以甘油为碳源发酵Pichia pastoris组成型表达人血管生成抑制素

Constitutive Expression of Human Angiostatin in Pichia pastoris Using Glycerol as only Carbon Source

摘要:

在Pichia pastoris组成型表达外源蛋白中碳源起着重要的调控作用。分别以葡萄糖、甘油、甲醇和油酸为碳源研究它们对摇瓶发酵GS115（pGAP9K-AS）表达hAS的影响。结果如下：油酸（163mg/L），甘油（83mg/L），葡萄糖 （76mg/L）,甲醇（57mg/L）。 根据以上结果，以甘油为碳源在30L生物反应器中进行GS115（pGAP9K-AS）工程菌的高密度发酵。48h后测得hAS的产量为169mg/L。产物hAS具有免疫活性并能抑制bFGF 诱导的CAM血管生成和实验小鼠黑色素瘤的生长。治疗12d后，抑瘤率达到90%，统计学分析结果表明hAS治疗组和PBS治疗组小鼠的肿瘤体积呈现显著性差异(P<0.01)。

Abstract:

Carbon source plays an important role in the constitutive expression of foreign proteins in Pichia pastoris. In present study, glucose , glycerol , methanol and oil acid, was used respectively as the only carbon source to constitutively express hAS in Pichia pastoris GS115 （pGAP9K-AS）in shaking flask. The result shows that oleic acid is the best（163mg/L）compared with glycerol （83mg/L）,glucose（76mg/L）and methanol （57mg/L）。Since oleic acid is insoluble in water, glycerol was used as the carbon source in the high-density cell culture of GS115 （pGAP9K-AS）in a 30 liter bioreactor and 169 mg/L of angiostatin was obtained after 48h of culture. The expressed angiostatin is immunologically active as shown by Western blotting. The recombinant hAS inhibits bFGF induced CAM angiogenesis and suppresses the growth of B16 melanoma in C57BL/6J mice. The tumor inhibition rate is 90% after 12 days of treatment. Statistics analysis revealed that the tumor volume difference of mice between the hAS group and PBS group is prominent (P<0.01).