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

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.

Abstract: 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 Escherichia coli BL21(DE)3,but 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 overexpressing Pfu-sHSP were disrupted by ultrasonication,and the sediment was re-suspended and wa...