| Cancer is a kind of serious disease that jeopardizes the health and life of human beings.Although anti-tumor pharmaceuticals have been developed in recent years,their use is limited because of their low efficacy and adverse effect of toxicity.Therefore,the development of cytotoxic drugs that can specifically target tumors with minimal toxicity to normal cells continues to be a challenge in cancer therapy.In our laboratory,we have constructed and expressed a new recombinant protein, EGF-E4orf4,based on the targeting of EGF and cytotoxicity of adenovirus E4orf4.In the previous experiments,it was indicated that this fusion protein inhibited human breast adenocarcinoma cell line MDA-MB-231,human Glioblastoma BT325 and human osteosarcoma cell line Saos-2 growth.There are some problems waiting for overcome such as how to increase the fusion protein's expression level in Pichia pastoris yeast and low-purity with existing purification procedure.The present study focused on the expression,purification and function of the fusion protein,EGF-E4orf4.Firstly,the experiments were carried out on increasing the expression level of in Pp yeast and the purity of EGF-E4orf4.Improvement of EGF-E4orf4 expression level was done in bioreactor by fed-batch fermentation.The expression level of EGF-E4orf4 was increased compared with the shaking incubator.The fusion protein was purified by anion-exchange chromatography and gel exclusion chromatography.As EGF-E4orf4 was identified as polymer,we used two methods to reconstruct the fusion proteins:(1) adding 6xHis tag to the C terminal of EGF-E4orf4 to facilitate the purification process.(2) constructing cycling 7 peptides-E4orf4-His protein to reduce the number of disulfide bonds. The two fusion proteins were purified by His-tag affinity chromatography and gel exclusion chromatography.Secondly,important part was to investigate the possible biological functions of the fusion protein EGF-E4orf4 using MDA-MB-231 and BGC823 cells that were characteristics for EGFR expression.The fusion protein stimulated EGFR phosphorylation in a dose- and time-dependent manner by western blotting analysis.Likewise,the results obtained from confocal microscopy confirmed the internalization and showed the differences between EGF-E4orf4 and EGF afterwards,EGF-E4orf4 significantly inhibited the ability of colony formation of BGC823.In vivo,EGF-E4orf4 suppressed tumor growth in a dose-dependent fashion with an inhibition ratio of 79%in MDA-MB-231 and 49%in BGC 823,respectively(P＜0.05).No adverse effects were observed in the nude mice even at the dose of 10mg/kg of EGF-E4orf4.Final part was to create stable cell lines expressing E4orf4 and by MTT colorimetric assays,it was found that inducible expressing E4orf4 could inhibit cell growth. The first batch of microarray was carried out to identify changes in RNA levels resulting from E4orf4 expressing and the further independent experiments are still needed.Our experiment results demonstrate that EGF-E4orf4 could be successfully introduced into the tumor cells by interaction between EGF and EGFR,and the antitumor efficacy of EGF-E4orf4 was evaluated involving clonogenic-cell-growth inhibition and the EGF-E4orf4-treated xenografts in nude mice.The fusion protein,EGF-E4orf4,with its high killing cancer cell potency and special selectivity,could be a promising drug for cancer therapy. |
