Expression of a functional recombinant vascular endothelial growth factor 165 (VEGF 165 ) in Arabidopsis thaliana

Objective: Targeting the protein of interest to a particular tissue to achieve high-level expression is an important strategy to increase expression efficiency. The use of the plant seed oil body as a bioreactor can not only increase the amount of target protein, but also reduce the cost of downstream processing. Methods: VEGF165 was expressed in Arabidopsis thaliana seeds via oilbody fusion technology. The pKO-VEGF165 vector was construted and transformed into A. thaliana seeds. T3 transgenic seeds was detected by SDS-PAGE and western blot methods. The cell activity was tested by MTT methods. Result: The phaseolin promoter was used to drive seedspecific expression of the VEGF165 gene in transgenic A. thaliana. The coding region of VEGF165 was fused to the Arabidopsis oleosin sequence to target the protein to the oil bodies in the seeds of transgenic plants. The T-DNA region of recombinant plasmid pKO-VEGF165 was shifted to A. thaliana seeds via the floral-dip method. Protein was analyzed by electrophoresis and protein hybridization analyses. Finally, MTT assays showed that the oleosinVEGF165 fusion protein played a part in the proliferation of HUVEC cells in vitro. Conclusion: Oleosin-VEGF165 was successfully expressed and it had stimulated HUVEC cell proliferation activity.


Introduction
Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein that induces the production of mitogen in endothelial cells [1]. It plays a role in an basal regulator of vasculogenesis and hematopoiesis, wound healing [2][3][4] and pathological status such as rheumatoid arthritis, cancer, cardiovascular disease and psoriasis [5][6][7][8]. VEGF can promote vasopermeability, stimulate endothelial cell proliferation and migration [9], and drive neovascularization [10][11][12]. The alternative splicing of VEGF-mRNA generated five isoforms (VEGF 121 , VEGF 145 , VEGF 165 , VEGF 189 , and VEGF 206 ) [13]. A variety of VEGF isoforms are possible to carry out different effects and they synergize in the tumor angiogenesis process. VEGF 165 is the most effective angiogenic factor in the VEGF family [13]. The literature reported that increased tumor angiogenesis and poor outcomes correlate with the overexpression of VEGF 165 in colon cancer, osteosarcoma, renal cell carcinoma and non-small cell lung cancer [13][14][15].
Plants have been used as a source of natural products throughout history, dating back thousands of years. The development of plant transformation technology has led to the use of various plant hosts for the expression of human therapeutics [16,17], nutraceuticals [18], antibodies [19,20], industrial enzymes [21,22], vaccine antigens [23,24], and biopolymers [25,26]. The bioactive proteins are produced in the plant bioreactor that is most safe, non-toxic, simple and lowest cost. Although they have some limitations, such as appear to be the carriers for exogenous proteins in seeds, these proteins are produced more stable in plant seeds than other tissues [27]. The purification of the exogenous protein from the wide spectrum is a major bottleneck, because it requires many tedious processes [27]. However, it is easier to extract a recombinant protein located on the surface of an oil body, since oil bodies can be separated easily from other cellular components by flotation centrifugation [27][28][29].
After the target protein is anchored on the surface of oil body, the oil bodies can be separated via flotation centrifugation and the oleosin-target protein can be easily extracted [27,30]. Oil body expressed hirudin was extracted from transgenic seeds and the recombinant hirudin showed anti-thrombin activity [27,31]. The β-glucuronidase linked to the oil body was detected to be active and remained active after it was separated from the surface of the oil body [27,32]. Human epidermal growth factor that fused with oleosin and inserted into a chymosin cleavage factor between oleosin-hEGF has been expressed in Arabidopsis thaliana. Here, we expressed VEGF 165 targeted to the oil body in A. thaliana. The expression vector pKO-VEGF 165 was constructed and the T-DNA region of the recombinant plasmid was transformed into plant genome. The oleosin-VEGF 165 fusion protein expressed in the transgenic A. thaliana seeds showed good antigenicity, and it stimulated HUVEC cell proliferation.

Reagents
Escherichia coli DH5α and Agrobacterium tumefaciens EHA105 were obtained from the team of XK Li professor in Jilin Agricultural University, China. The restriction enzymes such as NcoI and Hind III, ExTaq DNA polymerase were purchased from TaKaRa (Dalian, China). Streptomycin (Str) and Kanamycin (Kam) were obtained from Sigma (Hong Kong, China). Glufosinate was from Boehringer Mannheim Corp [33]. (Mannheim, Germany). The rabbit anti-VEGF polyclonal antibody was acquire from Beijing Bioss Co., Ltd. Goat anti-rabbit IgG was obtained from Promega (Madison, WI, USA). Human umbilical vein endothelial (HUVEC) cells were stored in liquid nitrogen until use. Methylthiazol tetrazolium (MTT) was obtained from Gold Biotechnology (St. Louis, MO, USA) [33,34]. The M199 medium was purchased from Gibco BRL (Grand Island, NY, USA).

Transformation into Arabidopsis thaliana
Arabidopsis thaliana (Columbia ecotype) were used for infection. The floral-dip liquid medium which included 1% (w/v) B5 (200 ×) medium, 100 g/L sucrose, 2 mg/L 6-BA, 1 M sodium hydroxide, and 200 μL Silwet L-77 was prepared as previously described [34]. Plants' aerial parts were dipped into the medium for 5 min, and then culture dark light for 16-24 h. Then the plants were cultured in light condition. The dry seeds were (T1) were collected in a sample bag.

Protein expression analysis
Arabidopsis thaliana seeds (1 mg) were ground with 40 μL Tris-HCl (50 mM) in 1.5 mL ep tubes. The mixtures that included oil body liquid supernatant and 5 × loading buffer were boiled for 10 min, and they were detected on two 12% polyacrylamide gels under reducing conditions. The polyacrylamide gels were colored overnight using Coomassie blue. In the meantime, the gels were electroblotted onto 0.45-μm polyvinylidene difluoride (PVDF) membranes. Electroblotting was carried out at 100 V for 75 min which contained 2.9 g/L glycine, 5.8 g/L Tris, 0.37 g/L SDS and 20% (v/v) methanol [36]. The PVDF membranes were incubated with a rabbit anti-VEGF polyclonal antibody antiserum (1:1000 dilution) and then washed 4 times with TBST. Then the secondary antibody which is goat anti-rabbit IgG/AP antibody (1:5000) incubated for 120 min at 25°C [36]. The PVDF membranes were colored Alkaline Phosphatase.

Activity assay of VEGF 165
Seeds (20 mg) were ground in 200 μL Tris-Cl (pH 7.5), and then the mixture was centrifuged at 10,000 × g and 4°C for 30 min. The oil body was collected at resuspended in 200 μL Tris-Cl (pH 7.5) [33], and centrifuged again. After centrifugation, the oil body was mixed again with 10 μL Tris-Cl buffer [33]. The biological activity on the proliferation of HUVEC cells was analyzed from transgenic oil body suspension. The HUVEC cells were cultured in M199 medium containing 20% fetal bovine serum (FBS) until they achieved the logarithmic phase. Then, the cells were removed and cultured into a new 96-well plate (8 × 10 3 /well) with 1% FBS for 24 h. The HUVEC cells were incubated with various dilutions of oleosin-VEGF 165 (250, 500, 1000, 2000, 4000, and 8000-times dilutions) or wildtype oil bodies (negative control, diluted 250, 500, 1000, 2000, 4000, and 8000 times) for 48 h. To determine the activity of the cells, 20 μL MTT was added, the cells were incubated for 4 h and 100 μL DMSO was added to each well [37]. After 10 min, the absorbance was measured at 570/630 nm in a Microplate Reader model 450.

Result Expression Oleosin-VEGF 165 in Arabidopsis thaliana and screening for transgenic lines
We constructed the pKO vector cassette linking to the VEGF 165 gene to express VEGF 165 and VEGF 165 gene was inserted into pKO vector (Figure 1). The pKO-VEGF 165 plasmid was detected using PCR method and transformed successfully into Agrobacterium EHA105 competent cells by the freeze-thaw method. The VEGF 165 gene was anchored on the surface of oil body and it was specifically expressed in A. thaliana seeds [34]. The pKO-VEGF 165 plasmid was transformed into A. thaliana by floral-dip method. The infection process was as shown in Figure 2. The T1 transgenic seeds were sown until the plants grew 6-8 leaves. Afterwards the transformants were chosen by 1% (w/v) glufosinate. The cotyledons of non-transformed plants became bleached, while transgenic seedings grew normally. Positively transformed lines were grown and ultimately homozygous seeds were obtained [36]. Four lines (T3-1, T3-2, T3-3, T3-4) were confirmed as positive transformants and were used for further analysis.

Oleosin-VEGF 165 fusion protein expression analysis
To detect whether VEGF 165 genes was expressed in the transgenic T3 A. thaliana seeds, oil bodies were extracted from T3 transgenic A. thaliana seeds. Four independent transgenic lines was evaluated by extraction of the oil bodies which expressed oleosin-VEGF 165 protein ( Figure 3A and B). The accumulate of oleosin-VEGF 165 was calculated. The fusion protein were analyzed by SDS-PAGE. The SDS-PAGE results showed that the target band of oleosin-VEGF 165 fusion protein was about 36 kDa from T3 transgenic plant T3-1, T3-2, T3-3. The protein from wild type seeds had no band at this position and there was no target band at this position from T3-4 transgenic plant ( Figure 3A). To further confirm the presence of 36 kDa oleosin-VEGF 165 in the oil body, the immunodetection by rabbit anti-VEGF 165 polyclonal antibody was carried out in the experiment. The Oleosin-VEGF 165 fusion protein was identified from T3 transgenic plant T3-1, T3-2, T3-3 by the anti-VEGF 165 polyclonal antibody ( Figure 3B). The hybridization band appeared at 36 kDa from T3 transgenic plant T3-1, T3-2, T3-3 in accordance with the predicted oleosin-VEGF 165 fusion protein. However, there was no band at this position in the analysis of the total proteins from wild-type seeds. The VEGF 165 gene was expressed in A. thaliana and its product accumulated in the oil bodies in the transgenic seeds.

Activity assay of VEGF 165
According to western blot result, transgenic line T3-1, T3-2, T3-3 were chosen to evaluate the bioactivity of the oleosin-VEGF 165 fusion proteins. Because the oil bodies expressed oleosin-VEGF 165 was obtained from T3-1, T3-2, T3-3 seeds, these seeds were used to detect the proliferative activity by the MTT method with HUVEC cells. The oil bodies extracted from Line T3-1, T3-2, T3-3 were analyzed. In Figure 4, the x-axis shows the dilution ratio of the oil bodies and the y-axis shows the OD value (Figure 4). A gradient of VEGF 165 dilution ratio was used and the dilution ratio was set as a gradient from 250 to 8000 times ( Figure 4). The results showed that the oil bodies that expressed oleosin-VEGF 165 fusion protein from the T3-1, T3-2, T3-3 had a dose-dependent proliferative effect on HUVEC cells. The wild-type oil bodies used as the negative control did not obviously show this dose-response relationship with cell proliferation.

Discussion
The plant expression systems can produce active exogenous proteins in many studies [36]. Such systems show great advantages in terms of cost-efficiency, product quality and safety [36]. Oil bodies can also serve as an efficient system for the expression of cellulolytic enzymes [27], because the enzyme is restricted to the cell surface, it is prevented from contacting its substrates (cell wall constituents) in plants [38]. The target protein is expressed in the seed after being linked with the oleosin and oleosin accounts for 2%-10% of the total protein in the seeds. So the expression level of target protein is lower. But the advantage of oil body system is that the target protein need not be purified and the oil body is directly applied to the skin surface. So the oil body expression system represents a perfect system for the production of therapeutic proteins. The oleosin-haFGF protein was expressed in A. thaliana seeds via oleosin fusion technologyand it had biological activity which stimulated NIH/3T3 cell proliferation activity [33]. Human fibroblast growth factor 9 was expressed in A. thaliana oil bodies [34]. Fibroblast growth factor 10 was expressed in Carthamus tinctorius L. seeds [39]. The process of purifying VEGF 165 from E. coli cells was more complicated than purifying the protein from the oil body. VEGF 165 was targeted to the C-terminus of oleosin and the process of purification was easier. The purification process was simple and refolding of proteins was not required [34,40]. Oleosins can act as a natural emulsifying and stabilizing agent at oil/water interfaces; therefore, there are many potential applications [34]. It is important to have an economical, reliable, and up-scalable method to synthesize bioactive oleosin-VEGF 165 to evaluate such applications. The oil body expression system represents a perfect system for the production of therapeutic proteins. This system has the potential to produce safe, biologically active proteins with low purification costs. When the dilution ratio of oil bodies was 250-4000 times, the absorbance of oil bodies harboring VEGF 165 was higher than that of wild-type oil bodies. When the dilution ratio was 8000 times, the absorbance of the transgenic oil bodies began to decline slowly. The oil bodies harboring VEGF 165 promoted the proliferation of HUVEC cells in a dose-dependent manner.