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获得具有催化活性的人巨噬细胞弹性蛋白酶的催化区(hMECD),并建立有效的高通量筛选方法筛选其仰制剂.方法:在大肠杆菌中表达hMECD,并根据酶活性以比色法建立高通量筛选模型.对一批共8560个纯化合物和混合物进行了高通量筛选.结果:构建了有效的大肠杆菌表达系统.存在于包涵体中的表达蛋白在体外重折叠复性,经阴离子交 换柱层析的方法纯化,1L大肠杆菌培养物可得到23mg纯化的活性蛋白.该蛋白的重折叠复性和酶活性需要钙锌离子,但高浓度的锌离子则抑制其重折叠复性和活性.hMECD剪切合成底物包括一个硫酯和几个荧光发生的肽类底物,并在pH8.0显示最强的活性.对8560个化合物和混合物的高通量筛选发现了27个纯化合物和14个天然产物在20mg/L的浓度下具有大于80％的抑制活性.结论:建立了有效的人巨噬细胞弹性蛋白酶的催化区表达和纯化的方法.该重组蛋白抑制剂的高通量筛选模型具有有效、可信和快速的特点.
(HMECD) of human macrophage elastase with catalytic activity was obtained and an effective high-throughput screening method was established to screen its preparation.Methods: hMECD was expressed in E.coli and was established by colorimetry High-throughput screening model.A total of 8560 pure compounds and mixtures were screened by high throughput.RESULTS: An effective E.coli expression system was constructed.Expression proteins present in inclusion bodies were refolded in vitro, Purification by anion exchange column chromatography yielded 23 mg of purified active protein in 1 L of E. coli cultures, requiring both calcium and zinc ions for refolding and enzymatic activity, whereas high concentrations of zinc inhibited its refolding And activity The hMECD cleavage synthetic substrate includes a thioester and several fluorescently occurring peptide substrates and shows the strongest activity at pH 8.0 High-throughput screening of 8560 compounds and mixtures found 27 Pure compounds and 14 natural products had greater than 80% inhibitory activity at a concentration of 20 mg / L.CONCLUSION: An efficient method for the expression and purification of the catalytic domain of human macrophage elastase has been established. The high-throughput screening model of the formulation is effective, reliable and fast.