重组羧肽酶原B在大肠杆菌中的可溶性表达及活性羧肽酶B的纯化

Soluble Expression of Recombinant Pro-Carboxypeptidase B in E.coli and Purification of Active Carboxypeptidase B

实现羧肽酶原B表达质粒在大肠杆菌中的可溶性表达,并对表达产物进行激活和纯化,得到活性CPB。采用摇瓶发酵,对诱导温度,诱导时间,诱导剂IPTG的浓度进行优化,促成其可溶性表达;优化酶解激活条件,提高粗酶液比活。粗酶液用DEAE-FF离子交换柱和Octyl-FF疏水柱两步纯化。结果:实现可溶性表达的最佳条件为15℃下,0.1 mmol/L IPTG诱导20 h。此条件下得到的粗酶液经激活后两步纯化,得到比活为128.2 u/mg protein的较纯的活性CPB,活性回收率达39%。

The expression condition of recombinant pCPB was optimized to achieve the soluble expression in E. coli; the recombinant pCPB was enzymatically cleaved and subsequently purified to obtain active carboxypeptidase B. Through adjusting the inducing temperature, inducing time, and inducer concentration, pCPB was expressed in supernatant in E. coli; the special activity of CPB was increased by improving the enzymatic cleavage condition. The rude CPB was purified by DEAE FF chromatography and Octyl-FF chromatography. The highest soluble expression level of recombinant pCPB was obtained after being induced at 15℃ for 20 h and the concentration of IPTG was 0.1 mmol/L. The active CPB was purified subsequently by DEAE-FF and Octyl FF chromatography to obtain the active carboxypepti dase B with special activity about 128. 2 u/mg protein, and total activity recovery was 39%. The soluble expression of recombinant procarboxypeptidase B was achieved in E. coli. We got the active CPB after enzymatical cleavage and purification.