埃兹蛋白(Ezrin)及其突变体的原核表达与纯化

Thr567位点磷酸化是ezrin活化的必需条件。利用定点突变引物将T567突变为A或D,通过PCR扩增出相应的基因片段,将其通过T4连接酶连接至含His标签的原核表达载体pET-20b(+),构建了原核重组质粒pET-20b(+)-Ez WT,pET-20b(+)-EzT567A和pET-20b(+)-EzT567D。转化表达宿主大肠杆菌Rosseta后,用异丙基-β-硫代半乳糖诱导重组蛋白的表达。重组蛋白经亲和镍柱纯化以后,应用western blot鉴定纯化的融合蛋白。Ezrin野生型及其组成型激活和显性负突变质粒的成功构建及其目的蛋白His-Ez WT,His-EzT576A和His-EzT576D的成功表达纯化,为更深入地研究ezrin生物学功能奠定基础。

Prokaryotic Expression and Purification of Ezrin and Its Mutants

The phosphorylation of threonine 567 is required for the activation of ezrin.T567A mutant and T567D mutant were generated using mutant primers.Genes were amplified by polymerase chain reaction(PCR),and cloned into the expression vector pET-20b(+) containing His tag to construct the recombinant plasmids pET-20b(+)-Ez WT,pET-20b(+)-EzT567A and pET-20b(+)-EzT567D.The recombinant plasmids were transformed into E.coli Rosseta to express the fusion proteins after induction with Isopropyl-β-D-1-thiogalactopyranoside.The recombinant proteins were purified by a nickel affinity column and confirmed by western blot.The construction of ezrin wild type,constitutively active mutant and dominant-negative mutant,and the expression and purification of His-Ez WT,His-EzT576A and His-EzT576D are useful for the further study on ezrin function.