蓝氏贾第鞭毛虫沉默信息调节因子2重组蛋白复性方法比较

Comparison of methods of refolding by recombinant silent information regulator 2 of Giardia lamblia

目的采用3种不同复性方法获得具有生物活性的重组蛋白蓝氏贾第鞭毛虫(Giardia lamblia,Gl)沉默信息调节因子2(GlSir2),并比较3种方法的复性效率,为研究其活性和生物学功能奠定基础。方法表达并纯化GlSir2的包涵体,分别采用稀释复性、透析复性和柱上复性等3种方法对GlSir2包涵体进行复性,并通过检测复性蛋白的活性,比较3种复性方法的特点。结果蛋白活性回收以柱上复性较高,为1 712Flu,透析复性和稀释复性法分别为758Flu和206Flu。结论 3种复性方法均能获得有活性的融合蛋白GlSir2,其中以柱上复性方法处理的蛋白活性回收值最高。

Objective To compare the refolding efficiency of recombinant silent information regulator 2 （G1Sir2） protein from Giardia larnblia by three different methods of refolding and to determine the most effective way to obtain biological- ly active G1Sir2 protein. Methods Recombinant G1Sir2 was expressed in Escherichia. coZi. The inclusion bodies were collected and dissolved with 8 mol/L urea. The supernatant was used in Ni~＋ affinity purification or directly in on-column refolding. The purified G1Sir2 was refolded by two different methods, dialysis refolding or dilution refolding. G1Sir2 pro- teins refolded by three different methods were analyzed with 12~ SD^PAGE and deacetylase activity was determined with a Histone Deacetylase Assay Kit （Fluorometric） in vitro. The rate of G1Sir2 refolding was measured and rate of re- covery was calculated. Results G1Sir2 protein refolded by on-column-refolding had the highest deacetylase-specifie ac- tivity, followed by the protein refolded by dilution and dialysis. The highest rate of G1Sir2 activity was achieved by oncolumn refolding, followed by dialysis and dilution; the level of activity was 1 712, 758, and 206 Flu, respectively. Conclusion Active GISir2 protein can be obtained by all three methods of refolding though on-column refolding is preferable to the other two.