绿盲蝽膜结合型海藻糖酶ALTre-2基因表达、纯化及酶学特性

Expression, Purification and Enzymatic Activity of a Membrane-bound Trehalase Gene from Apolygus lucorum

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1.1 mg·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60 kD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。

Using a previously obtained cDNA sequence encoding a membrane-bound trehalase from Apolygus lucorum, a pET28a vector containing ALTre-2 gene was constructed and transformed into BL21 competent of E.coli. The target recombinant protein was overexpressed, and purified using Ni-NTA （Nickel-nitrilotriacetic acid） agarose, and then its activity and enzymatic characteristics were studied. The results showed that the ALTre-2 gene could be overexpressed in E.coli, and the target recombinant protein had a higher trehalase activity （84.747±3.299 nmol· μg^-1·min^-1） when using trehalose as substrates; the most suitable reaction temperature was 50 ℃, and the ideal pH was 7.0. Our results facilitate further molecular research on the membrane-bound trehalase from Apolygus lucorum.