重组人血清白蛋白突变体干扰素2b融合蛋白的纯化

Purification of Recombinant Modified Human Serum Albumin Interfon 2b Fusion Protein

在成功构建人血清白蛋白突变体干扰素α2b融合蛋白(rmHSA-IFN-α2b)毕赤酵母工程菌株PicZαA/rmHSA-IFN-α2b/X33的基础上,建立了发酵分泌表达rmHSA-IFN-α2b的纯化工艺。对工程菌进行9 L罐高密度发酵后,离心收集发酵液上清,rmHSA-IFN-α2b的表达量为421 mg/L。对发酵液上清,依次进行3步纯化。第一步采用SP sepharose FF除去大部分色素和部分杂蛋白。第二步采用Blue sepharose FF根据亲和作用原理除去部分杂质。第三步采用Q sepharose FF除去溶剂和其他杂质以及类毒素。最终得到高纯度、有活性的rmHSA-IFN-α2b。通过HPLC检测和SDS-PAGE检测,最终产品的纯度都大于95%;通过活性检测,rmHSA-IFN-α2b的比活为1.5×106IU/mg。纯化总收率为25.3%。连续重复纯化6批,结果稳定。该工艺简单稳定,容易放大,适合规模化生产。简单高效纯化工艺的建立,为rmHSA-IFN-α2b后续研究奠定了坚实的基础。

To explore the purification of recombinant modified human serum Albumin interferon α2b fusion protein（rmHSA-IFN-α2b） secretely expressed by Pichia Pastoris in high density fermentation,the recombinant Pichia pastoris strain PicZA/ rmHSA-IFN-α2b/X33 was grown in 9-litre fermentor and induced with methanol to express rmHSA-IFN-α2b.The supernatant was collected by centrifugation.rmHSA-IFN-α2b in the supernatant was purified by SP SepharoseTM FF,Blue SepharoseTM FF and Q SepharoseTM FF sequence.The existence of rmHSA-IFN-α2b during the processes was determined by SDS-PAGE.The purity of the final product was detected by HPLC and SDS-PAGE.The specific activity of rmHSA-IFN-α2b is determined by Cytopathic effect inhibition assay.The results showed that the expression of rmHSA-IFN-α2b reached 0.421 grams per litre supernatant.The rmHSA-IFN-α2b in the supernatant was purified in three steps.The first step is SP SepharoseTM FF and after this step,majority of yeast pigments and parts of other proteins were removed.The second step is Blue SepharoseTM FF and after this step,parts of yeast protein were removed.The third step is Q SepharoseTM FF,and after this step,parts of yeast protein,solvent and Endotoxin were removed.The purified rmHSA-IFN-α2b retaining its bioactivity was obtained.The final product exceeded 95% purity by HPLC and SDS-PAGE.The specific activity of rmHSA-IFN-α2b was 1.5×106 IU/mg.The total purification recovery rate was 25.3%.The purification route of rmHSA-IFN-α2b secretely expressed by Pichia Pastoris in high density fermentation was successfully constructed.The purification process is simple,well reproducible and suitable for large-scale production of rmHSA-IFN-α2b.