新型集成干扰素-人血清白蛋白融合蛋白在毕赤酵母中的分泌表达和纯化

将含编码HSA天然信号肽、HSA和新型集成干扰素(NIFN)的融合基因,插入毕赤酵母表达载体pPIC3.5,转染毕赤酵母GS115,用于分泌表达融合蛋白HSA-NIFN的酵母工程菌。诱导表达后,产物经SDS-PAGE、Western blot和MALDI-TOF-MS分析表明该蛋白分子量为86369Da,且能被抗HSA单抗特异性结合;表达的HSA-NIFN经超滤、Blue亲和层析和CM阳离子交换层析纯化后,HSA-NIFN的纯度大于95%。用细胞病变抑制法测定其比活性为7.75±0.39×106IU/mg。

Secretory Expression and Purification of HSA-NIFN Fusion Protein in Pichia pastoris

Abstract HSA-NIFN gene was got by total gene synthesis technology in vitro. The NIFN-HSA gene was inserted into Pichia pastoris express vector pPIC3.5. The recombinant plasmid pPIC3.5/HSA-NIFN was linearized by restriction enzyme Sal I and then transformed into Pichia pastoris GS115 by electroporation. The recombinant strains confirmed by PCR analysis and were induced by methanol to express fusion protein HSA-NIFN. SDS-PAGE , Western blot and MALDI-TOF-MS analysis of the fusion protein showed that the expressed fusion protein HSA-NIFN with 86369Da molecular weight had the antigenicity of HSA.The putity of HSA-NIFN was higher than 90% after Blue Sepharose FF and CM Sepharose FF, and the biological activity determined by WISH-VSV system was more than 7.75±0.39×106 IU/mg.