基于磁蛋白标签的黄曲霉毒素氧化酶的分离及应用

Isolation and application of aflatoxin⁃oxidase based on magnetic protein label

建立了以家鸽磁蛋白(clMagR)分离标签为基础的黄曲霉毒素氧化酶(AFO)分离纯化策略及黄曲霉毒素B1(AFB1)清除体系。将具有AFB1降解活性的AFO与clMagR基因共编码,采用pET 28a(+)载体构建了pET 28a(+)AFO clMagR重组质粒,转入E.coli BL21(DE3)宿主异源表达融合蛋白,并优化表达条件。随后,利用磁性纳米颗粒与磁蛋白的物理吸附,进行AFO的快速分离纯化及其对黄曲霉毒素B1的去毒率测定。结果表明:clMagR标签可增加AFO的可溶蛋白表达量;同时,融合clMagR的AFO具有良好的磁感应性,可通过与磁珠的吸附而被高效分离,纯化后的AFO对于AFB1的去毒率为75.94%,可循环使用13次。本研究为AFO的分离纯化及AFB1的生物去毒化研究提供了新策略,也为其他蛋白的分离纯化及应用提供了良好借鉴。

The separation of aflatoxin-oxidase(AFO)based on pigeon magnetic protein(clMagR)label,and the aflatoxin B1(AFB1) scavenging system were established.The gene AFO was co-encoded with the clMagR gene,then inserted into pET-28 a(+)and transformed into E. coli BL21(DE3) for heterologously expression of fusion protein.After optimization of the expression conditions,we explored rapid separation and purification of AFO based on the physical adsorption of magnetic nanoparticles and magnetic proteins.In addition,the purified AFO was applied to the detoxification assay of AFB1.The magnetic protein label could slightly increase the soluble protein expression of AFO.At the same time,the magnetic protein-labeled AFO had good magnetic induction and was efficiently separated by adsorption with magnetic nanoparticles.The detoxification rate of AFB1 was 75.94% using purified AFO,which could be reused for 13 times.We developed a novel strategy for the separation of aflatoxin-oxidase,and applied into biological detoxification of aflatoxin.This strategy provides promising utilization for further protein separation research and applications.