Study On Candidate Drug Recombinant Fusion Protein VAS-TRAIL

There are two main strategies for solid tumor treatment, one is the direct killing of tumor cells, the other is the inhibition of tumor tissue blood supply. In our research, a bifunctional fusion protein, VAS-TRAIL, was designed for superior therapeutic efficacy by combining anti-angiogenesis activity with tumor-selective apoptosis activity. The fusion protein was constructed by two anti-tumor drug candidates, tumor necrosis factor related apoptosis ligand (TRAIL) and endogenous vascular growth inhibition factor, vasostatin. Our research mainly focused on the expression and activity assay of VAS-TRAIL fusion protein. First, we exploited the recombinant expression and preparation process of VAS-TRAIL fusion protein. Then the refolding and biological activity of the fusion protein was improved by protein engineering. The oligomerization and apoptotic activity of VAS-TRAIL fusion protein was further identified. The main research contents and conclusions of this work were introduced as follows:1) Cloning and expression of VAS-TRAIL fusion geneE. coli system was used as the host for fusion protein expression. TRAIL and vasostatin gene were fused in different fusion order and named as TRAIL-VAS, and VAS-TRAIL, respectively. We compared the effect of MBP and SUMO fusion tags on the soluble expression of VAS-TRAIL protein. The results showed that only MBP tag can achieve soluble expression of fusion protein in high efficiency. However, degradation of target protein was observed during the removal of MBP tag. Therefore, TRAIL-VAS and VAS-TRAIL fusion proteins were expressed without fusion tags, and in the form of inclusion bodies in E. coli.2) Refolding of fusion proteinThe refolding process TRAIL-VAS and VAS-TRAIL fusion protein was preliminary established by experiment of single factor. The inclusion bodies were solubilized by 8M urea, and then refolded by stepwise dialysis against refolding buffer containing L-arginine, Zn2+, and β-mercaptoethanol.The cytotoxic activity of TRAIL-VAS and VAS-TRAIL fusion protein on pancreatic cancer cell line SW1990, non-small cell lung cancer cell line NCI-H460, and endothelial cell FBHE were examined. TRAIL-VAS fusion protein showed no significant effect on the three cells. VAS-TRAIL showed a dose-dependent cytoxic effect on SW1990 and NCI-H460 cells, but no obvious effect on FBHE cells. Therefore, VAS-TRAIL is better than TRAIL-VAS in fusion order.3) Strategy for linker selection based on inclusion body conformation and activityIn order to improve the refolding yield and activity of VAS-TRAIL fusion protein,3 most common linkers, flexible linker (GGGGS)3, rigid linker (PAPAP)3 and helix-forming linker (EAAAK)3, were inserted between the two domains of VAS-TRAIL fusion protein. In addition, we propose a novel linker strategy for fusion protein, which was based on inclusion body conformation and activity. According to ATR-FTIR assay, inclusion body of fusion protein VRT with rigid linker was proved to contain the highest native-like structure content. Inclusion body of VRT also showed cytotoxic activity on NCI-H460 cells but not on FBHE cells, which indicated that inclusion body of VRT retained part of its activity. Therefore, rigid linker (PAPAP)3 was predicted to be the most suitable linker for VAS-TRAIL fusion protein.4) Efficient refolding of VAS-TRAIL fusion protein by a triple agent solutionMild solubilization of VRT inclusion body was achieved by a triple agent solution which contains 2 M urea,0.4 M arginine, and pH 11.5. Although more protein was solubilized at higher urea concentrations, protein solubilized by triple agent solution with 2 M urea was proved to retain the existing structure, and higher purity. Solubilized inclusion body of VRT was refolded by stepwise dialysis with a recovery at 46%. By contrast, VRT solubilized by 8 M urea only showed a recovery at 0.81%.In addition, the role of L-arginine in the triple agent solution was studied by comparison with alkaline solution without L-arginine. It was found that the addition of arginine could protect the structure of solubilized protein. The refolding of protein solubilized by triple agent solution (46%) was also higher than that of alkaline solution without L-arginine (22%). Therefore, L-arginine plays an important role more than providing alkaline pH.5) Verification of linker selection strategy based on inclusion body conformation and activityThe VAS-TRAIL fusion proteins with different linkers were solubilized by triple agent soluions and then refolded. The refolding recovery of all the VAS-TRAIL proteins with linkers (no less than 40%) was significantly higher than that of VT (23%), fusion protein without linker. VHT, fusion protein with helix forming linker (EAAAK)3 showed highest refolding recovery, while VRT and VFT showed similar refolding recovery. Activity assay showed that VRT had the highest activity as cytotoxic activity on NCI-H460 and FBHE cells. The cytotoxic activity of VRT on NCI-H460 cells was equal to that of sTRAIL, while the cytotoxic activity of VRT on FBHE cells was higher than that of sTRAIL and vasostatin. VRT killed NCI-H460 and FBHE cells by inducing apoptosis. CD spectra assay showed refolded VRT showed β sheets structure, which was expected. The refolded VRT also formed homotrimer.The above results show that the VRT fusion protein has been correctly refolded, and the rigid linker (PAPAP)3 is obviously superior to the other candidates. This conclusion confirms the validity of the proposed linker selection strategy of the fusion protein. Since the strategy could eliminate the time-consuming refolding process, the linker screening flux could be improved for aggregated fusion protein. Therefore, our strategy may be helpful for accelerating the development of novel therapeutic fusion proteins.