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[rBmB] 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