Zhu Wen he, Sun Miao nan, Wang Yong sheng, Sun De Jun, Zhang Shao xuan
Department of Biomedicine, Institute of Frontier Medical Sciences, Jilin University, Changchun, Jilin, China.
Protein Expr Purif. 2011 Apr;76(2):184-9. doi: 10.1016/j.pep.2010.12.001. Epub 2010 Dec 7.
A recombinant targeted toxin (Disintegrin-Conj-Mel) was developed that contained a disintegrin connected to cytotoxic melittin by a urokinase plasminogen activator (uPA)-cleavable linker. This recombinant targeted toxin was designed to target tumor cells expressing integrin αvβ3. The fusion gene was expressed under the control of the promoter AOX1 in Pichia pastoris. Electrophoresis by SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain, demonstrated that an approximately 13 kDa fusion protein was secreted into the culture medium. The molecular weight was that calculated from the predicted amino acid sequence. After optimizing the growth and expression conditions of the transformant strain, about 160 mg/L of the recombinant protein was achieved. The recombinant protein was purified to more than 95% purity by SP Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The hemolysis bioactivity test revealed that the fusion had no hemolytic activity or cytotoxicity against uPA non-expressing 293 cells, but exerted dose-dependent inhibition on uPA-expressing A549 cell proliferation.
开发了一种重组靶向毒素（Disintegrin-Conj-Mel），其包含通过尿激酶纤溶酶原激活剂（uPA）可裂解连接子与细胞毒性蜂毒素相连的整合素。这种重组靶向毒素旨在靶向表达整合素αvβ3的肿瘤细胞。融合基因在毕赤酵母中AOX1启动子的控制下表达。对甲醇诱导表达菌株的培养液进行SDS-PAGE电泳和蛋白质印迹分析，结果表明约13 kDa的融合蛋白分泌到了培养基中。该分子量与根据预测氨基酸序列计算得出的分子量一致。优化转化菌株的生长和表达条件后，重组蛋白产量达到约160 mg/L。通过SP Sepharose离子交换色谱和Sephadex G-75凝胶过滤色谱将重组蛋白纯化至纯度超过95%。溶血生物活性测试表明，该融合蛋白对不表达uPA的293细胞无溶血活性或细胞毒性，但对表达uPA的A549细胞增殖具有剂量依赖性抑制作用。
