极端嗜热菌Pyrococcus furiosus热休克蛋白HSP60的克隆表达和纯化  

Cloning Expression and Purification of Heat Shock Protein HSP60 from Hyperthermophilic Archaeon Pyrococcus furiosus

作　　者：陈华友[1,2] 张志燕[1] 吴岩[1] 张毅[2] 

机构地区：[1]江苏大学生命科学研究院,江苏镇江212013 [2]中国科学院上海生命科学研究院,上海200233

出　　处：《安徽农业科学》2009年第4期1483-1486,1501,共5页Journal of Anhui Agricultural Sciences

基　　金：国家基础研究973计划(2004CB719606);江苏大学校基金资助项目(08jdg011)资助

摘　　要：[目的]研究极端嗜热菌Pyrococcus furiosus的热休克蛋白HSP60(Pfu-HSP60)的克隆表达与纯化方法。[方法]应用加拿大HSP60数据库及其BLAST和FASTA序列比较工具对Pfu-HSP60基因序列进行同源性分析,对HSP60基因进行了体外扩增并在大肠杆菌中成功克隆与表达,还进行了Pfu-HSP60蛋白的纯化。[结果]以Taq酶为DNA聚合酶,用PCR方法扩增出预期的Pfu-HSP60基因,插入质粒pET-21a,转化入大肠杆菌受体菌BL21-Codonplus(DE)3-RIL,0.1mmol/LIPTG诱导2 h,得到可溶性高表达,经超声、离心、上清85℃加热、再离心得到初步纯化的Pfu-HSP60蛋白,再经DEAE Sepharose Fast Flow阴离子交换层析纯化,收集峰超滤浓缩后经SephacrylS-200凝胶过滤柱,收集Pfu-HSP60蛋白峰,结果获得纯度为94.5%以上的重组Pfu-HSP60蛋白,总得率为28.0%。[结论]该研究为Pfu-HSP60蛋白的结构和功能及其他热休克蛋白相互作用机制等的研究奠定基础。[ Objective ] The aim was to study the cloning, expression and purification method of heat shock protein HSP60 from Hyperthermophilic Archaeon Pyrococcus furiosus. [ Method ] The homology analysis of Pfu-HSP60 gene sequence was taken by using Canada HSP60 database and sequence comparative tools of BLAST and FASTA. HSP60 gene was amplified in vitro and cloned and expressed in Escherichia coll. Pfu-HSP60 protein was also purified. [ Result] With the Taq enzyme as DNA polymerase, the anticipative Pfu-HSP60 gene was amplified by PCR method, then it was inserted in plasmid pET-21a and transformed into E. coli BL21 -Codonplus（ DE）3-RIL. The soluble high expressive protein was obtained after induced by 0.1 mmol/L IPTG for 2 h. After ultrasonic and centrifugation, the supernate was heated at 85 ℃ and centrifuged again, then the primary purified Pfu-HSP60 protein was obtained and then it was purified by DEAE Sepharose Fast Flow anion exchange chromatography. After the collected peak was filtered and concentrated, the Pfu-HSP60 protein peak was collected through Sephacryl S-200 gel filtration column. The purity of the recombinant protein Pfu-HSP60 was 94.5% and the total yield was 28.0%. [ Conclusion] The research laid the foundation for the study of the structure and function of Pfu-HSP60 protein and the study of the interaction mechanism with other HSP.

关 键 词：热休克蛋白 PYROCOCCUS furiosus 纯化 极端嗜热菌 

分 类 号：S188[农业科学—农业基础科学]