黄粉甲抗冻蛋白AFP84a在大肠杆菌中的高效表达及活性检测

为表达和纯化黄粉甲Tenebrio molitor抗冻蛋白, 采用RT-PCR方法扩增得到黄粉甲抗冻蛋白基因afp84a cDNA, 将其连接到pMAL-p2X质粒上, 构建分泌型融合表达载体pMAL-p2X-afp84a, 并在大肠杆菌Escherichia coli TBI中表达; 进一步利用Amylose柱亲和纯化出该重组蛋白, 后利用细菌抗寒性检测重组蛋白的生物活性。结果显示: 融合蛋白含量占总可溶蛋白的40%。SDS-PAGE分析表明, 用MgSO4处理法与超声波细胞破碎法均可使融合蛋白从细胞中释放; 融合蛋白经Amylose柱亲和纯化, Factor Xa因子酶切, 电泳显示获得的目的蛋白呈单一条带。细菌抗寒性检测表明该重组蛋白具有较高的抗冻活性。黄粉甲抗冻蛋白基因afp84a cDNA的克隆、 原核表达为进一步研究抗冻蛋白的性质和应用提供了有用的实验材料。

Highly efficient expression and activity detection of Tenebrio molitor antifreeze protein AFP84a in Escherichia coli

For the expression and purification of Tenebrio molitor antifreeze proteins, RT-PCR method was adopted to obtain the cDNA of antifreeze protein gene afp84a which was then cloned into prokaryotic plasmid pMAL-p2X and expressed in Escherichia coli TBI strain. The recombination protein was purified through an amylose affinity column and assayed for antifreeze activity by observing bacterial survival rate in the presence of purified antifreeze protein following incubation for various intervals at 0℃. The results showed that the content of fusion protein was 40% of total dissolved proteins. SDS-PAGE analysis indicated that fusion protein could be released from cells treated with MgSO₄ or sonicate. A single band of target protein was acquired after the fusion protein was purified through amylose affinity column and incised by Factor Xa. Furthermore, the antifreeze protein AFP84a could increase the low temperature resistance of bacteria as shown in the biological activity analysis. The cloning and prokaryotic expression of afp84a from T. molitor could provide useful experimental materials for the further study of the nature and function of antifreeze protein AFP84a.