2. MATERIALS AND METHODS
2.1. Bacterial strains, plasmids, and reagents
E. coli XL1-Blue (endA1 gyrA96 [nalR] thi-1 recA1 relA1
lacglnV44 F‘[::Tn10 proAB+ lacIqΔ(lacZ)M15] hsdR17[rK-mK+] ) was
used as a host for the cloning of the recombinant gene, and protein
expression was induced in the host E. coli BL21(DE3) (ompT
gal dcm lon hsdSB[rB–mB–]
λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12[λS] ).
pET24a_hFGF7 was provided by the Korea Institute of Marine Science and
Technology (Busan, Korea) and used as the template for PCR with a set of
primers specially designed for the duplication of the gene encoding
hFGF7 (Table S1). The vector pSCT528 was used to
insert the duplicated hFGF7 gene for the generation of CP variants by
PCR, and the pCold Ⅰ vector (TaKaRa, Japan) was used to construct a
series of CP hFGF7 variants (cp-hFGF7). nPfu special polymerase
(Enzynomics, Daejeon, Korea) was used for the amplification of the
target gene by PCR. The restriction enzymes used were purchased from New
England Biolabs (Ipswich, MA, USA) and Takara Bio Inc. (Shiga,
Japan). T4 DNA ligase (Thermo Fisher, MA, USA) or the Infusion HD
Cloning Kit (Clontech, Shiga, Japan) was used for the subcloning of the
target gene into the plasmids according to the supplier’s recommended
protocol. Recombinant hFGF7 (indicated as wild type in this work) was
purchased from PeproTech (Cranbury, NJ, USA) and used as a positive
control for the structure and activity analyses of the purified
cp-hFGF7.
2.2. Prediction of CP cleavage sites in hFGF7
CP cleavage sites were predicted by a web-based tool, CPred
().26 Among residues with a probability score of
greater than 0.8, the candidate positions for creating new termini were
rationally selected from surface loops that predicted also without
significant loss of structural traits by the secondary structure
prediction using PSIPRED.29
2.3. Construction and analyses of the expression patterns of circularly
permuted hFGF7 variants
For the construction of diverse CP variants, the gene encoding hFGF7
without an innate signal sequence (31 amino acids) was primarily
duplicated by artificial fusion using PCR as follows. The DNA fragment
encoding 163 amino acid residues of hFGF7 was amplified by PCR using
pET24a_hFGF7 as the template and two sets of primers
(pSCT5_hFGF7-Infu-F and hFGF7-Infu-R, hFGF7(×2)-Infu-F and
pSCT5_hFGF7(×2)-Infu-R) under the typical conditions. Then, the
resulting DNA fragment was cloned into a linearized pSCT5 vector to
prepare the construct pSCT5_hFGF7(×2) containing the duplicate gene
(Figure 1A). Using the pSCT5_hFGF7(×2) vector as the template, CP
variants were amplified by PCR using nine sets of primers
(pSCold_CP1-hFGF7-F/R ~ pSCold_CP9-hFGF7-F/R), and
subcloned into the same restriction enzyme site (Spe I andHin dIII) to prepare pSCold_cp-hFGF7 constructs expressing each
of the nine CP variants (Figure 1B, C). Prior to the subcloning of
cp-hFGF7, the removal of TEE-6×His-fXa cleavage sequence from the
expression vector pCold I and incorporation of theSpe I-recognizing sequence ACTAGT (between 5’UTR and start codon)
were simultaneously carried out by PCR using a pair of primer
(pSCold_Vec-Infu-R and pSCold_Vec-Infu-F).28 The
resulting vector pSCold could express cp-hFGF7 without any additional
amino acids at N-terminal region. All primer sequences used in this
study are provided in Table S1.
To analyze the expression level and solubility of the cp-hFGF7 variants,
each of the recombinant plasmid pSCold_cp-hFGF7 variants
(CP1–CP9) was transformed intoE. coli BL21(DE3) cells. Subsequently, a single colony was
inoculated into 3.5 mL of LB medium containing ampicillin (100 µg/mL)
and cultured at 37℃ under constant shaking (200 rpm). When the
absorbance (OD600) of the culture reached 2.0, an
aliquot of culture broth was reseeded (2%, v/v) into the same LB
medium. The resulting cells were cultured to an OD600 of
0.6 and treated with isopropyl-β-D-thiogalactoside (IPTG, 0.2 mM) to
induce protein expression at 16℃ and 200 rpm for 36 h. The induced cells
were then harvested by centrifugation at 12,000 × g for 10 min and
resuspended in 10 mM sodium phosphate buffer (pH 6.5), then disrupted by
irradiation with ultrasonic waves three times for 2 seconds. The
resulting cell lysate (total fraction, T) was centrifuged at 4℃ and
12,000 × g for 25 min to obtain a supernatant (soluble fraction, S) from
which insoluble aggregates had been removed. Both total and soluble
fractions were loaded onto a Tricine-SDS-PAGE (10%) gel, and the
expression level and solubility were analyzed under the same conditions
previously reported.30 The resulting gels were also
subjected to western blot analyses as follows. The transfer of resolved
proteins from gels onto nitrocellulose membrane (GenDEPOT, Texas, USA)
was conducted using a Power Blotter-Semi-dry transfer system
(Thermo-Fisher Scientific, MA, USA). The membrane was then blocked using
5% skim milk for 1 h at room temperature (RT), followed by incubating
overnight at 4°C with anti-human FGF7 monoclonal antibody (1:5000,
Abcam, US). Subsequently, the membrane was incubated with horseradish
peroxidase-linked goat anti-mouse immunoglobulin G (1:5000, Enzo Life
Sciences, US) at RT for 1 h. The proteins on the membrane were
visualized with ECL detection kit system (Bio-Rad, US).
2.4. Purification of cp-hFGF7
The recombinant plasmid, pSCold_cp-hFGF7115-114, was
transformed into E. coli BL21(DE3) cells, and cultured at 37℃
until the cell density (OD600) reached 2.0 in 4 mL of LB
medium containing ampicillin (100 µg/mL). Then, 1 mL of the cultured
cells was reseeded into fresh LB medium (100 mL) and cultured to an
OD600 of 0.6–0.8, then induced with 0.2 mM IPTG at 16℃
and 200 rpm for 36 h. The induced cells were harvested by
centrifugation, resuspended in 20 mM sodium phosphate (pH 6.5) buffer,
lysed by sonication, and centrifuged at 4℃ and 10,000 × g for 60 min to
remove the cell debris. Using the resulting supernatant,
cp-hFGF7115-114 was purified via a successive step
consisting of heparin affinity, cation exchange, and size exclusion
chromatography (SEC). Considering the functional structure for cofactor
binding, the heparin HP column (1 mL, GE Healthcare, IL, USA) was
selected for the primary step. The supernatant was then loaded onto a
heparin column equilibrated with buffer A (20 mM sodium phosphate, pH
6.5). After binding, the column was thoroughly washed with the same
buffer containing 0.2 M NaCl. Thereafter, a linear gradient was induced
with 30 column volumes (CV) of buffer A and B (20 mM sodium phosphate, 1
M NaCl, pH 6.5). Fractions containing cp-hFGF7115-114were collected and diluted 2-fold with a buffer (20 mM sodium phosphate,
pH 7.3). Next, the eluted fractions from the heparin column were loaded
onto a HiTrap SP HP column (5 mL, GE Hewlett, IL, USA) equilibrated with
the same buffer. After complete washing with the same buffer, the bound
proteins were eluted with 20 CV of the linear salt (NaCl) gradient
buffer from 0 to 1.0 M. The final step of the purification was conducted
using a Superdex 200 increase 10/300 GL column (GE Healthcare, Chicago,
USA) with 20 mM sodium phosphate buffer (pH 7.3) containing 0.4 M NaCl.
The purity and yield of the protein in the eluted fraction were
determine by 10% Tricine-SDS-PAGE and western blot.
2.5. Spectroscopic property analyses of cp-hFGF7
To analyze the structural property of cp-hFGF7115-114,
UV-vis absorption scanning was performed by using a buffer (20 mM sodium
phosphate, 0.4 M NaCl, pH 7.3) under the specified conditions. The
protein solution (100 μL) was placed into a 1.0 cm quartz cuvette and
the absorbance spectrum was measured by changing the wavelength from 260
to 600 nm at 5 nm intervals.31 Fluorescence emission
scanning was also performed by using the same concentration (150 µg/mL)
of proteins. The change in fluorescence wavelength emitted from 280 to
500 nm was measured at 3 nm intervals by an excitation wavelength of 250
nm.32
The secondary structure analysis of cp-hFGF7115-114was performed using a circular dichroism (CD) spectropolarimeter (Model
J-1500, Jasco, Tokyo, Japan). Prior to CD measurement, the purified
cp-hFGF7115-114 from the size exclusion column was
completely desalted in a 10 mM sodium phosphate (pH 7.3) buffer by using
a PD-10 (GE Healthcare, IL, USA) column. The far-UV CD spectra of
cp-hFGF7115-114 (300 µg/mL) was recorded from 190 to
260 nm using a 0.1 cm path length cell at room temperature (25℃). Each
spectrum was obtained three times at a scan rate of 100 nm/min, and then
corrected by subtracting the spectral contribution of the buffer. The
commercially available hFGF7 was used as a control for all spectral
experiments.
2.6. Biological activity analyses of cp-hFGF7
Extracellular signal-regulated kinase (ERK) phosphorylation
assay . The embryonic mouse fibroblast cell line NIH3T3 was obtained
from the lab of Professor Tae-Hoon Lee at Chonnam National University
and routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% bovine calf serum (BCS) and
penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) in an
incubator (5% CO2) at 37℃. For immunoblotting,
2×105 NIH3T3 cells were seeded onto 6-well plates in
the same medium and cultured overnight. The cells were then
serum-starved for 24 h prior to treatment with
cp-hFGF7115-114. After time- and dose-dependent
treatment with cp-hFGF7115-114, the treated cells were
harvested and lysed using RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM
NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing
protease and phosphatase inhibitor cocktails. The lysed cells were
centrifuged at 4℃ and 12,000 × g for 20 min to obtain the supernatant.
The protein concentration in the supernatant was measured using the BCA
protein assay kit (Thermo Fisher, MA, USA). Then, the same amount of
protein from each cell was separated by 10% Tricine-SDS-PAGE and
transferred to a nitrocellulose membrane. The membrane was blocked in
TBS-Tween 20 (0.1%) containing 5% skim milk at room temperature.
Specific proteins on the membranes were detected by probing with
specific primary antibodies, anti-phospho-specific ERK-1/2
(Thr202/Tyr204) and anti-ERK-1/2 antibodies from Cell Signaling
Technology Inc. (Beverly, MA, USA) and the α-tubulin antibody from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA, USA), followed by incubation
with the secondary antibodies conjugated to HRP (Enzo Life Sciences, MI,
USA). The resulting specific binding was visualized by the ChemiDoc
image analyzer (Bio-Rad, CA, USA) using an ECL chemiluminescence
substrate (Bio-Rad, CA, USA).
Cytotoxicity and cell proliferation assay . Cytotoxicity
analysis was carried out by live cell counting using NIH3T3 cells and an
assay kit (Abcam, Cambridge, UK). Cells grown in the same medium
described above were inoculated into 24-well plates and incubated for
24–36 h in a humidified incubator (37℃) containing 5%
CO2. When the cell confluence reached 70–80%, a new
medium containing commercial hFGF7 and cp-hFGF7115-114was added. After incubation for 24 to 72 h, 10 μL of WST-8 dye was added
and an additional incubation was performed for 3 h. Afterward, the
degree of color change was measured at 450 nm using a spectral
microplate reader (SpectraMax ABS, Molecular devices, CA, USA). The
results were expressed as a percentage of the control where the
absorbance value of the untreated cells was normalized to 100%. After
the cell proliferation proceeded in the same manner, the degree of color
change of the WST-8 dye was measured at 460 nm. All assays were
performed in triplicate.
Scratch wound healing assay . NIH3T3 cells used in the wound
healing assay were inoculated into 24-well plates and cultured until
90% to 100% cell confluency was reached. A scratch wound was
introduced with a 10 μL pipette tip. After washing with serum-free DMEM
medium for cell debris removal after scratch formation, the cells were
treated with rhFGF7 and cp-hFGF7115-114 and incubated
for 72 h. During the incubation period, sutures for wound closing were
monitored and imaged with an optical microscope (Eclipse TE2000-E,
Nikon, Tokyo, Japan).33