N-豆蔻酰基转移酶在大肠埃希氏菌中的表达、纯化及活性检测

Expression of Recombinant N-myristoyltransferase in E.coli and Characterization of Activity

为表达具有活性的N-豆蔻酰基转移酶(N-myristoyltransferase,NMT),用于口蹄疫病毒样颗粒组装效率的优化,将hNMT1基因克隆至pRSFDuet1表达载体,将其转化至大肠埃希氏菌BL21-CodenPlus(DE3)-RIL中,经IPTG诱导,并对诱导前细菌浓度、诱导剂浓度、诱导温度进行优化。目标蛋白经Ni2+亲和层析纯化后进行免疫印迹验证,荧光法测定其活性。结果显示,成功表达出hNMT1,在OD 600 nm值为1.0、IPTG浓度为0.5 mmol/L、表达温度为16℃时,hNMT1蛋白可溶性表达量最高,镍柱纯化后蛋白与抗His标签单克隆抗体在46 ku处特异性结合,与预测大小一致。确定了hNMT1表达的最适条件并成功制备出具有催化豆蔻酰化活性的hNMT1,为研究豆蔻酰化修饰对小RNA病毒蛋白衣壳装配的影响奠定了基础。

To express the N-myristoyltransferase(NMT)from Escherichia coli for enhancing the assembly efficiency of foot and mouth disease virus-like particles(FMDV VLPs),the hNMT1 gene was cloned into pRSFDuet1 expression vector and the recombinant plasmid was transformed into E.coli BL21 CodenPlus(DE3)-RIL competent cells followed by induction with IPTG and optimization of induction conditions.The purified hNMT1 was identified by Western blot and its activity was determined by fluorescence assay.The results showed that hNMT1 was successfully expressed and the recombinant hNMT1 reached to the maximal soluble production when the parameters set as follows:OD600value was 1.0 mmol/L and 0.5 mmol/L IPTG concentration,incubation at 16℃.The results of Western blot showed that the purified hNMT1 represented high reactogenicity,fluorescence test showed that hNMT1 showed catalytic activity.In this study,the optimal conditions for the expression of hNMT1 and the purified hNMT1 with catalytic activity of myristoylation were defined,which may contribute to further research of the effect of myristoylation modification on assembly of picornavirus protein capsid.