利用his-tag纯化和复性基因工程产物肠激酶

Purification and Refolding Escherichia coli Produced Enterokinase Inclusion Bodies Immobilized by Nickel Chelating Chromatography

构建了融合表达肠激酶轻链（EKLC）的基因工程大肠杆菌，将该菌于37℃在含30mg／L卡那霉素的LB培养基中培养，当细胞密度（OD600）达到0．5～0．6时加入最终浓度为0．4mmol／L的IPTG并降温至26℃进行诱导表达，4h后目的蛋白质的表达量可达菌体总蛋白质的40％以上，但所表达的重组产物大多以包涵体形式存在于菌体中。利用产物N端携带6个组氨酸标签与金属螯合层析介质中镍离子的亲和性，应用镍离子金属螯和层析对样品包涵体进行了纯化和复性研究。实验结果表明，在变性条件下纯化的样品在柱上不经洗脱，直接用复性液洗涤6h，可以使EKLC在得到提纯的同时实现复性，得到了电泳纯度为96％的具有生物活性的EKLC，最高活性为712U。

A genetic strain E. coli BL21 （DE3）, which over- expression enterokinase gene, was constructed. When cell density （OD 600 ） reached at 0.5-0.6 in LB medium which contained 30 mg/L kanamycin, IPTG was feed to the fermentation broth to 0.4 mmol/L and then temperature changed to 26 ℃. After induction for 4 h, the expressed fusion protein concentration was 40% of the total bacteria proteins and in the form of inclusion bodies. The inclusion bodies were further purified under denaturing conditions with affinity of 6 histidine residues at its N-terminal to metal chelate chromographic matrixes. The results showed that the purified recombinant protein under denaturing conditions was refolded in Ni-NTA column by using refolding buffer. The enterokinase activity and purity was 712 U and 96 %, respectively.