Pyrococcus furiosus小分子热休克蛋白Pfu-sHSP的克隆表达和纯化  

Cloning,Expression and Purification of a Small Heat Shock Protein from Pyrococcus furiosus

作　　者：陈华友[1,2] 谭小力[1] 张志燕[1] 褚仲梅[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：《信阳师范学院学报（自然科学版）》2009年第1期71-73,共3页Journal of Xinyang Normal University(Natural Science Edition)

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

摘　　要：用PCR扩增嗜高温菌Pyrococcus furiosus的小分子热休克蛋白基因后,克隆于质粒pT7473中.该小分子热休克蛋白Pfu-sHSP含有较多的稀有密码子,在大肠杆菌BL21(DE)3几乎无表达,而在带有大肠杆菌稀有密码子AGA(arg)AGG(arg),AUA(ile)和CUA(leu)的BL21—Codonplus(DE)3—RIL中能大量表达.大量表达Pfu-sHSP蛋白的细胞用超声波破碎后,离心沉淀用85℃水浴20 min,再离心,上清液再以Q Sepha-rose Fast Flow阴离子交换和S-200凝胶过滤层析进一步纯化,可以得到90%以上的蛋白.The gene encoding a small heat shock protein of the hyperthermophile Pyrococcus furiosus （Pfu-sHSP） was cloned into plasmid pT7473. The Pfu-sHSP that had a high content of rare codons was hardly expressed in Esche-richia coli BL21 （DE） 3, bat could be overexpressed in Escherichia coli BL21 （DE）3 --Codonplus--RIL , a host containing extra copies of tRNA genes of the rare codon AGA （arg） , AGG （arg）, AUA （ile） and CUA （leu）. Cells over- expressing Pfu-sHSP were disrupted by ultrasonieation, and the sediment was re-suspended and was heated at 85 ℃ for 20 min. After centrifugation, the supematant protein was further purified with anion exchange chromatography Q Sepharose Fast Flow and gel fdtration chromatography S-200 column. As a result, the purity of Pfu-sHSP was about 90%.

关 键 词：小分子热休克蛋白 PYROCOCCUS furiosus 分子伴侣 

分 类 号：Q556[生物学—生物化学]