应用分子伴侣共表达系统表达pfu基因及酶活性测定

Expression and Enzyme Activity Assay of pfu with Molecular Chaperone

将通过In-fution方法构建的pET32a-pfu质粒与可以促进可溶性表达的HG-PGRO7质粒一起转入大肠杆菌BL21(DE3)共表达,以pET32a-pfu单独在BL21(DE3)中表达作为对照。用热变性和(NH4)2SO4沉淀去除部分杂蛋白,再经Ni-NAT亲和层析柱纯化分离pfu蛋白,SDS-PAGE检测结果表明目的蛋白大小约为90kD,与预计的分子量大小一致。最后对其酶活性测定结果表明分子伴侣能够促进pfu基因表达,并能够提高酶活性。

Co-expressing the plasmid pE332a-pfu which was constructed using In-fution technique with chaperone plasmid HG-PGRO7 at the same time in E. coli. BL2.1 （DE3）. The expression system, which expressed pfu gene alone in E. coll. BL21 （DE3）, was used as control. Most of unnecessary proteins were exenterated by heat treatment and （NH4）2 SO4 precipitation. The purified fusion protein was obtained by the Ni chelating resin affinity chromatography. The SDS-PAGE analysis showed that the molecular mass of the purified fusion protein was about 90 kD, conforming to the expected molecular mass of Pfu protein. The results of enzyme activity assay of pfu demonstrated that molecular chaperone was able to activate pfu gene expression and its enzyme activity.