重组猪羧肽酶原B在大肠杆菌中的表达与复性研究  

Cloning and Overexpression of Porcine Procarboxypeptidase B Gene in Escherichia coli and Research on Renaturation of Expression Product

作　　者：张笑岩[1] 张会勇[2] 范豪 李泰明[1] 刘景晶[1] 曹荣月[1] 

机构地区：[1]中国药科大学生命科学与技术学院微基因实验室,江苏南京210009 [2]新乡医学院生命科学技术系,河南新乡453003 [3]浙江一就生物医药有限公司,浙江平湖314200

出　　处：《药物生物技术》2010年第1期39-44,共6页Pharmaceutical Biotechnology

基　　金：国家自然科学基金资助项目(No.30672464;30872393);江苏省自然科学基金资助项目(No.BK2007170)

摘　　要：利用大肠杆菌表达系统,表达猪源羧肽酶原B蛋白包涵体,并对包涵体的体外复性条件进行优化。对猪源羧肽酶原B基因进行设计,优化稀有密码子,并将基因片段插入到pET28a载体中,构建表达质粒pET28a-proCPB,并转化入BL21(DE3)中。通过乳糖诱导表达得到包涵体。通过检测酶的比活力来考查复性液的pH、蛋白终浓度、氧化还原对比例、复性时间、复性温度对复性程度的影响。最终确定的体外稀释复性条件为:复性液成份为100mmol/L Gly-NaOH、pH=9.5、GSH与GSSG的比例为1∶0.8(GSH为1mmol/L)、蛋白终浓度为200μg/ml、复性温度为25℃、复性时间24h。通过检测,复性液中酶的比活力为11.7u/mg,经过DEAE纯化后酶的比活力可达到73u/mg。To express the inclusion body of procarboxypeptidase B in E. coli expression system and to optimize conditions of renaturation of the inclusion body, synthetic porcine-derived procarboxypeptidase B gene,of which the rare codon was substituted, was inserted into pET28a and then transformed into BL21（DE3）. Inclusion body was obtained in the inducement of lactose. Renaturation conditions, including pH value, temperature, time, protein concentration, and the ratio of GSH and GSSG were optimized. After optimizing,the finalized conditions of renaturation are. 100 mmol/L Gly-NaOH pH value 9.5, GSH ： GSSG=1 ： 0. 8, 25℃, renaturation time is 24h, the protein concentration is 200μg/ml. The specific activity was 11.7 u/mg after the dilution method and the specific activity achieved 73 u/mg after purification with DEAE.

关 键 词：重组羧肽酶B 大肠杆菌 稀释复性 

分 类 号：Q55[生物学—生物化学] Q786