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棉花学报  2013 

绿盲蝽膜结合型海藻糖酶ALTre-2基因表达、纯化及酶学特性

DOI: 1002-7807(2013)05-0396-07, PP. 396-402

Keywords: 绿盲蝽,膜结合型海藻糖酶,原核表达,酶学特性

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Abstract:

以绿盲蝽为材料,在前期获得绿盲蝽膜结合型海藻糖酶(ALTre-2)基因的基础上,构建了ALTre-2原核表达载体(pET28a-ALTre-2),经IPTG诱导表达、变性、复性和蛋白纯化后,获得具海藻糖酶活力的重组蛋白,最后在以海藻糖为底物及重组蛋白浓度为1.1mg·mL-1的条件下,对纯化得到的重组ALTre-2蛋白进行活性检测,确定了其酶促反应的最佳pH和最适温度。试验结果表明ALTre-2基因在大肠杆菌BL21(DE3)中能够高效表达一个约60kD大小的蛋白,SDS-PAGE显示该蛋白以包涵体形式存在,经变性、复性及蛋白纯化获得了具海藻糖酶活力的重组蛋白;该重组ALTre-2蛋白在pH为7.0时活性最高,同时重组ALTre-2蛋白酶活性最适温度是50℃。研究结果为深入探讨绿盲蝽膜结合型海藻糖酶分子调控机制奠定基础。

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