Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis through binding to its specific receptors, which mainly occurs to VEGF receptor 2 (VEGFR-2), a kinase insert domain-containing receptor. Therefore, the disruption of VEGFR-2 signaling provides a promising therapeutic approach for the treatment of cancer by inhibiting abnormal or tumorinduced angiogenesis. To explore this potential, we expressed the catalytic domain of VEGFR-2 (VEGFR-2-CD) as a soluble active kinase in
. The recombinant protein was purified and the VEGFR-2-CD activity was investigated. The obtained VEGFR-2-CD showed autophosphorylation activity and phosphate transfer activity comparable to the commercial enzyme. Furthermore, the IC
value of known VEGFR-2 inhibitor was determined using the purified VEGFR-2-CD. These results indicated a possibility for functional and economical VEGFR-2-CD expression in
to use for inhibitor screening.
Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis
binding to specific VEGFRs (VEGF receptors) on the surface of vascular endothelial cells
. Among these VEGFRs, VEGFR-2 (also known as kinase insert domain-containing receptor, KDR) plays an important role in tumor angiogenesis
. Although VEGFR-2 has seven extracellular immunoglobulin-like or ligandbinding domains, a single transmembrane region and a cytoplasmic domain, only its cytoplasmic domain (catalytic domain) has the tyrosine kinase catalytic activity
. Since abnormal tyrosine kinase activity is associated with many diseases, discovering specific inhibitors of tyrosine kinase, including VEGFR-2, represents one of the most promising methods in the development of new drugs
Several VEGFR-2 inhibitors have been studied as anticancer agents, such as SU11248, Bay43-9006, and PTK787, based on the fact that angiogenesis is crucial to the malignancy of tumors
. In order to screen more inhibitors targeting VEGFR-2, large amounts of purified and active VEGFR-2 are required. Hence, it is necessary to develop a simple and cost-effective method for VEGFR-2 production. Up to now, there have been several reported studies on the expression of the catalytic domain of VEGFR-2 (VEGFR-2-CD) in many different systems. For example, the catalytic domain of VEGFR-2 has been expressed in the
insect cell/baculovirus expression system
system is widely recognized as a more economical system for recombinant protein expression because of its ease in culture and the relatively cheap medium. The cytosolic domain of VEGFR-2 has been expressed in
; however, the major product was inclusion bodies, even on decreasing the temperature from 37℃ to room temperature
. As far as we know, there is no report on the expression of VEGFR-2 as a soluble active kinase in
. Because the intracellular catalytic domain of VEGFR-2 can be used as a substitute of the whole receptor as a target for inhibitor screening
, here, the recombinant VEGFR-2-CD was selected and this domain was expressed in
as a soluble active kinase. The recombinant VEGFR-2-CD was purified and characterized. With the recombinant protein obtained, a convenient and simple screening model for the VEGFR-2 inhibitors was established.
Materials and Methods
The tissue of pancreatic cancer was provided by the Shanghai First Peoples Hospital. Trizol for extracted RNA was obtained from Invitrogen (Shanghai, China). Restriction enzymes, T4 ligase, and Taq DNA Polymerase were purchased from TaKaRa (Dalian, China). The kits for the PCR product cleaning, DNA recovery from the gel after electrophoresis, and plasmid extraction were purchased from Sangon (Shanghai, China).
DH5α from Invitrogen and
BL21 (DE3) from Novagen (Shanghai, China) were used for the plasmid propagation and protein overexpression, respectively. The plasmid pGEX-4T-2 was purchased from Invitrogen. The Bradford assay kit for measuring protein concentrations was purchased from Sangon. The anti-VEGFR-2 antibody and mouse monoclonal antibody against phosphotyrosine (PY99) were purchased from BD (Becton, Dickinson and Company, Shanghai, China). All purified chromatography columns were obtained from GE Healthcare (Shanghai, China). VEGFR-2 expressed in
) insect cells was purchased from Abcam (Shanghai, China). PTK787, a known VEGFR-2 inhibitor, was obtained from MedChemExpress (Shanghai, China). The isopropyl β-D-1-thiogalactopyranoside (IPTG) and ampicillin were obtained from Sigma-Aldrich (Shanghai, China). All the solutions were made up with MilliQ water.
- RT (Reverse Transcription)-PCR and Construction of the Expression Plasmid
Total RNA was extracted from tissue of pancreatic cancer with the Trizol reagent and the reverse transcription of RNA to cDNA used oligo(dT)
as the primer. Synthetic oligonucleotide primers 5’-ACGC
TAAA TGG GAA TTC CCC AGAGACCGGC-3’ (forward) and 5’-ATAAGAATG
TTAATTA GCTTGC AAGAGA-3’ (reverse) were designed on the basis of the sequence of the reported catalytic domain (amino acids 826-1169) of human VEGFR-2 (GenBank Accession No. AF063658). The restriction sites are underlined. Using the two-step RT-PCR Kit (TaKaRa, Dalian, China), the cDNA region coding for VEGFR-2-CD was amplified from the total RNA with the following regime: 30℃ for 10 min, then 42℃ for 30 min, and a final 95℃ for 5 min. Next, with the cDNA from RT (reverse transcription)-PCR, PCRs were performed in a Peltier Thermal Cycler-200 (Applied Biosystems, Foster City, CA, USA) with DNA polymerase using the following conditions: 94℃ for 2 min, then 30 cycles of 94℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 1 min, followed by a final elongation at 72℃ for 10 min. The amplified PCR product was digested with
I, and subcloned into the corresponding restriction sites of vector pGEX-4T-2, resulting in the plasmid pGEX-4T-2/VEGFR-2-CD. The recombinant plasmid was then transformed into protoplasts of
DH5α and the sequence of VEGFR-2-CD was confirmed by DNA sequencing (Invitrogen Biotechnology). The scheme for construction of the expression plasmid is shown in
. All the operations were conducted based on classic molecular cloning methods
Scheme of VEGFR-2-CD fusion expression plasmid pGEX-VEGFR-2-CD, which was derived from plasmid pGEX-4T-2 by inserting the gene of VEGFR-2-CD into the SalI/NotI sites of the plasmid.
- Expression of VEGFR-2-CD inEscherichia coli
The plasmid pGEX-4T-2/VEGFR-2-CD was transformed into
BL21 (DE3) for production of VEGFR-2-CD. One single positive colony was selected, inoculated in 5 ml of LB with ampicillin (100 µg/ml), and grown overnight at 37℃. The overnight culture was diluted 1:100 in 100 ml of fresh LB medium with ampicillin (50 µg/ml) and further incubated at 37℃ up to an OD
of 0.5. To check the effects of the inducer (IPTG) concentration and culture growth temperature on the expression of soluble GST-VEGFR-2-CD recombinant proteins, each host strain culture was induced with four IPTG concentrations (0.25, 0.5, 0.75, and 1 mM) and at temperatures of 16℃, 22℃, 30℃, and 37℃ at different culture times (3, 5, 7, and 9 h), respectively. The cells were harvested by centrifugation at 10,000 ×
for 10 min and the cell pellets were suspended in PB (20 mM sodium phosphate and 0.15 M NaCl, pH 7.4) at 4℃ and disrupted under a pressure of 120 MP five times at a flow rate of 10 ml/min at 4℃ using a high pressure homogenizer (JNBIO, Guangzhou, China). The supernatant was collected by centrifugation at 10,000 ×
for 10 min.
- Purification of Recombinant VEGFR-2-CD fromEscherichia coli
The AKTA prime plus (GE Healthcare) was used for the purification of recombinant VEGFR-2-CD. The supernatant was filtered with a 0.22-µm-pore size filter (Merck Millipore, Darmstadt, Germany) before being loaded on a 1 ml GSTrap Fast Flow column (GE Healthcare) pre-equilibrated with at least 3 column volumes of buffer A (20 mM sodium phosphate and 0.15 M NaCl, pH 7.4). The column was then washed with 2 column volumes of buffer A, and the absorbed materials were eluted with buffer B (50 mM Tris-HCl and 10 mM reduced glutathione, pH 8.0) at a flow rate of 0.5 ml/min. The recombinant VEGFR-2-CD was eluted from the GSTrap Fast Flow column at about 20 min in buffer B. Fractions were monitored by a UV detector at 280 nm. The fractions containing VEGFR-2-CD were collected and subjected to dialysis steps (7 kDa cut-off) with a dialysis tube in buffer C (20 mM Tris-HCl, pH 8.0) at 4℃ for 12 h to remove reduced glutathione. The sample was dissolved in buffer C and loaded onto a 1 ml anion-exchange-resin DEAE-Sepharose Fast Flow column (GE Healthcare) pre-equilibrated with the same buffer. After washing with 2 column volumes of buffer C, the column was eluted with a linear gradient of 0–1.0 M NaCl in buffer C at a flow rate of 0.5 ml/min, monitored again by a UV detector at 280 nm. The fractions containing recombinant VEGFR-2-CD, eluted from the anion-exchange column at approximate 0.4 M NaCl in buffer C, were collected, dialyzed as described above, freeze-dried, and then stored at -20℃.
- SDS-PAGE and Western-Blot Analysis of Recombinant VEGFR-2-CD
After purification, the samples were diluted in SDS-PAGE loading buffer and boiled for 10 min. Afterwards, the samples were loaded onto SDS polyacrylamide gels (12%) and protein bands were visualized with Coomassie Blue R-250 staining after electrophoresis. The purity of the protein was measured using ImageJ software (
). Proteins were transferred onto nitrocellulose membranes using a Trans-Blot semi-dry transfer cell (Bio-Rad, Hercules, CA, USA) for western blotting. The membranes were then blocked with 5% (w/v) non-fat dried skimmed milk powder in TBST (Tris-buffered saline: 100 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) for 2 h at 25℃. Afterwards, one membrane was incubated overnight at 4℃ with 5% (w/v) non-fat dried skimmed milk powder in TBST containing a 1:1,000 (v/v) dilution of mouse monoclonal antibody against phosphotyrosine (PY99), and the other one was incubated with anti-VEGFR-2 antibody. After washing with TBST for 5 min five times, the membranes were incubated for 2 h at 25℃ in TBST containing secondary antibody. Following incubation with the second antibody, the membranes were washed again as described above and visualized with an enhanced chemiluminescence detection reagent (TianGen, Beijing, China).
- Mass Spectrometric Analysis of Purified VEGFR-2-CD
For the identification of the purified protein as VEGFR-2-CD, a purified preparation was subjected to SDS-PAGE analysis, stained with Coomassie blue, and the band containing VEGFR-2-CD was excised, washed, and destained. The protein was reduced and alkylated with iodoacetamide, followed by trypsin digestion. The tryptic peptides were extracted from the gel and guanidinated with O-methylisourea, followed by desalting on a ZipTip (Merck Millipore, Darmstadt, Germany). The desalted peptides were analyzed by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (Applied Biosystems).
- Protein Kinase Assays
Protein concentrations were measured using the Bradford method. VEGFR-2-CD tyrosine kinase activity was determined with an EnzyChrom Kinase Assay Kit (a generic fluorimetric high-throughput kinase assay; BioAssay Systems, USA). This homogeneous microplate-based assay involves incubating the kinase with a single working reagent, in which ADP is enzymatically converted to ATP and pyruvate, which are quantified using a fluorimetric (530 nm/590 nm) assay method. The kinase activity assay was performed in a 384-well plate according to the manufacturer’s instruction. Briefly, 20 µl of reaction mixture contained 2 µl of recombinant VEGFR-2-CD (50 µg/ml), 50 µM ATP, and substrate tyrosine [poly (Glu, Tyr)
] of 50 µg/ml in the reaction buffer containing Mg
. Reactions that contained ATP and substrate tyrosine [poly (Glu, Tyr)
] without purified VEGFR-2-CD or with commercial VEGFR-2 were used as controls. The plate was incubated at 30℃ for 30 min, followed by adding 40 µl of working reagent to each assay well and incubation at room temperature for another 10 min. The fluorescence intensity was detected with a multifunction microplate reader (BioTek, USA) using excitation at 530 nm and emission detection at 590 nm.
- Tyrosine Kinase Inhibition Rate Assay
The tyrosine kinase inhibition rate assay to VEGFR-2-CD was assayed using the EnzyChrom Kinase Assay Kit. Test compounds are usually pre-incubated with VEGFR-2-CD for 30 min, prior to adding ATP and substrate tyrosine [poly (Glu, Tyr)
] to initiate the kinase reaction. After a 30 min kinase reaction, the produced ADP was quantified using the fluorimetric assay. A known VEGFR-2 inhibitor, PTK787, was used as a positive control, and 0.1% (v/v) DMSO was utilized as the negative control. Each concentration was performed in triplicate. The reaction was initiated by adding 2 µl of VEGFR-2-CD, and the fluorescence intensity was detected with a multifunction microplate reader (BioTek) using excitation at 530 nm and emission detection at 590 nm. The inhibition rate (%) was calculated using the following equation:
- Expression and Purification of Human VEGFR-2-CD
To obtain enough high quality and inexpensive VEGFR-2 for inhibitor screening, the cDNA of human VEGFR-2-CD obtained from total RNA of pancreatic cancer cells by RT– PCR was cloned into plasmid pGEX-4T-2 (
). After confirming the DNA sequence, the resulting plasmid was transformed into
BL21 (DE3) to generate a GST-tagged fusion protein. Analysis by SDS-PAGE with Coomassie Blue staining indicated that VEGFR-2-CD was expressed by
BL21 (DE3) (pGEX-4T-2/VEGFR-2-CD) as the soluble protein with a molecular mass in agreement with the expected size (67 kDa) for GST-VEGFR-2-CD (
). The effect of temperature and inducer concentration (IPTG) on the expression pattern of GST-VEGFR-2 was also studied by SDS-PAGE analysis. The GST-VEGFR-2-CD expression at 22℃ with 0.5 mM IPTG for induction achieved the maximum level of soluble protein production. The analysis of the time-course of VEGFR-2-CD expression showed that the amount of the recombinant VEGFR-2-CD reached a maximum level at 7 h. After the recombinant VEGFR-2-CD was produced using the optimized protocol, it was purified through two steps that included GST-tagged protein purification and anion-exchange chromatography. The recombinant VEGFR-2-CD accounted for 7.15% of the total protein in the supernatant of disrupted cell pellet, 32.81% in the pooled fractions from the GSTrap Fast Flow column, and 92.35% in the pooled fractions from the DEAE-Sepharose column (
). Using this scheme, we obtained approximately 3 mg of protein (50 µg/ml) from 1 L of
cell culture overexpressing the protein (
SDS-PAGE analysis of recombinant VEGFR-2-CD. The sizes of marker proteins are indicated on the left (kDa). Lane 1, E. coli [pGEX-4T-2/VEGFR-2-CD] without induction; lane 2, E. coli [pGEX-4T-2/VEGFR-2-CD] induced with IPTG; lane 3, Soluble fraction of VEGFR-2-CD; lane 4, Fraction purified by GST fast flow column; and lane 5, Fraction purified by DEAE-Sepharose fast flow anion-exchange column.
Purification of recombinant VEGFR-2-CD fromE. coli.
aThe purification was carried out with 1 L of induced culture of fermentation. bThe amount of total protein was determined using the Bradford method (Sangon, Shanghai, China).
- Western Blot Analysis of Recombinant VEGFR-2-CD
To verify the purified VEGFR-2-CD, western blot analysis with an antibody probe specifically recognizing VEGFR-2 was performed. The single immunoreactive band displayed an apparent molecular mass of 67 kDa, which corresponds to the expected size of the GST-VEGFR-2-CD protein (
A). To detecte whether the purified VEGFR-2-CD was enzymatically active and had been phosphorylated in
, the mouse anti-phosphotyrosine PY99 monoclonal antibody was used. A reactive band showed on the nitrocellulose membrane with the expected molecular mass (67 kDa) (
B), indicating that the purified recombinant VEGFR-2-CD had been autophosphorylated in
and existed mainly in a phosphorylated state.
Western-blot analysis of purified recombinant VEGFR-2-CD. (A) Probed with anti-VEGFR-2; (B) probed with mouse monoclonal antibody against phosphotyrosine (PY99). Lanes 1 and 2 are parallel classes.
- Mass Spectrometric Analysis of Purified VEGFR-2-CD
To further confirm the identity of the purified protein, we carried out a mass spectrometric analysis. Because the purified VEGFR-2-CD contained an N-terminal GST tag, the tag was removed by subjecting the protein to thrombin digestion (a thrombin cleavage site was engineered between the GST tag and the VEGFR-2-CD sequence) (Invitrogen). The result showed that the purified protein prepared after thrombin digestion contained the peptides that matched only the VEGFR-2-CD sequence (Supplementary data 1 and 2).
- Protein Kinase Assays
activity of purified VEGFR-2-CD was detected by Kinase Assay Kit (BioAssaySystems, USA). The synthetic peptide substrate tyrosine [poly (Glu, Tyr)
] was used in this study
. The activity of recombinant VEGFR-2-CD produced by
(this study) was comparable to that of commercial VEGFR-2 from
insect cells (Abcam) (
). To test the optimum concentration required for the kinase assay, various amounts of the purified VEGFR-2-CD were added. The phosphorylation state of the substrate escalated with increasing concentrations of VEGFR-2-CD and reached a plateau at 75 ng per well of VEGFR-2-CD (
). These results indicated that the VEGFR-2-CD fusion protein expressed in
kinase catalytic activity and could catalyze the phosphorylation of peptide substrate in an enzyme concentration-dependent manner when the amount was lower than 75 ng/well.
The activity of VEGFR-2-CD detected by the Kinase Assay Kit (BioAssaySystems, USA) from different expression systems. The recombinant VEGFR-2-CD produced from E. coli and the commercial VEGFR-2 generated by Sf9 insect cell (Abcam) were used in this assay.
Kinase assay (BioAssaySystems, USA) determining the tyrosine kinase catalytic activity of the GST-VEGFR-2-CD fusion protein. The sample of VEGFR-2-CD fusion protein was purified by GST and DEAE chromatography. Various quantities of purified GST-VEGFR-2-CD were added to 384-well plates, and then the tyrosine kinase activity was quantified using a fluorimetric (530nm/590nm) assay method.
- Inhibition Rate Assay by VEGFR-2 Inhibitors
The goal of production of VEGFR-2-CD is to apply it as a target in anticancer drugs screening. A well-known VEGFR-2 small-molecule inhibitor, PTK787, was used to evaluate the biological activity of the VEGFR-2-CD protein. An inhibition rate assay was performed using varying concentrations of PTK787, which inhibited VEGFR-2-CD activity with an IC
value of 39.7 nM (
), which is similar to the value reported in the literature
. These results suggested that the purified GST-VEGFR-2-CD fusion protein was functionally active and could be used for VEGFR-2 inhibitor screening. In addition, our results also indicated that the GST part did not affect the biological kinase catalytic activity of VEGFR-2.
Inhibition of PTK787 to tyrosine kinase activity of GST-VEGFR-2-CD, which was purified by GST and DEAE chromatography.
Inhibition of PTK787 to tyrosine kinase activity of GST-VEGFR-2-CD, which was purified by GST and DEAE chromatography.
, whose genetics are far better characterized than those of any other microorganism, is one of the most widely used hosts for the production of heterologous proteins
. Nevertheless, most of the gained eukaryotic proteins are typically insoluble and misfolded inclusion bodies that need further solubilization and refolding
. Moreover, many families of proteins that are most biochemically interesting, such as kinase phosphatases, membrane-associated proteins, and many other enzymes
, can hardly be produced as soluble proteins in
. The reason is unknown so far. However, there are two possibilities - the rate of translation and the rate of protein folding, both of which are almost ten times faster in
than in eukaryotic systems
. At present, domestic and foreign researchers mainly adopt eukaryotic expression hosts, especially
insect cells, for the expression of human VEGFR-2-CD
. However, eukaryotic expression hosts have their own difficulties in terms of ease of use, time, cost, and experimental flexibility.
In this study, we first constructed a recombinant expression plasmid (pGEX-VEGFR-2-CD), in which we merged the VEGFR-2-CD cDNA with the glutathione S-transferase (GST) coding sequence downstream of the
-inducible promoter to achieve high-level expression of soluble recombinant VEGFR-2-CD protein in
). The overwhelming evidence has shown that GST is, at best, a poor solubility enhancer
. Using this plasmid, we have achieved expression of soluble VEGFR-2-CD as a GST fusion protein using
BL21 (DE3) (
), under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. Western blot and mass spectrometric analyses confirmed the sequence of VEGFR-2-CD and indicated that the purified VEGFR-2-CD was enzymatically active and existed in a phosphorylated state (
). The recombinant protein prepared in this study contained amino acid residues 826–1169 of VEGFR-2, similar in length to residues 816–1175 and 834–1162 reported by Liu
, respectively. Both the
biological activity assay and the inhibition rate assay showed that the GST-VEGFR-2 fusion protein exhibited high kinase catalytic activity comparable to the commercial enzyme (
) and could be inhibited sensitively by the specific VEGFR-2 small molecule inhibitor PTK787 (
). In general, the inhibition rate would be lower if the expressed VEGFR-2 was not folded correctly or not biologically active. In addition, these results demonstrated that the GST part did not affect the biological kinase catalytic activity of VEGFR-2.
In conclusion, we demonstrated a simple but effective design and production protocol for the production of soluble VEGFR-2-CD for inhibitor screening. With that, the VEGFR-2-CD was expressed successfully in recombinant
. The process enabled the production of a high-purity product at a level of 3 mg protein/l. Western blot analysis and
biological activity assay demonstrated the purified VEGFR-2-CD had autophosphorylation activity and phosphate transfer activity. Moreover, a convenient and simple screening model for VEGFR-2 inhibitors was established, suggesting that the GST-VEGFR-2 fusion protein expressed in
could be used as a target for anticancer drug screening.
This work was supported by the China Postdoctoral Science Foundation Grant (2013M540341), Key grant cultivating interdisciplinary studies of Chinese Education Ministry (WF1113014), National Natural Science Foundation of China (21303050 and 31471659), Pujiang Talent Program of Shanghai Municipality (13PJD012), and National “Thousand Talents Program” of China.
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