重组肠激酶融合蛋白纯化工艺的研究

Study on purification of fusion protein enterokinase

目的研究建立重组肠激酶融合蛋白纯化工艺的方法。方法利用大肠杆菌发酵表达肠激酶轻链融合蛋白DsbA-rEKL,超声破碎法破碎菌体,离心后收集包涵体。6 mol盐酸胍、5 mmol EDTA溶解包涵体,在胱氨酸存在的情况下,以脉冲方式复性。经IMAC层析及DEAE层析纯化,复性的融合蛋白做酶切实验。结果融合蛋白复性在5 mmol/L胱氨酸存在下、脉冲加量0.03 mg/ml和复性终蛋白浓度0.3 mg/ml为最佳复性方案。经IMAC层析及DEAE层析纯化,rEKL纯度可达90%以上。每升发酵液经复性及纯化后,可得rEKL 60 mg以上。结论实现了大规模生产rEKL的目的,使以融合蛋白表达rPA等药用蛋白成为现实。

Objective To establish a purification method for recombinant enterokinase fusion protein. E. coli fermentation technology was used to express the light chain fusion protein of enterokinase, DsbA - rEKL , bacteria was broken by ultra- sound and then centrifuge to collect the inclusion bodies, the inclusion bodies were dissolved with 6 mol hydrochloric acid guanidine and 5 mmol EDTA, refolding in the presence of cystine by the pulse mode. Purification was made with IMAC and DEAE chromatography, digestion experiment was used to identify the refolded fusion protein. Results Optimum reaction conditions was 5 mmol/L cystine and 0.03 mg./ml pulse. Purity of rEKL was up to 90% with IMAC and DEAE chromatog- raphy. After refolding and purification, at least 60 mg rEKL was acquired from 1 liter fermentation. Conclusion The large scale production of rEKL makes it come true to use fusion protein technology express medical protein.