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Activity and Integrating Expression of HumanEndostatin Produced by Pichia pastoris

机译：毕赤酵母产生的人类内皮抑素的活性及其整合表达

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Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P. pastoris occurs in simple minimal defined media making this system attractive for production of expressed proteins for purification. Endostatin is a potent and specific antiangiogenic protein capable of inhibiting the growth of murine and xenotransplanted human tumors. Thus far, however, recombinant endostatin prepared from Escherichia coli has been insoluble after purification and therefore inappropriate for clinical settings. In this study, human endostatin gene was integrated into the chromosome of host pichia pastoris by using the yeast inserted plasmid pPICZaA-endo including human endostatin gene, native Saccharomyces cerevisiae a-factor secretion signal, zeocin resistant gene and the AOX1 promoter and transcription terminator (TT) of pichia pastoris electroporation. The recombinant clones were then selected by the plates containing antibiocin-zeocin (100ug/ml), and finally one highly expressed clone was selected by PCR, SDS-PAGE and Western-blot. The yield of expressed endostatin from P. pastoris depended critically on growth conditions, and attainment of high cell densities by fermentation had been shown to improve protein yields up to 15mg/L estimated. Moreover, the protein was easily purified by using a heparin-agarose column. A strengthened antiangiogenic activity in vivo has been identified by anovel and simple CAM (chorioallantoic membrane) technique.
机译：巴斯德毕赤酵母中的异源表达具有真核表达，适当折叠和二硫键形成，糖基化和分泌的许多优点。与其他真核系统相反，巴斯德毕赤酵母的蛋白质生产发生在简单的最低限度定义的培养基中，这使得该系统对表达的蛋白质的纯化具有吸引力。内皮抑素是一种有效且特异性的抗血管生成蛋白，能够抑制鼠类和异种移植人类肿瘤的生长。然而，到目前为止，由大肠杆菌制备的重组内皮抑素在纯化后已经不溶，因此不适用于临床环境。在这项研究中，通过使用酵母插入的质粒pPICZaA-endo将人类内皮抑素基因整合到宿主毕赤酵母的染色体中，该质粒包括人内皮抑素基因，天然酿酒酵母a因子分泌信号，zeocin抗性基因以及AOX1启动子和转录终止子（巴斯德毕赤酵母电穿孔的TT）。然后通过含有抗生物素-zeocin（100ug / ml）的平板选择重组克隆，最后通过PCR，SDS-PAGE和Western-blot选择一个高表达克隆。巴斯德毕赤酵母表达的内皮抑素的产量关键取决于生长条件，并且通过发酵获得高细胞密度已显示可将蛋白产量提高至估计的15mg / L。而且，通过使用肝素-琼脂糖柱容易地纯化蛋白质。通过厌食和简单的CAM（脉管尿囊膜）技术已经确定了体内增强的抗血管生成活性。

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