用金属螯合柱一步纯化和复性重组人血管生长抑制因子(rhB)

Single-step Purification and Refolding of Recombinant Human Angiogensis Inhibitor(rhB)

rhB基因表达产物在大肠杆菌中以不溶性包涵体形式存在 .利用其产物N端携带 6个连接的组氨酸与金属螯合层析介质中的镍离子的亲和性 ,应用Ni -NTA金属螯合层析在蛋白质变性条件下进行纯化 ,并对纯化样品的复性进行了研究 ,分析和比较了不同复性方法和复性条件对其复性率的影响 .实验结果表明 ,在变性条件下经纯化的样品在Ni柱上不经洗脱 ,用 8.0mol/L～ 1.0mol/L尿素梯度洗涤可使样品在柱上直接复性 .用该法除了可有效地提高复性率外 ,还可使整个纯化过程变得简单、快速和高效 .一步层析即可得到高纯度的且具有生物活性的rhB .

The gene expression product rhB existed in E.Coli in inclusion bodies. The recombinant protein was yurified under denaturing conditions with affinity of 6 histidine residues at its N-terminal to metal chelate chromographic matrixes. The experimential results showed that the purified recombinant protein under denaturing conditions was immediately refolded in Ni-NTA column using 8.0mol/L～1.0mol/L urea at a linear gradient. The method effectuvely improved the protein renature, which is simple,rapid and efficie...