Xu Cui-Xiang, Hu Zhi-Ming, Li Jing-Long, Gao Ji-Min
Institute of Biological Treatment, School of Biotechnology, Southern Medical University, Guangzhou, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2009 Mar;29(3):412-5.
To study the purification, refolding and bioactivity of streptavidin-tagged human tumor necrosis factor-alpha (SA-TNF-alpha) bi-functional fusion protein.
SA-TNF-alpha fusion protein was expressed in BL21(DE3) host bacteria, purified using Ni-NTA affinity chromatography and refolded by dilution and dialysis followed by identification using Western blotting. The effect of SA-TNF-alpha fusion protein against L929 cells was evaluated by MTT assay. Flow cytometry was used to analyze the surface modification of biotinylated MB49 tumor cells by SA-TNF-alpha fusion protein.
Recombinant SA- TNF-alpha fusion protein was expressed in BL21(DE3) at about 30% of total bacterial protein, with a purity of about 95% after purification. The SA-TNF-alpha fusion protein existed as dimmers, tetramers and higher order structures after refolding. The fusion protein exhibited a bi-functionality by inhibiting L929 cells and SA-mediated high-affinity binding to biotinylated cell surfaces, with an anchor modification rate of above 90%.
The dimmers, tetramers and higher order structures of the obtained SA-TNF-alpha fusion protein all exhibit a bi-functionality, and may serve as a potential candidate therapeutic agent for tumors.
研究链霉亲和素标记的人肿瘤坏死因子-α（SA-TNF-α）双功能融合蛋白的纯化、复性及生物活性。
SA-TNF-α融合蛋白在BL21(DE3)宿主菌中表达，采用镍-氮三乙酸亲和层析法纯化，经稀释和透析复性，然后用蛋白质印迹法进行鉴定。采用MTT法评估SA-TNF-α融合蛋白对L929细胞的作用。利用流式细胞术分析SA-TNF-α融合蛋白对生物素化MB49肿瘤细胞的表面修饰。
重组SA-TNF-α融合蛋白在BL21(DE3)中表达量约占细菌总蛋白的30%，纯化后纯度约为95%。复性后的SA-TNF-α融合蛋白以二聚体、四聚体及更高阶结构形式存在。该融合蛋白通过抑制L929细胞以及SA介导的与生物素化细胞表面的高亲和力结合表现出双功能性，锚定修饰率高于90%。
所获得的SA-TNF-α融合蛋白的二聚体、四聚体及更高阶结构均表现出双功能性，可能成为一种潜在的肿瘤治疗候选药物。
