MTX USA). PD10 desalting column was purchased

MTX and isopropyl-?-D-thiogalactoside (IPTG) was purchased from Sigma (St. Louis, MO, USA). Dulbecco’s
modified Eagle’s medium (DMEM), penicillin and streptomycin antibiotic
mixtures; sodium pyruvate and fetal bovine serum were from Invitrogen
(Carlsbad, CA, USA). Escherichia coli
strain BL21 (DE3) was obtained from IBRC (Tehran, Iran). Nitrilotriacetic acid sepharose superflow (Ni-NTA) was
purchased from Qiagen (Valencia, CA, USA). PD10 desalting column was
purchased from GE Healthcare Life Sciences (USA). Pierce
FITC Antibody Labeling Kit was from Thermo Fisher Scientific (USA).
Anti-His6-peroxidase antibody was purchased from Roche (Switzerland). PE Annexin V
Apoptosis Detection Kit I was from BD
Biosciences (USA).
All other chemicals and reagents were of the highest commercial grade
available.

 

2.2. Construction of the TAT-CPG2 expression vector

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To evaluate if
the fusion TAT segment is exposed and does not have any interference with the
CPG2 domain, a 3D protein model by homology modeling using phyer2 server was
created (Kelley and Sternberg, 2009). For
expression in E. coli, the amino acid sequence of CPG2 from Pseudomonas
Sp. Strain RS-16 was codon-optimized by codon usage wrangler server
(https://www.mrc-lmb.cam.ac.uk/ms/methods/codon.html) and evaluated using genscript server (https://www.genscript.com/tools/rare-codon-analysis). A TAT-CPG2 expression vector was
constructed to express the basic domain
(YGRKKRRQRRR) of HIV-1 TAT fused with CPG2 in frame with an
N terminal 6xHis tag. The synthetic
DNA sequence encoding TAT-CPG2 was subcloned into the BamHI and NdeI
sites of pET-14b. The control vector expressed CPG2, was constructed by inserting
sequence encoding CPG2 coding sequence in the same sites into pET-14b without
the TAT domain. Expression vectors
were transformed into E. coli BL21 (DE3) by heat shock at 42 ?C for 1 min and chilled on ice for 2 min.

 

2.3. Expression and purification of TAT-CPG2 fusion
proteins

Transformed bacteria with CPG2 and TAT-CPG2 constructs
were grown in the LB medium containing 100 µg/ml ampicillin at 37 ?C to reach an optical density of
0.6 at 600 nm. Expression of the fusion proteins was induced at 0.5 mM and 1 mM
of IPTG. Cells were grown at either 37 ?C or 28 ?C for 4 h. Purification of
CPG2 and TAT-CPG2 fusion proteins were carried out under native and denaturing
conditions by the batch method of Qiagen. For purification of recombinant
proteins under native condition the bacterial cells
were harvested by centrifugation at 10,000×g and resuspended in binding buffer
(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole,
and 1 mM PMSF at pH 8). Lysozyme was added at a concentration of 0.1 mg/ml and
incubated on ice for 30 min. Cells were then disrupted by sonication (6 ×10 s
bursts at 200–300 W with a 10 s cooling period
in-between) on ice. The bacterial lysates were centrifuged (10,000×g at 4 °C
for 30 min) and the supernatants were added to a 50% Ni-NTA resin
pre-equilibrated with binding buffer and mixed gently by shaking (200 rpm on a
rotary shaker) at 4 °C for 60 min. The
lysate–Ni-NTA mixture was loaded onto an empty PD10 column. The column was
washed twice with 4 ml wash buffer (50 mM NaH2PO4, 300 mM
NaCl, 20 mM imidazole, pH 8). The CPG2 and TAT-CPG2 fusion proteins were eluted
with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM
imidazole, pH 8).  Eluted proteins were
desalted on a PD10 desalting column. For purification under denaturing condition, pellets
from purification under native condition were resuspended in buffer B (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M
urea, pH 8) and stirred for 60 min at room temperature. The mixture was
then centrifuged at 10,000×g for 30 min at room temperature to pellet the
cellular debris. Supernatant was mixed with 50% Ni-NTA resin and gently shaked
(200 rpm on a rotary shaker) at room temperature for 60 min. The lysate–Ni-NTA
mixture was added to a column and washed twice with 4 ml buffer C (100 mM NaH2PO4,
10 mM Tris-HCl, 8 M urea, pH 6.3). The recombinant proteins were eluted 4 times
with 0.5 ml buffer D (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M
urea, pH 5.9), followed by 4 times elution with 0.5 ml buffer E (100 mM NaH2PO4,
10 mM Tris-H, 8 M urea, pH 4.5). Monomers generally elute in buffer D, while
multimers and aggregates elute in buffer E. The eluted proteins were desalted
by PD10 desalting column. The purified fusion proteins were verified by
SDS/PAGE, coomassie brilliant blue staining and western blot analysis with
anti-His6-peroxidase antibody (1:500; Roche). The protein concentrations were
estimated by the Bradford method (Bradford,
1976).

 

2.4.
Fluorescence microscopy analysis

For direct detection of protein transduction, FITC-labeled TAT-CPG2
and CPG2 fusion proteins were generated using Pierce FITC
Antibody Labeling Kit. HepG2 cells
were grown on glass coverslips and treated with 2 ?M of TAT-CPG2 and control
CPG2 proteins. After incubation for 2 h at 37 °C, cells were washed twice with
the PBS buffer and fixed with 4% paraformaldehyde
for 10 min at room temperature. To evaluate the transduction efficiency, HepG2
cells were incubated with 2 ?M of native and denatured TAT-CPG2 for various
periods of time (15–120 min). Then, cells were treated with trypsin–EDTA, washed with PBS and
fixed with 4% paraformaldehyde. 
Fluorescence analysis was performed by inverted fluorescence microscope (CETI, UK)

 

2.5. Transduction of TAT–CPG2 fusion
protein into the HepG2 cells

HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium
containing 20 mM HEPES/NaOH (pH 7.4), 5 mM NaHCO3, 10% fetal bovine
serum, and antibiotics (100 ?g/ml streptomycin, 100 U/ml penicillin) at 37 °C
under humidified conditions of 95% air and 5% CO2. To evaluate
concentration and time dependency of TAT-CPG2 transduction, treatments were
carried out as follows: HepG2 cells were grown to
confluence on a six well plate and incubated with different
concentrations (0.5-4 ?M) of native
and denatured proteins for 2 h or with 2 ?M of native and denatured
proteins for various periods of time (15–120 min). Cells were incubated with 2
?M CPG2 for 2 h as the control group. To examine the intracellular stability of TAT-CPG2 protein, cells were treated with 2 µM TAT-CPG2 for 2 h.  Cells were then washed with PBS to remove non-transduced TAT-CPG2. Cells were further incubated in fresh culture medium for
2, 6, 12, 24, 36 and 48 h. After the above treatments, cells were treated
with trypsin-EDTA and washed with PBS. Cells were lysed with the lysis buffer (250 mM Tris-HCl, 10% v/v glycerol, 1% triton X-100, 1 mM PMSF, and 10 µg/ml leupeptin at pH 7.4).
After centrifugation at 15,000 × g for 30 min at 4
°C, cell extracts were
prepared for western blot and enzyme assay analyses.

 

2.6. Inhibitory effect of TAT-CPG2 against MTX- induced
toxicity in HepG2 cells

2.6.1. Cell viability assay

Cell viability was evaluated using the MTT assay (Mosmann, 1983). In brief, HepG2
cells were seeded in 96-well plates in
a volume of 100 µl at a density of 1.0 × 104 cells/well. After 24 h
of incubation for growing and adherence, the medium was
replaced with the fresh medium containing various concentrations of
CPG2 and TAT-CPG2 for 2 h. After
treatment, culture medium was removed and cells were washed twice with ice-cold
PBS. Cells with or without TAT-CPG2 pretreatment were exposed
to different concentrations of MTX (0.1-100 µM) and incubated for 24 and 48 h.
In addition, effect of TAT-CPG2 on the cell viability was carried out at
concentrations of 0.5 to 4 µM. To examine the inhibitory effect of different protein concentrations against MTX-induced cell death,
HepG2 cells were treated with various concentrations of TAT-CPG2 for 2 h and
then exposed to 10 µM MTX for 48 h. Following all above treatments, cells were incubated
with MTT (0.5 mg/ ml) at 37 ?C for 4 h. The
medium was then removed and 100 µl of DMSO was added into each well and mixed
thoroughly on a shaker for 15 min to dissolve formazan crystals.
The absorbance was measured at 570 nm using a Microplate
Spectrophotometer (BioTek PowerWave XS2, USA). Cell viability was
calculated as a percentage of absorbance of sample compared to that of the
control.

 

2.6.2. Assessment of apoptotic cell
death

HepG2 cells were seeded into 6-well culture
plates at a density of 5×105 cells per well
and incubated overnight at 37 °C in 5% CO2. After 24 h of
incubation, medium was discarded and
replaced with fresh serum free culture medium containing 2 µM of control CPG2
and TAT-CPG2 (native and denatured forms) for 2 h. After 2 h of incubation, cells were washed three times
with PBS to remove non-transduced proteins then treated with 10 µM MTX and
incubated for 24 and 48 h. Apoptosis was assessed by using PE Annexin V
Apoptosis Detection Kit according to the manufacturer’s protocol.
Briefly, cells were harvested by trypsin and washed twice with the cold PBS
buffer and then resuspended in 1X binding buffer
at a concentration of 1×106 cells/ml. Then, 100 ?l of the solution (1 × 105 cells) was transferred
to a 5-ml culture tube. Five microliter of
AnnexinV-FITC and 5 ?l of 7-Amino-Actinomycin (7-AAD) were added to each tube. Cells were vortexed gently and incubated for 15 min at
room temperature in the dark.
Finally, 400 ?l of 1X binding buffer was added to each tube and analyzed by
flow cytometer (FACSCalibur, BD Biosciences).

 

2.7. Western blot analysis and enzyme assay

For
western blot analysis, equal amount of each cell lysate was resolved by 12%
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The
protein on the gel was transferred onto a PVDF membrane and then blocked
over-night with 1% skim milk in PBS at 4 ?C. The membrane was incubated with
anti-His tag-peroxidase antibody (1:500; Roche) for 1 h at 25 ?C. After three
times washing the membrane with the PBS containing 0.1% tween
20, and once with PBS without tween 20 (5
min for each), the target protein was visualized with 3,3′-Diaminobenzidine
(DAB) as the substrate.

Enzyme
activity was assayed by a modified method of McCullough et al. (McCullough et
al., 1971). The reaction mixture containing 100 mM Tris-HCl at pH
7.3, 0.2 mM ZnSO4 and 50 µM MTX was
equilibrated at 37 ?C for 10 min. 
Decrease in the absorbance at 320 nm was measured using
Spectrophotometer (T80 UV/VIS Spectrometer, PG
Instruments). CPG2 activity was assessed using an extinction coefficient of
8300 L mol-1 cm-1 for MTX. The activity was calculated in
units per milliliter (U/mL). One Unit is defined as the amount of the
enzyme required to catalyze the hydrolysis of 1 µmol MTX per minute at
37 ?C.

2.8. Quantification of
intracellular ROS production, GSH content and catalase (CAT) activity

The production of intracellular ROS was measured using
DCFH–DA (Jia
et al., 2006). DCFH–DA passively enters the cell, where
it reacts with ROS to form a highly fluorescent compound, dichlorofluorescein
(DCF). HepG2 cells were seeded in a 6-well plate at 3 ×105 cells for
24 h.  Cells were treated with CPG2 and
TAT-CPG2 for 2 h. Cells with or without TAT-CPG2
pre-treatment were exposed to 10 µM MTX, and
ROS detection was evaluated at 12, 24 and 48 h after treatment. Following
exposure, cells were trypsinized and washed with the ice-cold PBS. Then, cells
were co-incubated with serum-free DMEM containing10 µM DCFH-DA for 30 min at 37
?C in the dark. Subsequently, cells were harvested and
rinsed with the PBS buffer. Fluorescent intensities were measured using a
spectrofluorometer (FluoStar Omega, BMG labtech) at 485 nm excitation/530 nm
emission at the end of exposure times.

For determining GSH content and CAT activity, HepG2 cell
line was seeded in 6-well culture plates at 3 ×105 cells/well. Then,
cells were exposed to 10 µM MTX with or without pretreatment by TAT-CPG2 for
12, 24, and 48 h. Finally, cells were harvested and washed twice with PBS at 4 ?C.
Cells were then lysed
in the cell lysis buffer (250 mM Tric-HCl, 10% v/v
glycerol, 1% triton X-100, 1 mM PMSF and 10 µg/ml leupeptin at pH 7.4) and
centrifuged at 10,000×g for 15 min. The supernatant was used for determination
of the GSH level and CAT activity.

GSH content was determined using the Ellman method (Ellman, 1959). For assay, 0.1 ml of the supernatant
was added to 0.9 ml of 5%
trichloroacetic acid (TCA), and centrifuged at 2300 g for 15 min at room
temperature. Then, 0.5 ml of the supernatant was added into 1.5 ml of 0.01%
DTNB and the reaction was monitored at 412 nm using spectrophotometer. The GSH
level was expressed as ?g GSH/mg protein. CAT activity was determined based on
the decomposition of H2O2 (Aebi,
1984).  Decrease in the absorbance
at 240 nm was monitored for 2 min using spectrophotometer. Enzyme activity was
calculated using the extinction coefficient of H2O2 (43.6
mM?1 cm?1) and expressed as units/mg protein.

 

2.9. Statistical analysis

All experiments were performed three times independently. All data were reported as
the mean ± standard deviation (SD). Statistical significance was determined by
one-way and two-way analysis of variance (ANOVA) by Grappad prism 7 software. p
< 0.05, p < 0.01 and p < 0.001 considered to be significant.

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