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

[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.