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

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.