豆豉纤溶酶基因在大肠杆菌中的可溶性表达及纯化(英文)

豆豉纤溶酶是从豆豉中发现的新型纤溶酶.从枯草杆菌DC-12中提取总DNA,PCR法扩增pro-DFE基因.将pro-DFE基因插入载体pET-32a,构建表达质粒pET-pro-DFE,转化大肠杆菌BL21(DE3),对重组菌株进行变温诱导表达,重组菌株于37℃培养至OD600为0.6时,加入终浓度为0.4mmol/L的IPTG于24℃进行诱导,SDS-PAGE显示可溶性重组融合蛋白约占重组蛋白的60%.且少量融合重组蛋白发生自切割,形成成熟的豆豉纤溶酶,破菌上清中含有成熟的豆豉溶栓酶,纤溶活性为200IU/mL.重组酶经纯化后比酶活为1280IU/mg.

Soluble Expression and Purification of Douchi Fibrinolytic Enzyme Gene in Escherichia coli

DFE(Douchi fibrinolytic enzyme) is a new fibrinolytic enzyme which was discovered in Douchi. The pro-DFE gene from Bacillus subtiilis DC-12 was cloned and expressed as a soluble protein in Escherichia coli.According to SDS-PAGE, about 60% of the recombinant protein was produced in a soluble form. This solubility was obtained by using a temperature shift strategy comprising of growth at 37 ℃ and a postinduction temperature of 24 ℃ in LB medium and 0.4 mmol/L IPTG as the inductor. The strong fibrinolytic activity was detected in the supernatants and reached 200 IU/mL, which indicates that a few soluble Trxpro-DFE in the supernatants has been cleaved into mature DFE. The recombinant DFE was purified and the specific activity was 1 280 IU/mg.