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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。
Based on the successful construction of the recombinant Pichia pastoris strain PicZαA / rmHSA-IFN-α2b / X33 with human serum albumin mutant interferon α2b fusion protein (rmHSA-IFN-α2b), the expression of rmHSA-IFN-α2b Purification process. The engineered bacteria were subjected to high-density fermentation in a 9-L canister. The supernatant of the fermentation broth was collected by centrifugation and the expression level of rmHSA-IFN-α2b was 421 mg / L. The fermentation broth supernatant, followed by 3-step purification. The first step uses SP sepharose FF to remove most of the pigment and some of the extra proteins. The second step using Blue sepharose FF according to the principle of affinity to remove some impurities. The third step uses Q sepharose FF to remove solvents and other impurities and toxoids. Finally, high purity, active rmHSA-IFN-α2b was obtained. The purity of the final product was more than 95% by HPLC and SDS-PAGE. The specific activity of rmHSA-IFN-α2b was 1.5 × 106 IU / mg. The total yield of purification was 25.3%. Six consecutive batches were purified and the results were stable. The process is simple and stable, easy to enlarge, suitable for large-scale production. The establishment of a simple and efficient purification process laid a solid foundation for the subsequent study of rmHSA-IFN-α2b.