Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli
© The Author(s) 2017
Received: 1 December 2016
Accepted: 7 December 2016
Published: 6 February 2017
Vascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF165 in convenient yield. In this study, recombinant human VEGF165 was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment. Inclusion bodies were also isolated from the cell lysate and subjected to a simple protocol of solubilisation and refolding. Single-step purification was performed using nickel affinity chromatography and the purified proteins were able to recognize monoclonal Anti-poly-His antibody. The biological activity of the VEGF165 was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).
KeywordsRT-PCR Soluble VEGF165 expression Inclusion bodies Refolding Purification Cell migration and proliferation CAM assay
Angiogenesis is considered as a complex multistep process involving the growth of blood vessels from the existing vasculature (Adair and Montani 2010). Formation of new blood vessels can takes place under both normal physiological conditions such as embryonic development, endometrial and placental proliferation, growth and tissue repair, as well as pathological ones, including cancer vascularization. The promotion of tumour growth is dependent on the expression of growth factors in the microenvironment like vascular endothelial growth factor (VEGF), heparin-binding fibroblast growth factor (FGF), and platelet-derived endothelial cell growth factor (PD-ECGF) (Niu and Chen 2010). VEGF ranks as key inducer of angiogenesis and central mediator that promotes vascular permeability (Schmitz et al. 2006). Several proteins including VEGF-A to D and placental growth factor (PlGF) compose the VEGF family. They do not share high homology but they share cysteine “knot motif” comprising eight conserved cysteine residues. VEGF-A binds to VEGFR-1 and -2, mediating the activation of all pathways required in angiogenesis. VEGF-A which commonly referred to as VEGF, was firstly isolated in 1989 from medium conditioned by bovine pituitary follicular cells (Ferrara and Henzel 1989) and described in highly vascularized tumours where its expression is stimulated by hypoxia (Shweiki et al. 1992). The VEGF pre-mRNA is transcribed from a single gene containing 8 exons, is spliced and expressed as various isoforms owing to alternative splicing of exon 6 and 7. These two exons determine the VEGF fate, either being associated to cell surface or being secreted and associated to the extracellular matrix (Roodink and Leenders 2010). There are at least 4 principal variants VEGF121, VEGF165, VEGF189 and VEGF206 with the numerals denoting the number of amino acids in the mature peptide (Roskoski 2007; Ferrara et al. 1992).
VEGF165, the most abundant isoform with VEGF121, is secreted and represents the most relevant promoter of tumour vascularization as it exerts several effects in different pathways required in angiogenesis such as endothelial cell migration, proliferation, tube formation and survival (Papetti and Herman 2002) and is therefore the focus of intense investigation. It has been reported that the quantification of the total VEGF mRNA expression by real-time reverse transcription PCR revealed that VEGF121 and VEGF165 mRNA were up-regulated in various neoplasm compared to normal tissue (Zygalaki et al. 2007; Hervé et al. 2008). VEGF121 and VEGF165 were also found to be the most over-expressed isoforms in both colonic and lung carcinoma (Cheung et al. 1998). VEGF189 and VEGF206 are poorly secreted and are essentially cell associated although their peptide signal sequence is identical to that found in VEGF121 and VEGF165 (Houck et al. 1991).
In 1971, Folkman suggested the idea that anti-angiogenic therapies could be used as a highly promising and effective approach in cancer treatment (Folkman 1971). VEGF165 showing strong mitogenic potency to vascular endothelial cells is used to direct therapy in a wide range of cancers. On the basis of this pioneering hypothesis, numerous studies were carried out to provide a fast and easy way to produce this therapeutic protein. VEGF is a highly conserved disulfide-bonded glycoprotein with a molecular mass of 43 kDa consisting of an anti-parallel homodimer structure (Vicari et al. 2011). The VEGF belonging to the PDGF family is characterized by the presence of eight conserved cysteine residues implicated in intra- and inter-chain disulfide bonds (Keyt et al. 1996).
Since the functional potency of VEGF165 is not dependent on the N-linked glycosylation at Asn75 residue, eukaryotic expression platform is not required for VEGF recombinant protein production (Claffey et al. 1995). Many bacterial expression systems have been developed to achieve high yield as well as high quality and functional potency of the VEGF165. It was generally reported that expression resulted in most of cases in the formation of inclusion bodies which represented the primary source of the expressed protein (Gast et al. 2011). Escherichia coli which remains one of the most attractive cell hosts, have been widely utilized for production of recombinant His-tagged proteins.
In the current study, we report on soluble His-tagged VEGF165 protein that was successfully expressed in E. coli (BL21)-DE3. Key factors for efficient production were assessed and optimization of cell growth conditions and media were sought. Several practical methods have been implemented to ensure high cell-density cultivation. Our methods allow us to consistently obtain high yield of biological active VEGF165. More importantly, different protein extraction procedures with optimized conditions were performed to achieve high solubility of the expressed protein. An economically and fast protein extraction protocol combining sonication and lysozyme treatment was used to facilitate soluble VEGF165 extraction. Moreover, expression of VEGF165 in E. coli (BL21)-DE3 often results in accumulation of the recombinant protein as insoluble aggregates. We describe here an economic and efficient process for solubilisation and refolding of the VEGF165 aggregates.
Materials and methods
Strains and culture conditions
pGEMT-easy and pET-21a (+) vectors were used respectively to clone and to express the VEGF splice variants proteins. E. coli strains Top10 (F-mcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara–leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-) and BL21(DE3) (F–ompT gal dcmlonhsdSB(rB-mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) were used as recipient for cloning and expression vectors respectively.
Culture media were LB (Luria-Bertani):10 g/l bacto-tryptone, 5 g/l yeast extract, 5 g/l NaCl; 2YT: 17 g/l bacto-tryptone, 10 g/l yeast extract, 5 g/l NaCl. LBA and 2YTA: LB and 2YT containing 100 µg/ml ampicillin.
The VEGF165 coding sequence, isolated from MCF7 cell lines, was 100% identical to the human VEGF165, already published (Piotrowski et al. 2015) under the accession number NM_001287044.1, was cloned downstream of the T7lac promoter and transformed into E. coli BL21-(DE3). Recombinant strains were cultivated on 2YTA medium and induced by the addition of 1 mM of IPTG at 37 °C for 20 h.
Amplification of VEGF splices variants
Primers used in this work
pGEMT easy vector
F (ex2) 5′GGATCCGCACCCATGGCAGAAGGAGGA
R (ex8) 5′CTCGAGTCAGTGGTGGTGGTGGTGGTGCCGCCTCGGCTTGTCACATCT
Cloning and DNA sequencing of RT-PCR products
The PCR fragment, amplified on cDNA from MCF7 cell line, was firstly cloned into the pGEMT-easy vector (Promega). Ligation product was transformed into competent E. coli Top10 cells and plated on LBA. A fraction (1/20) of each colony-plasmid was amplified with Dream Taq DNA polymerase using F1 and R1 primers. As these primers can detect all VEGF splice variants, each variant was identified by PCR screening and was verified by DNA sequencing using universal and reverse primers (Table 1). Thereafter, the VEGF165 variant was sub-cloned in pET-21a using the BamHI and XhoI restriction sites.
Fermentation of the recombinant strain expressing the VEGF165
A 7 l stirred tank bioreactor (Infos, AG GH-4103 Bottmingen, Switzerland) equipped with air flow, temperature, dissolved oxygen concentration, pH and agitation control was utilized to produce elevated levels of VEGF165. The fermentation was carried out with a working volume of 4 l. In the batch cultivation, the temperature was maintained at 37 °C and dissolved oxygen was kept above 20% of medium saturation by air supply and agitation rate variation (400–600 rpm). To decrease foam production Silicone 426 R antifoam (Prolabo, Paris, France) was added. The initiated pH of the medium was 7 and the pO2 was 98%. After 7 h, pO2 decreased to 43%. When the OD600nm of the culture reached 0.7, IPTG was added at a final concentration of 1 mM. The induction phase was maintained for more than 16 h. After 16 h, pO2 continue to decrease to 1%. When pO2 increased to 9% (18 h of induction phase) the growth rate was found to slow down. At this pO2, the growth of E. coli cells was stopped and the cell pellet was collected by centrifugation at 6000 rpm for 20 min and stored at −20 °C.
In order to optimize the protocol of extraction of the recombinant VEGF165 from the cell pellet, five methods were adopted: M1: Alumina treatment; M2: lysozyme treatment; M3: sonication in PBS; M4: sonication in Lysis Buffer; M5: lysozyme treatment followed by sonication in lysis Buffer treatment. Induced cultures were centrifuged at 6000 rpm for 20 min for each of these methods.
A mechanical cell shearing performed by the abrasive effect of the alumina powder was adopted in M1. In that case, after pelleting, cells frozen at −20 °C (1.5 g) and subsequently thawed in a chilled mortar were grinded energetically for 15 min with Alumina powder w/w (Sigma-Aldrich, Munich, Germany) using a pestle until the mixture formed a fairly stiff paste (Hughes 1950). The cell paste was re-suspended in 2 ml of PBS 1X buffer containing 2 mM PMSF. Alumina, unbroken cells as well as cell debris were decanted after a low speed centrifugation at 3000 rpm for 10 min and the supernatant (crude lysate) was saved.
Osmotic lysis of bacterial cells was adopted in M2. After washing with buffer A, containing 500 mM sucrose, 25 mM Tris–HCl pH 8, 10 mM EDTA, the cell pellet was re-suspended in this hypertonic solution and then treated with lysozyme (5 mg/ml). Protoplasts were harvested by centrifugation at 4000 rpm for 10 min and burst with an osmotic imbalance created by an hypotonic solution (buffer B) composed of 25 mM Tris–HCl pH 8, then phenyl methyl-sulfonyl fluoride (PMSF) was added. The remaining non-burst cells and cells debris were centrifuged at 3000 rpm for 10 min.
Additionally, a high operating pressure using a “Vibra cell VCX 750 sonicator was performed for 30 min at 60% amplitude with PBS in M3 and with lysis buffer composed of 50 mM Tris–HCl pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton, 1% PMSF in M4.
Alternatively, combining sonication and lysozyme treatment was carried out in M5 by first treating the pelleted protoplasts with lysis buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton, 1% PMSF) and then sonication for 30 min at 60% amplitude. The resulting protein extract was centrifuged at 3000 rpm for 10 min to remove unbroken cells and cell debris.
For all extraction protocols, after the mild centrifugation (3000 rpm for 10 min) the supernatant, called crude lysate, is clarified by centrifugation at 13,000 rpm for 10 min. The supernatant represented the total soluble protein extract while the pellet represented the IB (inclusion bodies).
Inclusion bodies isolation
The IB pellet material collected from the large scale production (700 ml of culture) was predominantly used for VEGF165 solubilisation and refolding experiments. It was re-suspended in 50 ml of buffer containing 50 mM Tris-HCl pH 8, 50 mM NaCl, 1 mM EDTA, 1% Triton X-100. After washing with the same buffer but without Triton X-100, the inclusion bodies were collected by centrifugation at 8000 rpm for 10 min and subsequently subjected to the solubilisation step by adding of 25 ml of 8M urea and 5% β-mercaptoethanol (βME). The suspension was stirred overnight at 4 °C and then centrifuged at 8000 rpm at 4 °C for 10 min. The solubilized proteins were then dialyzed at 4 °C against 2 l of buffer containing 25 mM Tris pH8, 50 mM NaCl. The dialysis buffer was changed four times to sufficiently allow VEGF165 refolding. The remaining insoluble material was eliminated by centrifugation at 8000 rpm for 10 min.
A nickel affinity chromatography was used to purify the VEGF proteins. The total protein extract (either from total soluble proteins or from solubilised IB) was loaded on His Trap™chelating HP 1 ml column (GE healthcare life sciences) with a flow rate of 1 ml/min. The resin was washed with 30 ml binding buffer (20 mM NaH2PO4 Na2HPO4, 500 mM NaCl, 10 mM Imidazole, pH 7.4) to enable elution of non-specifically-bound proteins. Finally, the His-tagged proteins were eluted from the resin with Imidazole linear gradient from 10 to 500 mM in Elution Buffer (20 mM NaH2PO4 Na2HPO4, 500 mM NaCl, pH 7.4). The purity of collected fractions was assessed by SDS-PAGE, and protein concentration was checked using Bradford’s method.
SDS-PAGE and western blot analysis
The expression of the recombinant VEGF165 protein in E. coli BL21 cells was evaluated by SDS-PAGE and Western Blot. The total protein extract (30 µg) and the purified protein were mixed to loading buffer, heated for 5 min and applied on the gel. 15% SDS-PAGE electrophoresis was conducted in buffer (25 mM Tris; 250 mM Glycine; 1‰ SDS) for 2 h. Separated proteins were directly electro-blotted onto a nitrocellulose membrane in buffer (39 mM Glycine; 48 mM Tris; 0.037% SDS; Methanol 20%) for 1 h at constant voltage (15 V). The membrane was stained with Ponceau S then distained using bi-distilled water, to verify protein-transfer efficiency. The membrane was blocked for 1 h at room temperature with 5% skim milk in phosphate-buffered saline (0.9% NaCl in 10 mM phosphate buffer, pH 7.4) with Tween-20. Immuno-blotting was carried out by incubating the membrane with primary antibody Anti-His Sigma-Aldrich diluted to 1:5000; then with the appropriate Horseradish Peroxidase conjugated secondary antibody diluted to 1:5000. Peroxidase activity was detected using the Amersham enhanced chemo-luminescence system and autoradiography or densitometric analysis performed by the Versadoc MP4000 imaging system (Bio-Rad).
In vitro endothelial cell proliferation assay
Human umbilical vein endothelial cells (HUVEC) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) in a humidified incubator with 5% CO2. Cells were seeded at a density of 5000 cells per well and allowed to grown over-night at 37 °C in a 96-well tissue culture plates until reaching a pre-established confluence.
Various concentrations of recombinant human VEGF165 (200 and 500 ng) were added and incubated with HUVEC cells for 72 h. Four duplicate wells were set up for each condition and three independent assays were performed. The proliferation of endothelial cells was evaluated with the MTT test; the treated cells were incubated with 0.5 mg/ml MTT for 2 h at 37 °C. Culture medium was removed carefully from each well and 100 µl of DMSO was added. The plate was then gently agitated until the color reaction was uniform and OD560nm was measured using a microplate reader.
Chicken chorioallantoic membrane assay
Chick embryos from 3-day-old eggs were opened and placed in double Petri dishes with added water to maintain eggs humidified. After 5 days at 37 °C, filter paper disks (diameter 6 mm) soaked in buffer (0.9% NaCl), 200 ng and 500 ng of recombinant human VEGF 165 were applied on the chicken chorioallantoic membrane (CAM). After 48 h, spontaneous and induced angiogenesis were observed and photographed with a digital camera at 10× magnification. The response was quantified by scoring the extent of vascularization using the software program ImageJ.
Wound-healing migration assay
Human umbilical vein endothelial cells were cultivated at 37 °C in 48-well plates in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and maintained overnight in a humidified incubator (5% CO2). Cells were seeded at a density of 5000 cells per well. The next day, monolayers created were carefully scratched using a 20-μl microtip. The cellular debris was subsequently removed by washing with PBS. The cells were thereafter treated with or without recombinant VEGF165 (200 ng) in serum free RPMI medium for an additional 12 h. Cell images for each condition were taken with a digital camera connected to an inverted microscope LEICA (×10 objective). The software program ImageJ was used to determine the percentage of wound healing for each condition.
Data is presented as the mean ± SEM of five independent experiments. Statistical significance was analyzed using unpaired Student’s t test using STATISTICA 6. p < 0.05 was considered statistically significant and is indicated with asterisks over the value (**p < 0.05 and ***p < 0.001).
Amplification of the VEGF splices variants
The PCR product mixture obtained after amplification of the MCF7 cDNA was ligated into pGEMT easy vector and cloned in E. coli Top10. Several clones were analysed by PCR using the same primers (Fig. 1b) and subsequently sequenced. As expected, the three types of VEGF are represented in the different clones and their sequences were completely identical to the published ones, i.e. the VEGF165 with accession number NM_001287044.1 (Piotrowski et al. 2015).
Heterologous expression VEGF165using the pET-21a(+) vector
It should be recalled that the recombinant VEGF165 is produced here as a His-Tagged fusion protein. The conditions of culture and induction were optimized at different levels: the culture medium (LB or 2YT), the inducer concentration (0.4 and 1 mM IPTG), the post-induction temperature (25, 30 or 37 °C) and the duration of the culture post-induction.
Optimization of the VEGF165 protein extraction
VEGF165 purification using Nickel-affinity chromatography
Being His-Tagged, the VEGF165 was purified on a His Trap column. The total protein extract, from the best method M5, was applied to the column and the fractions were eluted using Imidazole gradient (10–500 mM) and analyzed on 15% SDS-PAGE (Fig. 3b). The VEGF165 protein was eluted at 250 mM Imidazole. Western Blot analysis confirmed that this purified protein corresponded to VEGF165 (Fig. 3d, lane 2).
VEGF165 recovery from inclusion bodies
Solubilisation and refolding operations are the most important steps that could efficiently convert aggregated protein to bioactive form. In our study, a simple solubilisation method was performed using a high concentration of urea (8 M) and 1% of Triton X-100 to solubilize the pellet. To obtain reduced state of the cysteine residues, β-mercaptoethanol was used as reducing agent. 1 mM EDTA was also added to the solubilisation buffer to prevent metal-catalysed air oxidation of cysteine residues. Thereafter, an elaborate method of proteins aggregate refolding is needed to ensure a good amount of the bioactive VEGF165. Therefore, step-wise dialysis was used for the renaturation of the recombinant protein. The gradual removal of the denatured reagent is the most important step as to increase the refolding efficiency of the denatured VEGF165. Figure 3c shows that after renaturation, VEGF165 was successfully refolded, facilitating thereby its purification. Under reducing conditions, the molecular weight of the refolded VEGF165 was 23 kDa. Similarly to the soluble VEGF165, we found that elution with 250 mM Imidazole resulted in an increased quantity and purity of the recombinant protein. Both the monomer and the dimer were efficiently eluted.
Finally, we compared the eluted fraction at 250 mM Imidazole from the soluble VEGF165 to the refolded VEGF165; Fig. 3d shows that they behave similarly by the western blot analysis. The batch fermentation process yields approximately 1.5 mg/l of purified VEGF165 from both supernatant and inclusion bodies.
In vitro HUVEC cells proliferation assay
Chicken chorioallantoic membrane assay
In vitro scratch wound assay
The production of VEGF165 in significant amounts is an important prerequisite for the search, expansion of promising and effective anti-angiogenic drugs. Many attempts for producing VEGF165 in bacterial system have been made. They have mainly focused on the optimization of production conditions such as induction temperature, IPTG concentration and time incubation after induction (Kang et al. 2013). Generally, low temperature ensured the expression of less inclusion bodies and more soluble form of recombinant protein but some reports found that the soluble recombinant VEGF165 expression was increased when the cells were incubated at 37 °C (Lee et al. 2011). Interestingly, we showed that the expression of the VEGF165 induced with 1 mM IPTG in 2YT medium at 37 °C for 20 h increased the percentage of the soluble protein. In this expression assay, inclusion bodies occurred heavily when the inducing temperature was set at 37 °C. We also showed that VEGF protein was much more expressed in 2YTA than in LBA medium, probably because it was exclusively produced as inclusion bodies in LBA condition.
The common objective of a successful heterologous expression is generally to balance success rates with speed, ease, cost and breadth of use. For this reason, different parameters for VEGF165 expression are needed to ensure a high level and a good yield of the recombinant protein. The central interest of many works is to optimize production conditions such as shaking speed, medium, induction temperature, IPTG concentration, but the optimization of the protein extraction method remains a major challenge for a high percentage of the produced protein. It is apparent that the methods described here have, in many instances, to be quite similar especially with use of sonication in lysis buffer to extract target proteins. But combining different protein extraction method is not frequently used. In this study, we found that combining lysozyme treatment and sonication in lysis buffer (M5) could increase the level of the soluble VEGF165. More interestingly, the consequence of this method is the important clarification of the protein lysate, thereby facilitating the purification of the VEGF165. A single step of purification using affinity chromatography that does not need any organic solvent like acetonitrile was carried out.
Many procedures for producing recombinant human VEGF165 in bacterial system were described but resulted in most of cases in the production of insoluble inclusion bodies which represented the primary source of the target protein (Gast et al. 2011). With the fact that 30% of proteins from E. coli itself cannot be expressed in soluble form (Gräslund et al. 2008), it is meant that the main limitations of the recombinant protein expression from bacterial cells are the low production levels and low refolding yield of the inclusion bodies, leading to biologically inactive recombinant proteins (Bang et al. 2013).
It was reported that aggregation reactions of different proteins displayed certain common properties. A strong temperature dependence of unfolding enthalpy which increases rapidly with temperature was shown. The use of low temperature during induction phase could increase peptide stability and reduce inclusion bodies formation. In contrast to previous studies (Kim et al. 2007; Zhang et al. 2014) we found that maintaining induction temperature at 37 °C for 20 h led to a low aggregation level of the VEGF165 improving its solubility. Nevertheless, our protein was expressed largely in the form of inclusion bodies. The protein aggregates was solubilized using high concentration of Urea while β-Mercaptoethanol was added to split the disulfide bond (that are responsible of the inter-subunit aggregation) and to maintain cysteine residues in a reduced state. The step-wise dialyses used allow protein folding into their native form and decrease sufficiently the denaturant concentration.
This insoluble fraction was therefore subjected to a simple procedure that overcomes the need of multi-days refolding experiments (Lee et al. 2011). Indeed, these authors spent about 7 days of serial dialysis in order to recover a refolded protein while our procedure takes only 48 h.
A single step purification of the refolded VEGF165 was thereafter carried out using a Nickel affinity column. Similarly to the soluble VEGF165, the refolded protein was eluted at 250 mM Imidazole and detected by anti His-tag antibodies. On the other hand, the function of VEGF in wound repair has been extensively studied. Thereby, VEGF stimulates angiogenesis and also influences wound closure and epidermal repair. Here, the biological activity of the recombinant VEGF165 was verified by chicken chrioallantoic membrane assay, scratch wound healing and proliferation assay using HUVEC cells. Thus, VEGF165 potency and bioactivity were confirmed by its ability to promote endothelial cell migration and proliferation and to induce new capillaries and branching vessels in the CAM model.
In summary, this study described a simple approach for producing recombinant VEGF165 for therapeutic applications. The overall probability of expressing human VEGF successfully depends on the variation of the large-scale production parameters and also protein extraction methods that could increase the yield and the purity of the soluble protein. Protein extraction was a major challenge that could assess the purity of the VEGF165 and lead to a simplified purification procedure. Overall, a successfully VEGF165 expression in bacterial system within soluble and insoluble fraction with fast and low cost procedure was presented to produce efficiently a functional VEGF protein for therapeutic applications.
AT carried out the experiments. AG participated in the design of the study, and supervised the research work. AT and AG drafted the original manuscript. NM, MM and HB performed the angiogenesis in vitro and in vivo experiments. RA produced and provided the cDNA. MG participated in the fermentation process. NFM participated in the affinity chromatography experiment. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All datasets on which the conclusions of the manuscript rely are presented in the main paper.
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.
This work was funded by a grant of the Tunisian Ministry of Higher Education and Scientific Research.
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