Expression and Purification of Human Interferon-ω in Yeast Pichia pastoris
- 生命科学－已发表论文 
[中文文摘]ω干扰素(IFN-ω)是同IFN-α功能相近但抗原性不同的一类干扰素分子,它具有同IFN-α、IFN-γ以及肿瘤坏死因子α相似的抑制病毒复制的作用.采用5L发酵罐发酵表达IFN-ω,发酵培养液经超滤膜浓缩、SP层析柱梯度分离,得到糖基化的IFN-ω,用基质辅助激光解析电离飞行时间质谱(MALDI TOF-TOF MS)和N端蛋白序列分析仪分析其分子质量和N端序列,用细胞病变抑制法测定活性.结果表明,诱导约48h时,目的蛋白表达量最高,通过SP梯度洗脱,可将糖基化和非糖基化的IFN-ω分离,质谱分析糖基化蛋白样品,质谱峰为糖基化蛋白特有的峰.纯化后糖基化的IFN-ω抗病毒活性为1.46×108 U/mL. [英文文摘]IFN-wis a kind of interferon molecules,which has similar function but different antigenicity to IFN－α.It can inhibit virus replication like IFN－α,IFN-γ and tumor necrosis factor alpha.5 L fermenter was used to ferment and interferon-ω was expressed.Then,the glycosylated interferon-ω was purified by a procedure stepped by ultrafiltration and gradient elution SP chromatography column.Its molecular weight and N amino acid sequence were analyzed by MALD I TOF-TOF MS and the N protein sequence analyzer,respectively.In addition,IFN-ω activity was determined by cell changes suppression method.The results showed that IFN-ω expression level reached to highest in induced 48h.The glycosylated and unglycosylated IFN－ω could be separated by SP gradient elution completely. The glycosylated IFN-ω showed a series of continuous peaks by mass spectrometry,which is the characteristic peak of glycosyl protein.The antiviral activity of the purified glycosylated IFN-ω was 1.46×108 u/／mL.