重组融合蛋白Trx-IFN-CSP的肠激酶酶切及纯化  

Recombinant fusion protein Trx-IFN-CSP was digested by enterokinase and purification of IFN-CSP

作　　者：卢雪梅[1,2] 黄演婷[1,2] 金小宝[1,2] 朱家勇[1,2] 

机构地区：[1]广东药学院基础学院药用生物活性物质研究所,广东广州510006 [2]广东省生物活性药物研究重点实验室,广东广州510006

出　　处：《生物技术》2015年第5期491-494,共4页Biotechnology

基　　金：国家科技部新药创制重大专项项目("CSP I-plus修饰的肝靶向IFN抗HBV的成药性研究";No.2013ZX09103003-003)资助~~

摘　　要：[目的]重组融合蛋白Trx-IFN-CSP的肠激酶酶切及纯化研究。[方法]选用国产肠激酶,在不同反应条件下酶切重组融合蛋白,15%的SDS-PAGE检测切割产物,并利用Ni2+亲和层析色谱联合肝素亲和层析色谱对酶切产物进行分离纯化。[结果]0.8 U的肠激酶在温度为10℃下酶切72 h能够完全裂解50μg融合蛋白。酶切产物经亲和层析色谱纯化,可获得电泳纯达99%的目的蛋白。[结论]重组融合蛋白Trx-IFN-CSP经肠激酶酶切及纯化后,可获得纯度达99%的肝靶向干扰素IFN-CSP单体,为进一步研究肝靶向干扰素的生物学活性及产业化开发奠定了基础,也为融合蛋白后处理工艺提供了技术借鉴。[ Objective ] To investigate the enterokinase digestion of fusion protein Trx -IFN -CSP and the purification of IFN - CSP. [ Methods] Recombinant fusion protein Trx - IFN - CSP was digested by domestic enterokinase under different reaction conditions. The enterokinase digestion was analyzed by 15% SDS- PAGE. The enzyme digestion products were separated and purified by Ni2＋ affinity chromatography and heparin affinity chromatography. [ Results ] The results showed that 50 μg fusion protein was digested by 0.8 U enterokinase for 10 h at 10℃. The target protein IFN - CSP （ electrophoresis purity up to 99% ） was obtained by purification with Ni2 ＋ affinity chromatography and heparin affinity chromatography. [ Conclusion ] The novel liver - targeting interferon IFN - CSP was obtained by enterokinase digestion and affinity chromatography purification. These re- suits may provide foundation for further study of its biological activity and industrial development. It also provides a reference of technology for the fusion protein postprocessing.

关 键 词：肝靶向干扰素 融合蛋白 肠激酶 酶切条件 亲和纯化 

分 类 号：Q816[生物学—生物工程]