重组人Midkine真核表达载体的构建以及蛋白表达和纯化  

Expression and Purification of Mature Human Midkine (hMK) in Pichia pastoris

作　　者：徐杨琪 李京敬[2] 关文[1] 郜尽[2] 贾世香 屠平 韩伟[2] 俞雁[1] 

机构地区：[1]上海交通大学农业与生物学院,上海200240 [2]上海交通大学药学院,上海200240 [3]交晨生物医药技术(上海)有限公司,上海200240

出　　处：《基因组学与应用生物学》2014年第2期228-233,共6页Genomics and Applied Biology

基　　金：国家自然科学基金(No.81173113和No.81273573);上海市科学基金(No.11431921300)共同资助

摘　　要：Midkine(MK)是一种肝素结合型生长因子,在胚胎早期的分化发育、肿瘤、炎症、再生等方面发挥着重要的调控作用。本研究将人MK(hMK)全长基因构建到pPICZa(B)表达载体上,通过化学转染方法,转染质粒至不同酵母菌株中进行筛选,利用毕赤酵母的高密度发酵进行表达,纯化出高纯度的重组人Midkine(rhMK),并通过试验证明,纯化的rhMK具有趋化UMR106细胞的活性。rhMK的真核表达与纯化方法的建立为后续的药效学试验奠定了基础,并对其它蛋白的真核表达、纯化也有一定的参考意义。Midkine（MK） is one of the heparin-binding growth factors that are strongly expressed during embryogenesis. It is vigorously involved in development and regeneration of normal tissues, as well as in inflammation and tumorigenesis. To facilitate further studies of recombinant human MK（rhMK）, we developed a strategy for expressing and purifying rhMK using Pichia pastoris expression system. This strategy is simple and efficient with construction of expression vector, high-density fermentation and one-step of cation exchange chromatography. Furthermore, the biological activity of final product was verified with migration assay on UMR-106 cells. The strategy will facilitate the physiological and pathological research of MK and have reference values for the expression and purification of other proteins.

关 键 词：中期因子 毕赤酵母 发酵 离子交换层析 高效液相色谱 

分 类 号：Q78[生物学—分子生物学]