肺炎克雷伯菌乙酰基转移酶类毒素蛋白的表达与纯化

Expression and Purification of the Acetyltransferase Toxin Protein in Klebsiella pneumoniae

KacT是肺炎克雷伯菌乙酰基转移酶类毒素-抗毒素系统中的毒素蛋白。KacT对大肠杆菌有生长抑制作用,因而难以在大肠杆菌中对其进行过量表达。KacA是与KacT对应的抗毒素蛋白,可中和KacT的毒性。为获得高纯度的KacT蛋白,本研究首先在大肠杆菌中表达KacAT蛋白复合物,并在KacA蛋白的N端引入六聚组氨酸标签以进行镍亲和层析;在30℃、IPTG浓度为0.2 mmol/L时KacAT蛋白复合物的表达最优。然后使用含有5 mol/L盐酸胍的变性缓冲液来解离KacA与KacT蛋白之间的相互作用。最后,经分管收集和温和梯度复性获得纯度较高的KacT蛋白。结果显示,在大肠杆菌中通过表达纯化复合物蛋白再变性解离的方法,成功分离得到KacT毒素蛋白,建立了KacT分离纯化的完整流程。

KacT is a toxin protein in acetyltransferase toxin-antitoxin system of Klebsiella pneumoniae.Due to the lethal effect of KacT,it cannot be purified by direct expression in Escherichia coli.In order to obtain high-purity KacT in vitro,the expression strain of KacAT complex protein was successfully constructed.The N-terminus of KacA was introduced into the His tag.The expression of KacAT was optimal at 30℃and IPTG concentration of0.2 mmol/L.Finally,the KacT protein was obtained by denaturing dissociation of KacA and KacT interaction on a denaturing buffer column containing 5 mmol/L guanidine hydrochloride and gentle gradient renaturation.In this study,the purified complex protein KacAT was expressed in E.coli,and the KacT protein was successfully isolated on the column.The complete separation and purification method of KacT was established,which laid a foundation for further study of the function of KacT.