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

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%.