重组人胰岛素的制备—下游纯化工艺的研究

Preparation of Recombinant Human Insulin—Study of Downstream Process

建立了一套通过大肠杆菌表达 (His) 6 - Arg- Arg-人胰岛素原 [(His) 6 - Arg- Arg- human proinsulin,RRh PI]制备重组人胰岛素的工艺。将大肠杆菌中以包涵体形式表达的 RRh PI依次经过 DEAE-琼脂糖快流速阴离子交换层析 ,Sephadex G- 2 5层析 ,重组复性酶切转化和 Superdex75分子筛 ,制备重组人胰岛素。 SDS- PAGE、HPL C、氨基酸组成分析和小鼠惊厥实验结果证明 ,制备的重组人胰岛素具有天然生物活性 ,而且纯度较高。

This study was intended to establish a method of preparation of recombinant human insulin,with (His) 6-Arg-Arg-human proinsulin(RRhPI) expressed by Escherichia coli. After DEAE-Sepharose Fast Flow ion-exchange chromatography, Sephadex G-25 chromatography and refolding, enzyme cleavage and Superdex 75 size exclusion chromatography,the RRhPI expressed by Escherichia coli in inclusion body form was converted to human insulin. The obtained recombinant human insulin was analyzed by SDS-PAGE, HPLC, amino acid composition analysis and bioidentity test (mouse convulsion test). The results indicate that our obtained preparation is highly pureified, active recombinant human insulin.