Purification And Function Study Of The Anti-tumor Agent-Arresten Protein

ObjectiveTo explore the purification method of Arresten protein, in the same time to investigate the biological acticity of Arresten protein as the anti-tumor agent.Methods1. Expression of angiogenesis inhibitor Arresten in prokaryotic systemAccording to the mRNA sequence of Arresten gene from the GenBank, specific primers including the 6×His-tag (6 consecutive histidine residues) sequence in C-terminal for PCR were designed. Human Arresten gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from placenta tissue of a healthy puerpera, and cloned into the pMD19-T Simple Vector. The sequence of Arresten gene was analyzed. Digested with restriction endonuclease EcoR I and Sal I, inserted into plasmid pBV221. The constructed recombinant Arresten was transformed into E. coli JM109 and expressed primarily. The Western blotting method was used to examine whether the Arresten protein was expressed.2. Purification of angiogenesis inhibitor Arresten proteinPurification of Arresten protein was carried out with Ni2+-NTA agrose as there is a 6×His-tag at the C-terminal of the recombinant protein by immobilized metal affinity chromatography (IMAC). The His-Tag sequence binds to divalent cations (Ni2+) immobilized on NTA-based His-Bind resins. After unbound proteins are washed away, the target protein is recovered by elution with either imidazole or slight reduction in pH. Some factors influencing on the purification were discussed. This versatile system enables proteins to be purified under gentle conditions. The purification steps are wash the gel, binding of sample, wash the gel and elution sample. The purity and concentration were measured by 12% SDS-PAGE and Bradford method.3. The pharmacodynamics of Arresten protein(1) Arresten protein purified was incubated with HUVEC and HeLa cells in vitro:The inhibitory effect of Arresten protein on growth and adhesion of HUVEC and HeLa cells was examined by methyl thiazolyl tetrazolium (MTT) assay. The apoptosis of HUVEC and HeLa cell was monitored by FCM. The migration of the cultured cells was examined by Boyden Chamber.(2) Chicken chorioallantoic membrane (CAM) angiogenesis model was used to identify the influence of Arresten protein on neovascularization.(3) To set up colorectal cancer model with C57BL/6 inbreeding line mice. Recombination Arresten protein was used to treat C57BL/6 mice bearing subcutaneously implanted primary SL4 fibrosarcomas.20 mice were divided into negative control group and Arresten protein group. Arresten protein at the dose of 3.2mg/kg was subcutaneously injected once per 2 days in the tumor of mice, whereas subcutaneous tumors were growing in the midline dorsum of each mouse. The mice of negative control group were injected physiological saline 0.1ml once per 2 days under the same conditions. To observe the activity, eating behavior and tumor growth of the mice. The two groups of mice were sacrificed after 10 day treated, then, tumor were taken off. The tumor volume and tumor weight were measured, then the inhibitory rate have been calculated. The microvessel densities of the tumors were measured by immunohistochemical staining with anti-CD31 monoclonal antibody.Results1. The sequence analysis and restriction endonuclease identification indicated that the Arresten gene was inserted into the expression vector ssuccessfully. SDS-PAGE analysis indicated that expressed product was about 26000 and its amount was about 30% of total bacterial proteins. Western blotting indicated that the protein could react with His-tag antibody.2. The unbound proteins were washed away effectively with wash buffer including 15mM imidazole, and the target protein is recovered by elution in elution buffer pH value as 5.1. Under the condition, the expressed protein was one-step purified to 97% using Ni2+-NTA affinity chromatography method, and with a yield of 172.4μg/mL. The renaturation of Arresten protein was finished successfully by means of gradient dialysis.3. Arresten protein (0.8μg/ml) inhibits the proliferation and migration of HUVEC and promotes its apoptosis effectively; and the inhibition was dose-dependent. However, it had no significant effects on proliferation and apoptosis of HeLa cells. The migration of HeLa cells was also inhibited by Arresten protein, but increasing the protein dose, the inhibition was at a same level. Arresten protein could markedly decrease the adhesion rates of HUVEC and HeLa cells, and their adhesion inhibition rate reached to 42.7%. 4. Compared with PBS group, the vessels of Chicken chorioallantoic membrane (CAM) in Arresten protein (3.2μg/ml) treated groups were obviously damaged. So Arresten protein call significantly inhibit angiogenesis of CAM.5. The Arresten-treated group mice showed no weight loss or unusual behavior during the course of treatment and the growth of tumor was significantly slow; but the negative control group mice showed the expression that was angular reach cachexia to wait and tumors grew rapidly. Arresten protein evoked obvious antitumor effects when it was given 5 days. After treatment, compared with the control group (3461.20±927.53mm3, n=9), the average tumor volume of the Arresten-treated group mice was reduced in size (765.19±249.41mm3, n=10, P<0.01) with a relative inhibition rate of 80.81%. While the tumor weight also decreased (0.88±0.25g, n=10, P<0.01) in comparison with the control group (2.16±0.37g, n=9). To evaluate the suppressive effect of Arresten on tumor angiogenesis, tumor tissue sections were immunohistochemically stained with an endothelial specific antibody factor and microvessels in tumor tissues were randomly counted. A dramatically decreased microvessel density in tumor tissues was revealed in the Arresten-treated mice compared with control tumor tissues treated with physiological saline.ConclusionThrough of the gene engineering, the 6×His-tag (6 consecutive histidine residues) sequence was introduced in C-terminal of Arresten gene. The prokaryotic expression recombinant pBV221-Arresten was construct successfully and fusion protein was expressed in E. coli JM109. The optimal conditions were investigated, and the best conditions for separation and purification were established. Study on biological acticity shows that the Arresten protein can inhibit efficiently angiogensis and the growth and metastasis of tumor. Arresten protein is a powerful angiogenic inhibitor and is believed to have therapeutic potential in the treatment of solid tumors. The research may provide a theoretical basis for developing a new antitumor drug.