Construction of protein expression vectors and strains
Genes encoding the GFs with 6HFh8 were codon-optimized and synthesized by DNA 2.0 (ATUM, Menlo Park, CA, USA). The fusion proteins were constructed as following our previous report [37]. Briefly, hexa-histidine fused Fh8 (6HFh8) was fused to N-terminus of GFs via the S5N10 linker and TEV protease cleavage site (ENLYFQ-G/S) in the P1′ position. The genes were inserted into a pET-30a vector (Novagen, Madison, WI, USA) pre-digested with the restriction enzymes Nde I and Xho I using T4 DNA ligase (Takara Bio, Otsu, Japan). The resultant plasmids were heat-shocked to transform E. coli DH5α (RBC Bioscience, New Taipei City, Taiwan). For the expression of fusion proteins, the recombinant plasmids were used to transform E. coli BL21 (DE3) (NEB, Ipswich, MA, USA).
Bacterial cultivation for protein production and analysis of protein expression
The flask culture medium and 5-l fed-batch fermentation medium in the current study were described in our previous experiments [38]. Briefly, flask cultivations were performed in an auto-induction medium [per liter: 0.5 g glucose, 3 g glycerol, 2 g lactose, 0.15 g MgSO4·7H2O, 10 g yeast extract, 16 g tryptone, 3.3 g (NH4)2SO4, 6.8 g KH2PO4, and 7.1 g Na2HPO4·12H2O, and 1 ml of trace element solution containing 0.5 g/l CoCl2·6H2O, 65 g/l FeSO4·7H2O, 3 g/l MnSO4·5H2O, 5 ml/l H2SO4, 0.08 g/l KI, 6 g/l CuSO4·5H2O, 20 g/l ZnCl2, 0.02 g/l H3BO3, 0.2 g/l Na2MoO4·2H2O, and 0.2 g/l biotin) at three different temperatures (25 °C, 30 °C, and 37 °C). Recombinant E. coli cells harboring the GF expression plasmids were cultured in 2 ml of Luria-Bertani (LB) medium supplemented with 50 µg/ml kanamycin at 37 °C overnight. Then, 0.5 ml of culture was transferred to 50 ml of the auto-induction medium supplemented with 50 µg/ml kanamycin in a 250-ml baffled flask and incubated for 12 h at 37 °C, or 24 h at 30 °C or 25 °C, at 200 rpm.
In another set of experiments, 5-l fed-batch fermentations were performed in the following initial medium: 15 g/l glucose, 1 g/l MgSO4·7H2O, 10 g/l yeast extract, 10 g/l casein peptone, 10 g/l (NH4)2SO4, 0.5 g/l NaCl, 3 g/l Na2HPO4·12H2O, 3 g/l KH2PO4, and 1 ml/l trace element solution as described above. The inoculum for bioreactor cultures was prepared as follows. For the primary seed culture, a single colony from LB agar supplemented with 50 µg/ml kanamycin was inoculated into 50 ml of LB medium with 50 µg/ml kanamycin and cultured overnight at 37 °C and 200 rpm. For the secondary seed culture, 2 ml of the primary seed culture was inoculated into 200 ml of LB medium supplemented with kanamycin and incubated at 37 °C and 200 rpm for 5 h. The 5-l fed-batch fermentation was performed in a 2-l initial working volume in a 5-l bioreactor. The culture conditions were controlled and maintained as follows: cell growth temperature, 37 °C; pH adjusted to 7.0 by the addition of ammonium hydroxide; dissolved oxygen, above 30%; airflow, 1 vvm; and automatic agitation controlled between 200 rpm and 900 rpm. All the controlled conditions were monitored, and glucose levels were analyzed by a glucose analyzer (YSI 2700 Biochemistry Analyzer; Yellow Springs Instrument Co., Yellow Springs, OH, USA). When the glucose present in the initial medium was entirely consumed, additional glucose was fed at an initial feeding rate of 8 g/l/h. After adjusting the temperature to 25 °C or 30 °C, the feeding rate of glucose was decreased to 6 g/l/h or 4 g/l/h, respectively. Further, 15 g/l lactose was added to promote the expression of the recombinant protein gene, and the incubation continued for a total culture time of 23.5 h.
To analyze the expression level and solubility, 1 ml of cells with OD600 was harvested by centrifuging at 15,814 ⋅ g at 4 °C for 1 min, and remaining cells were harvested by centrifuging at 6,520 ⋅ g at 4 °C for 20 min for storage. After washing twice with PBS, the pellet was re-suspended in 1 ml PBS and disrupted by a sonicator (Cole-Parmer Instruments, Vernon Hills, IL, USA) at 40% amplitude, pulse 5 s on and 5 s off, for 10 min on ice. The debris was removed by centrifugation at 15,814 ⋅ g at 4 °C for 20 min. Protein concentration was determined by Pierce™ BCA protein assay kit (Thermo Scientific, Waltham, MA, USA), and absorbance was measured at 550 nm by the plate reader Infinite 200 PRO (TECAN, Männedorf, Switzerland). Protein expression was evaluated by loading the protein onto 4–12% Bis-Tris Plus SDS-PAGE gel (Thermo Scientific) and running it at 170 V, 500 mA, for 35 min, followed by staining with InstantBlue (Abcam, Cambridge, UK).
Purification of aFGF and VEGF165
All purification steps were performed using a ÄKTAprime plus chromatography system (GE Healthcare, Little Chalfont, UK). For purification, the cells were resuspended in 50 ml or 70 ml of each HisTrap binding buffer (1⋅ PBS with 150 mM NaCl or 50 mM Tris-HCl, pH 8.0, and 300 mM NaCl) and disrupted by sonication on ice at 40% amplitude, with pulse on for 5 s and pulse off for 5 s, for a total of 2 h. The sonicated samples were centrifuged at 14,810 ⋅ g and 4 °C for 20 min and filtered through 0.45-µm filters to remove the debris.
The soluble fraction containing recombinant aFGF (from 1.57 g of wet cells) was loaded on a HisTrap HP 5-ml column (GE Healthcare) pre-equilibrated with HisTrap binding buffer (1⋅ PBS with 150 mM NaCl) at a flow rate of 1 ml/min. The washing and elution steps were performed using HisTrap binding buffer supplemented with 25 mM and 500 mM imidazole, respectively, at a flow rate of 3 ml/min. The eluted fractions were pooled and conducted the cleavage reaction. The TEV protease and the β-mercaptoethanol were added in the pooled fraction with target protein to TEV protease ratio of 1:10 (w/w) and the final concentration of 10 mM, respectively, to cleave 6HFh8 and dialyzed against 1⋅ PBS with 150 mM NaCl at 4 °C overnight. The dialyzed sample was loaded onto the same column at a flow rate of 1 ml/min. Fractions eluted with HisTrap binding buffer with 50 mM imidazole at a flow rate of 3 ml/min were pooled and mixed with an equal volume of 20 mM sodium phosphate buffer (pH 6.0) to prevent protein aggregation. They were then dialyzed against the binding buffer (20 mM sodium phosphate buffer; pH 6.0) at 4 °C overnight. The dialyzed samples were loaded onto a HiTrap CM FF 5-mL column (GE Healthcare) pre-equilibrated with the binding buffer at a flow rate of 1 ml/min. The column was then washed with binding buffer supplemented with 160 mM NaCl. Recombinant aFGF was eluted with binding buffer supplemented with 300 mM NaCl at a flow rate of 3 ml/min.
The soluble fraction containing recombinant VEGF165 (from 0.9 g of wet cells) was applied onto a HisTrap HP 5-ml column pre-equilibrated with HisTrap binding buffer (50 mM Tris-HCl, pH 8.0, and 300 mM NaCl) at a flow rate of 1 ml/min. The column was washed with binding buffer supplemented with 75 mM imidazole, and the fusion protein was eluted in binding buffer supplemented with 500 mM imidazole at a flow rate of 3 ml/min. The fusion protein was dialyzed against 20 mM sodium phosphate, pH 6.0, with 10 mM β-mercaptoethanol at 4 °C overnight to remove imidazole and NaCl and to prevent protein aggregation. The TEV protease and the β-mercaptoethanol were added in the dialyzed protein with target protein to TEV protease ratio of 1:10 (w/w) and 10 mM, respectively, to cleave 6HFh8 at 4 °C overnight. The cleaved sample was applied onto a HiTrap SP FF 5-mL column (GE Healthcare, pre-equilibrated with 20 mM sodium phosphate, pH 6.0, with 50 mM NaCl) at a flow rate of 1 ml/min. The bound protein was eluted in binding buffer supplemented with 300 mM NaCl at a flow rate of 3 ml/min and dialyzed against 20 mM sodium phosphate, pH 6.0, with 50 mM NaCl. The dialyzed sample was loaded onto a HisTrap HP 5-ml column (pre-equilibrated with 20 mM sodium phosphate, pH 6.0, with 50 mM NaCl) at a flow rate of 1 ml/min. The bound protein was eluted using binding buffer supplemented with 150 mM imidazole at a flow rate of 3 ml/min and applied onto the HiTrap SP FF 5-ml column (pre-equilibrated with 20 mM sodium phosphate, pH 6.0, with 50 mM NaCl) at a flow rate of 1 ml/min. Recombinant VEGF165 was eluted in binding buffer supplemented with 500 mM NaCl at a flow rate of 3 ml/min. The purified aFGF and VEGF165 proteins were stored at 4 °C until further analysis.
Analysis of the purity of obtained aFGF and VEGF165 by HPLC
The purified recombinant aFGF and VEGF165 were analyzed by HPLC (1200 Series; Agilent Technologies, Santa Clara, CA, USA) with an UV detector at 214 nm. The C18 RP column (Zorbax Eclipse XDB, 80 Å C18, 4.6 ⋅ 150 mm, 5 µm; Agilent Technologies) connected to an HPLC system was maintained at 40 °C. The column was pre-equilibrated with buffer A (0.1% of trifluoroacetic acid in distilled water) and 5% (v/v) buffer B (0.1% of trifluoroacetic acid in acetonitrile). The flow rate was 0.5 ml/min; the sample volume was 20 µl, and the run time for each sample was 45 min.
N-terminal sequencing and LC-MS/MS
Protein N-terminal sequences were obtained after transferring the purified recombinant aFGF and VEGF165 proteins to a polyvinylidene difluoride membrane using a Procise ABI 492 protein sequencer (Applied Biosystems, Foster City, CA, USA). The authenticity of purified proteins was verified by native mass spectrometry at eMASS (Seoul, Republic of Korea). Samples were analyzed following the service provider’s protocol. Briefly, they were first resolved by UHPLC Ultimate 3000 (Thermo Scientific) on an ACQUITY-C8 column (2.3⋅130 mm, 1.7 µm; Waters, Milford, MA, USA). Mobile phases A [H2O/formic acid, 100/0.2 (v/v)] and B [acetonitrile/formic acid, 100/0.2 (v/v)] were used for analysis. Approximately 10 µl of sample was injected for analysis and separated using a gradient of B in A from 5–100% for 12 min. Protein native mass was detected by using TripleTOF 5600+ (AB SCIEX, Framingham, MA, USA).
Indirect analysis of disulfide bond formation by purified GFs
A 5⋅ SDS-PAGE loading dye was prepared: 250 mM Tris-HCl, pH 6.8, 10% (w/v) SDS, 0.25% (v/v) bromophenol blue, and 50% (v/v) glycerol, with or without 100 mM DTT. The purified aFGF and VEGF proteins were then mixed with the loading dye and boiled at 100 °C for 5 min. The protein was loaded onto 4–12% Bis-Tris Plus SDS-PAGE gel and run at 170 V, 500 mA, for 35 min, followed by staining with InstantBlue.
Proliferation assay with purified aFGF and VEGF165
The proliferative effect of purified aFGF and VEGF165 was investigated by the MTT assay using HDF (ATCC, Manassas, VA, USA) and HUVECs (ATCC), respectively. The cells were maintained in IMDM medium (Thermo Scientific) supplemented with 10% (v/v) fetal bovine serum at 37 °C with 5% CO2. The cells were seeded in a 96-well plate at a density of 1 ⋅ 104 cells/well. After 24 h incubation, the spent medium was removed, and 100 µl of serum-free medium with purified aFGF or VEGF165 (0–1 µg/ml protein) and commercial aFGF (Merck, Darmstadt, Germany) or VEGF165 (Merck) were added, and incubated at 72 h. Following this, 10 µl of CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan) was added, and sample absorbance was measured at 450 nm by the plate reader Infinite 200 PRO after 2–3 h incubation at 37 °C.
Statistical analysis
All data were obtained from the independent experiments are presented as the mean ± standard deviation. The data were analyzed with Student’s t-test. Analysis of variance (ANOVA) was performed for relevant data. Values of p ≤ 0.05 were considered statistically significant, and values of p ≤ 0.01 were considered highly significant.