RNase A及其链亲和素融合蛋白在pET系统中的表达

在大肠杆菌中用pET28a表达载体表达重组RNaseA。变性条件下,利用His-Resin亲和纯化,得到60mg／L电泳纯的RNaseA。纯化的RNaseA复性后,利用含大量RNA分子的碱法抽提质粒为底物,测定重组RNaseA活性,与商品化的RNaseA活性相当。同时在RNaseA活性测定体系中加入4mol／L尿素会使RNA分子切割效率提高10倍左右。在此基础上,成功表达RNaseA与链亲和素(streptavidjn)的融合蛋白,经纯化复性后,该融合蛋白同时具有核酸酶、biotin结合活性,在分子生物学中具有重要的应用价值。

Procaryotic Expression of RNase A by pET28a Expression System in E. coli

The recombinant RNase A was expressed in pET28a after IPTG induction. In denature buffer, the recombinant RNase A was purified by Ni-NTA Resin and obtained 60mg per liter. The denature-purified recombinant RNase A was refolded into active peptide in vitro through formation of four disulfide bonds. The RNA degradation activity of the purified recombinant RNase A on alkalic plasmids extracts including abundant RNA was comparable to commercial RNase A. 4 mol/L of urea can increase the degradation activity by 10 times, which may be resulted from the formation of single-stranded RNA by urea. The recombinant RNase A-streptavidn fusion protein was either expressed and purified. The purified fusion protein has both ribonuclease activity and biotin-binding capability. It was applicable since it had both RNase A and biotin binding characters.