背景:重组融合蛋白是根据凝血酶识别顺序将葡激酶与水蛭素串联而成的蛋白,具有双重功能,分子质量为23 ku,可在工程菌高密度发酵状态下获得高效表达.目的:通过对工程菌进行高密度发酵,构建重组葡激酶-水蛭素融合蛋白纯化工艺,并探讨其构建可行性及表达价值.方法:对工程菌进行高密度培养,并诱导表达重组葡激酶-水蛭素融合蛋白,对菌体进行离心收取,多次冻融后通过超滤、两部阴离子交换层析方法收集上清液,对重组葡激酶-水蛭素融合蛋白进行分离纯化,观察其纤溶比活性及在菌体中的高效表达价值.结果与结论:发酵培养工程菌,并在第17小时时予以诱导.结果发现,诱导0.5 h时,便能观察到重组葡激酶-水蛭素融合蛋白表达,且发酵时间越长重组葡激酶-水蛭素融合蛋白表达量相应越大;至发酵20 h时,表达达到顶峰.菌体干质量为32.20 g/L,目的蛋白表达量约为1.48 g/L.经过纯化处理后,重组葡激酶-水蛭素融合蛋白纯度高达98%,纤溶比活性约为2.6×104 IU/mg,回收活性的概率为56%.实验通过构建重组葡激酶-水蛭素融合蛋白纯化工艺,并分析其表达价值,提示该纯化工艺较为便捷,耗时较短,且能重复使用,可用于大规模生产.%BACKGROUND:Recombinant fusion protein is cascaded by staphylokinase and hirudin according to the thrombin cognition sequence, and has double functions and a molecular weight of 23 ku. The recombinant fusion protein can be highly expressed in the engineering bacteria at high-density fermentation. OBJECTIVE:To construct and purify recombinant staphylokinase-hirudin fusion protein in the engineering bacteria after high-density fermentation, and to explore the feasibility of construction and the expression value. METHODS:The engineering bacteria were cultured at high density and staphylokinase-hirudin fusion protein was induced to express. The bacteria were centrifuged and ultrafiltrated after repeated freezing and thawing. The supernatant was colected with ion exchange chromatography method. The staphylokinase-hirudin fusion protein was isolated and purified, then the fibrinolytic activity and expression in bacteria were observed. RESULTS AND CONCLUSION: The engineering bacteria were cultured and the fusion protein was induced at 17 hours. The results showed that, staphylokinase-hirudin fusion protein expression was detected at 0.5 hours after induction, and the expression levels were increased as the fermentation time; at 20 hours, the expression level reached the peak. The dried weight of the bacteria was 32.20 g/L and the expression level of target proteins was 1.48 g/L. After purification, the purity of recombinant staphylokinase-hirudin fusion protein was as high as 98%, fibrinolytic activity was about 2.6×104 IU/mg, the probability of activity recovery was 56%. The purification process of recombinant staphylokinase-hirudin fusion protein is convenient, less time, repeatable and alows large-scale production.
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