Aveneu Park, Starling, Australia

MTX fusion TAT segment is exposed and

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)

 

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