×10-1μg 550
and 4.64×10-1μg, respectively.Different molar ratio (5:1, 10:1, 20:1 and 40:1) of 551
6×His-PTD-TopoN and plasmid DNA (6×His-PTD-TopoN:DNA). 1.3 μg of bovine serum 552
albumin (BSA, molar ratio = 40:1) was used as a control protein. 5.8×10-1 μgprotease K 553
(ProK, molar ratio = 40:1) was added with 6×His-PTD-TopoN and plasmid DNA as control. 554
M, DNA marker. C, plasmid only as control. 555
Fig. 4. 6×His-pep1-TopoN binding to the linear form of DNA. Gel retardation electrophoresis
556
assay of 6×His-pep1-TopoN to two different sizes of the linear form of DNA. A. pBSK+ 557
(~3.0kb); in molar ratio of 10:1, 20:1 and 40:1 (protein: DNA), the amount of protein is 558
1.825×10-1μg, 3.65×10-1μg and 7.3×10-1μg, respectively.B. pF4.1Xhyg (~8.9kb); in molar 559
ratio of 10:1, 20:1 and 40:1 (protein: DNA), the amount of protein is 6.205×10-2μg, 560
1.241×10-1μg and 2.482×10-1μg. Different molar ratio (10:1, 20:1 and 40:1) of 561
6×His-pep1-TopoN and plasmid DNA (6×His-pep1-TopoN: DNA). 1.3 μg of bovine serum 562
34
albumin (BSA, molar ratio = 40:1) was used as a control protein. M, DNA marker. C, linear 563
form DNA. 564
Fig. 5. PTD-TopoN-mediated DNA delivery into mammalian cells. A. 6×His-PTD-TopoNs
565
(6×His-pep1-TopoN and 6×His-pep2-TopoN) concentration-dependent DNA delivery. Both 566
DNA (1 μg of pGL3-promoter [5kb]) were incubated with different concentrations of 567
6×His-PTD-TopoNs from 0.05 mM (ratio 5:1) to 5 mM (ratio 40:1), in serum-free cell culture 568
medium for 2 h. In molar ratio of 5:1, 10:1, 15:1, 20:1, 25:1 and 30:1 (protein: DNA), the 569
amount of protein is 5.5×10-2μg, 1.1×10-1μg, 1.65×10-1μg, 2.2×10-1μg, 2.75×10-1μg and 570
3.3×10-1μg, respectively. Following this transfection step, fresh DMEM supplemented with 571
serum was added for another 48 h. Cells were then extensively washed and examined by 572
luciferase activity. B–G. Comparison of lipofectamine and 6×His-pep-1-TopoN on delivery 573
of pEGFP-N1 into COS7 cells. B and E. COS7; C and F. Lipofectamine with pEGFP-N1; D 574
and G. 6×His-pep1-TopoN with pEGFP-N1 (10:1). B, C and D were excited with 490 nm 575
light and merged with bright field images. E, F and G were excited with 490 nm light. Bar = 576
20μm. *p < 0.005 when compared the transfection efficiency of 6×His-pep1-TopoN to 577
6×His-pep2-TopoN. 578
Fig. 6. Cell proliferation assays of exogenous proteins and lipids. A. GF-1. B. 3T3. C. COS7.
579
The amount of lipofectamine (total volume of 0, 12, 24 and 48 μl per experiment) reflected 580
the manufacture’s recommendation. For the other added peptides or protein (group a), the 581
35
concentrations were 0, 10, 100 and 1,000 μM. *p < 0.005 when compared the cytotoxicity of 582
lipofectamin to grouper a protein. 583
Fig. 7. Cytotoxicity of PTD-TopoNs to cells. 3T3 and COS-7 cells were incubated with 1 μM
584
to 1 mM of 6×His-PTD-TopoNs at 37 ºC in DMEM supplemented with 10% FCS. A. 585
6×His-PTD-TopoNs only. B. 6×His-PTD-TopoNs with pEGFP-N1 (6×His-pep1-TopoN: 586
DNA = 15:1; 6×His-pep2-TopoN: DNA = 20:1). Cytotoxicity was assessed using MTT. *p < 587
0.005 when compared the cell viability of control group to other experiment groups. 588
Fig. 8. PTD-TopoNs-mediated gene delivery is energy- and temperature-dependent. COS7
589
cells were incubated for 2 h in the presence of 6×His-pep1-TopoN/DNA and 590
6×His-pep2-TopoN/DNA complexes formed at a molar ratio of 15:1 and 20:1, respectively, 591
and 1 μg pGL3-Promoter plasmid encoding the reporter gene luciferase was added. Cells 592
were pre-incubated for 1 h at 4ºC, or with 10 mM sodium azide and 6 mM 2-deoxy- 593
D-glucose to deplete cellular ATP. *p < 0.005 when compared the ATP depletion group to 594
control group (37ºC). #p < 0.01 when compared the 4ºC group to control group (37ºC). 595
Fig. 9. Effect of hexahistidine tag on transfection activity. COS7 cells were incubated for 2 h
596
in the presence of 6×His-pep1-TopoN/DNA complexes (molar ratio = 15:1) and with 1 μg 597
pGL3-Promoter plasmid encoding the reporter gene luciferase. #p < 0.01 when compared the 598
transfection efficiency of 6×His-pep1-TopoN to pep1-TopoN. 599
Fig. 10. Effect of soluble and denatured pep1-TopoN on DNA binding. A. Gel retardation
36
electrophoresis assay of soluble and denature forms of 6×His-pep1-TopoN mixed with 601
pCMV-Mx-EGFP-N1 (~8.0 kb). Different molar ratios (10:1, 20:1, 40:1, 80:1 and 100:1) of 602
6×His-pep1-TopoN and plasmid DNA (6×His-pep1-TopoN:DNA) were used. In molar ratio 603
of 10:1, 20:1, 40:1, 80:1 and 100:1 (protein: DNA), the amount of protein is 7.0×10-2μg, 604
1.4×10-1μg, 2.8×10-1μg, 5.6×10-1μg and 7.0×10-1μg, respectively. B. A 15:1 molar ratio of 1 605
μg of 6×His-pep1-TopoN and pGL3-promoter plasmid were mixed and transfected to COS-7 606
cells. After 48 h, the cell lysate was collected to analysis the activity of luciferase. 3.25 μg of 607
bovine serum albumin (BSA, molar ratio = 100:1) was used as a control protein. M, DNA 608
marker. *p < 0.005 when compared the soluble or denature form of 6×His-pep1-TopoN to 609
native form. 610
37 Fig. 1
38 Fig. 2
39 Fig. 3
40 Fig. 4
41 Fig. 5
42 Fig. 6
43 Fig. 7
44 Fig. 8
45 Fig. 9
46 Fig. 10
47 Supplementary Data
Supplemental Table 1. The protein sequences used in this study.
Name Protein sequence*
6×His-pep1-TopoN HHHHHHMGKETWWETWWTEWSQPGRGDHLHNDSQIEADFRLNDSHKHKDK HKDREHRHKEHKKEKDREKSKHSNSEHKDSEKKHKEKEKTKHKDGSSEKHK DKHKDRDKEKRKEEKVRASGDAKIKKEKENGFSSPPQIKDEPEDDGYFVPPKE DIKPLKRPRDEDDADYKPKKIKTEDTKKEKKRKLEEEEDGKLKKPKNKDKDK KVPEPDNKKKKPKKEEE
6×His-pep2-TopoN HHHHHHMGKETWFETWFTEWSQPGRGDHLHNDSQIEADFRLNDSHKHKDKH KDREHRHKEHKKEKDREKSKHSNSEHKDSEKKHKEKEKTKHKDGSSEKHKD KHKDRDKEKRKEEKVRASGDAKIKKEKENGFSSPPQIKDEPEDDGYFVPPKEDI KPLKRPRDEDDADYKPKKIKTEDTKKEKKRKLEEEEDGKLKKPKNKDKDKKV PEPDNKKKKPKKEEE pep1-TopoN MGKETWWETWWTEWSQPGRGDHLHNDSQIEADFRLNDSHKHKDKHKDREH RHKEHKKEKDREKSKHSNSEHKDSEKKHKEKEKTKHKDGSSEKHKDKHKDR DKEKRKEEKVRASGDAKIKKEKENGFSSPPQIKDEPEDDGYFVPPKEDIKPLKR PRDEDDADYKPKKIKTEDTKKEKKRKLEEEEDGKLKKPKNKDKDKKVPEPDN KKKKPKKEEE 6×His-TopoN HHHHHHSQPGRGDHLHNDSQIEADFRLNDSHKHKDKHKDREHRHKEHKKEK DREKSKHSNSEHKDSEKKHKEKEKTKHKDGSSEKHKDKHKDRDKEKRKEEK VRASGDAKIKKEKENGFSSPPQIKDEPEDDGYFVPPKEDIKPLKRPRDEDDADY KPKKIKTEDTKKEKKRKLEEEEDGKLKKPKNKDKDKKVPEPDNKKKKPKKEE E
*Letters in red indicates the hexahistidine (6×His) tag at the N terminus of 6×His-pep1-TopoN; letters in purple indicates the pep1 sequences; letters in blue indicates the spacer domain; letters in orange indicates the N-terminal (3–200 amino acids.) domain of human topoisomerase I; letters in dark blue indicates the NLSs.
