[1]宋建民,孙爱友,魏东芝.融合型乳酸片球菌素在大肠杆菌中的表达与纯化[J].南京理工大学学报(自然科学版),2011,(02):268-272.
 SONG Jian-min,SUN Ai-you,WEI Dong-zhi.Expression and Purification of Fusion-typed Pediocin PA-1 in Escherichia Coli[J].Journal of Nanjing University of Science and Technology,2011,(02):268-272.
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融合型乳酸片球菌素在大肠杆菌中的表达与纯化
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《南京理工大学学报》(自然科学版)[ISSN:1005-9830/CN:32-1397/N]

卷:
期数:
2011年02期
页码:
268-272
栏目:
出版日期:
2011-04-30

文章信息/Info

Title:
Expression and Purification of Fusion-typed Pediocin PA-1 in Escherichia Coli
作者:
宋建民12孙爱友1魏东芝1
1. 华东理工大学生物反应器工程国家重点实验室,上海200237; 2. 合肥学院生物与环境工程系,安徽合肥230022
Author(s):
SONG Jian-min12SUN Ai-you1WEI Dong-zhi1
1.State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China;2.Department of Biological and EnvironmentalEngineering,Hefei University,Hefei 230022,China
关键词:
乳酸片球菌素 大肠杆菌 融合蛋白 表达 纯化
Keywords:
pediocin PA-1 escherichia coli fusion protein expression purification
分类号:
Q78
摘要:
为在大肠杆菌中高效表达乳酸片球菌素(PED),按照美国国立生物技术信息中心公布的PED基因序列设计合成引物,以乳酸片球菌基因组DNA为模板,用多聚酶链式反应扩增PED结构基因特异片段,构建编码组氨酸标记硫氧还原蛋白融合PED的重组表达质粒pET32c-PED,并转化大肠杆菌BL21(DE3)。在异丙基硫代半乳糖苷诱导后融合蛋白以包涵体形式表达,表达量占菌体总蛋白的28%。经镍亲合层析纯化的融合蛋白用肠激酶裂解,再经超滤纯化,可得78%的收率。融合蛋白没有抗粪肠球菌细菌素活性,被分开的PED恢复了抑菌活性。
Abstract:
To effectively express pediocin PA-1(PED)in Escherichia coli,the primers specific for PED coding sequence is designed and synthesized according to the National Center for Biotechnology Information.The gene fragment of PED is amplified from genomic DNA of pediococcus acidilacticii LH31 by polymeras chain reaction.The plasmid pET32c-PED,encoding PED fused with His-tagged thioredoxin protein,is constructed and introduced into escherichia coli BL21(DE3)strain.The fusion protein is expressed in inclusion bodies counting 28% of total cellular proteins in the strain after induction of isopropyl-β-D-thiogalactopyranoside(IPTG)and purified by nickel-nitrilotriacetic acid(Ni-NTA)metal affinity chromatography.For the recovery of biologically active PED,the purified fusion protein is cleaved by enterokinase and the liberated PED is finally purified by ultrafiltration with a 78% yield.The fusion protein,which does not have bactericidal activity against enterococcus faecalis,is cleaved by enterokinase and the cleaved PED recovers its own bactericidal activity.

参考文献/References:

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金( 20706017) 作者简介:宋建民( 1973 - ) ,男,博士生,讲师,主要研究方向: 发酵工程,E-mail: space116@ hfuu. edu. cn; 通讯作者: 魏东芝( 1963 - ) ,男,博士,教授,主要研究方向: 生物制药工程,E-mail: dzhwei@ ecust. edu. cn。
更新日期/Last Update: 2012-04-30