重组人脱氧核糖核酸酶Ⅰ的复性及活性鉴定

Refolding and Biological Activity Determination of Recombinant Human DNaseⅠ

建立尿素梯度凝胶过滤复性重组人脱氧核糖核酸酶Ⅰ的方法。将诱导表达的重组人脱氧核糖核酸酶Ⅰ包涵体通过初步纯化后变性,然后在尿素梯度凝胶过滤色谱柱Sephadex G-75中复性,洗脱流速0.4 mL/min,复性完毕后透析除去小分子复性剂,使用琼脂糖电泳法检验其有活性后,再用单向酶扩散法测定其酶活力为655.8 U/mg,复性得率为83.7%。最后通过LC-ESI-MS/MS从氨基酸序列组成上证明复性产物是重组人脱氧核糖核酸酶Ⅰ。结果表明,建立的方法能成功用于复性变性的重组人脱氧核糖核酸酶Ⅰ包涵体蛋白,获得了可用于结构和功能研究的具有生物学活性的重组人脱氧核糖核酸酶Ⅰ。

The on-column refolding of recombinant human DNase I was developed by urea gradient gelchromatography. After purification, recombinant human DNase I inclusion body was renatured by the urea gradient gel chromatography with 0.4 mL/min elution speed. Collect the target and then remove the small molecules by dialysis. Test by agarose electrophoresis and biological activity was 655.8 U/mg by the single-phase enzymes spread determining. The fragments from the target protein were sequenced by LC-ESI-MS/MS. The amino acid sequences of the fragments tested were matching well with the sequence of human DNase I from the database, by which the target protein was determined. These results demonstrated that the developed strategy here can be used successfully to produce biologically active recombinant human DNase I for the investigation of structure and function.