摘要
瑞替普酶(reteplase)是第三代溶血栓药物,其结构中含有9对二硫键,因而重组瑞替普酶在E.coli中表达时极易形成包涵体。研究引入二硫键异构酶Dsb C介导瑞替普酶折叠过程中错配二硫键的异构,从而促进其在E.coli中的可溶表达。首先分别构建了reteplase、Dsb C表达载体p BAD/His A-R及p ACYCDuet-1-Dsb C,并共转化入E.coli BL21菌株,获得了瑞替普酶的Dsb C共表达体系。在此基础上,考察了发酵过程中诱导方式及时间、培养温度、诱导剂浓度对瑞替普酶可溶表达量的影响。在37℃、200 r·min-1的条件下培养到OD600值为0.6左右时,加入0.1 mmol·L-1的IPTG诱导二硫键异构酶Dsb C的表达;在25℃、160 r·min-1的条件下培养1 h后,加入0.5 g·L-1的L-阿拉伯糖诱导目标蛋白瑞替普酶表达,继续培养10 h收获菌体,结果表明近60%的瑞替普酶实现了可溶表达,其产量约为70 mg·L-1发酵液,用平板溶圈法测得纯化后瑞替普酶的活性为2.35×105 IU·mg-1。通过共表达二硫键异构酶Dsb C实现了瑞替普酶在E.coli中的可溶表达,有助于瑞替普酶的临床及折叠机理研究,对其他富含二硫键的重组蛋白质生产具有一定的指导意义。
Reteplase has nine pairs of disulfide bonds which is the main obstacle for correct folding, and over-expressing recombinant reteplase in E. coli always accumulates as inclusion bodies. In order to achieve soluble expression of recombinant reteplase, disulfide bond isomerase DsbC was introduced to E. coli to help reshuffle of non-native disulfide bonds during folding process. Vectors of pBAD/HisA-R and pACYCDuet-I-DsbC were first constructed and co-transformed into E. coli BL21 to obtain reteplase/DsbC co-expression system. The influences of induction time, culture temperature and inducer concentration on soluble expression of reteplase were investigated. Host cells were cultured under conditions of 37℃and 200 r.min^-1, and 0.1 mmol.L^-1 IPTG was added to induce the expression of disulfide bond isomerase DsbC when OD600 reached 0.6. The culture conditions were then changed to 25℃ and 160 r·min^-1 for 1 h, and 0.5 g.L^-1 L-arabinose was added to induce the expression of target reteplase. Cells were harvested after culturing for 10 h. The results indicate that up to 60 percent of reteplase are soluble expressed in E. coli, and the yield is about 70 rng.L-1 fermentation broth with 2.35× 10^5 IU.mg^-1 thrombolytic activity. The soluble expression of recombinant reteplase in E. coil by co-expression with DsbC would contribute to further research in clinical application and folding mechanism, which also provides guidance for the production of other proteins with disulfide bonds.
出处
《高校化学工程学报》
EI
CAS
CSCD
北大核心
2015年第3期600-606,共7页
Journal of Chemical Engineering of Chinese Universities
基金
国家自然科学基金(21036005)
作者简介
禚孝发（1987-），男，山东临沂人，浙江大学硕士生。
通讯联系人：关怡新，E-mail：guanyx@zju．edu．cn
