重组牛肠激酶轻链在大肠杆菌中的表达和纯化  

Expression and Purification of Bovine Enterokinase Light Chain in Recombinant Escherichia coli

作　　者：黄磊[1] 阮红[2] 徐志南[1] 顾玮彦[1] 岑沛霖[1] 

机构地区：[1]浙江大学材料与化学工程学院,浙江杭州310027 [2]浙江大学城市学院,浙江杭州310015

出　　处：《江南大学学报（自然科学版）》2007年第4期478-482,共5页Joural of Jiangnan University (Natural Science Edition)　

基　　金：国家自然科学基金项目(30370039)

摘　　要：密码子优化全基因合成了牛肠激酶轻链基因(sBEKLC),采用pET-39b(+)构建了融合表达载体,在大肠杆菌中进行了成功表达.37℃在含30 mg/L卡那霉素的LB培养基中培养,当细胞密度(D600)达到0.5时,降低温度到28℃,加入终浓度为0.1 mM的异丙基硫代-βD-硫代半乳糖苷诱导外源蛋白表达,继续培养6 h后收集菌体,融合蛋白(DsbA-sBEKLC)产量达到151.2mg/L,相当于80.6 mg/L sBEKLC.使用渗透冲击技术从细胞周质中提取sBEKLC,经过亲和层析可从每升发酵液中得到6.8 mg sBEKLC,经检测sBEKLC具有生物活性.A codon optimized sequence coding light chain of bovine enterokinase gene （sBEKLC） was synthesized, and it was fused with DsbA to construct the expression vector （pET39- sBEKLC）. Then, the plasmid was transformed into E. coli BL21 （DE3） for expression. The strain was cultured in LB medium containing 30 μg/mL kanamycin at 37 ℃. When cell density （D 600） reached 0.5, IPTG was added to reach final concentration of 0.1 mM. After continued cultivation at 28 ℃ for 6 hours, cells were harvested, the volumetric productivity of fusion protein reached 151.2 mg/L, i.e. 80.6 mg/L sBEKLC. The cold osmotic shock technique was successfully used to extract sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8 mg of sBEKLC was purified from 1 liter fermentation broth and the enzyme activity of sBEKLC was well detected.

关 键 词：肠激酶 融合表达 纯化 生物活性 

分 类 号：Q819[生物学—生物工程]