人鼻病毒3C蛋白酶在大肠杆菌中的表达、纯化及活性分析  

Expression,purification and activity analysis of human rhinovirus 3C protease in Escherichia coli

作　　者：周瑜 徐虎 喻婵 翟超[1] ZHOU Yu;XU Hu;YU Chan;ZHAI Chao(Bio Resource Green Transformation Collaborative Innovation Center,College of Life Sciences,Hubei University,Wuhan 430062,China)

机构地区：[1]湖北大学生命科学学院,生物资源绿色转化协同创新中心,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2018年第1期18-23,共6页Journal of Hubei University：Natural Science

基　　金：中国农业科学院油料作物研究所油料脂质化学与营养湖北省重点实验室开放课题

摘　　要：人鼻病毒3C蛋白酶具有高特异性,且在低温条件下具有较高酶活,目前已被商品化.在大肠杆菌中利用麦芽糖结合蛋白(MBP)作为融合标签可促进人鼻病毒3C蛋白酶的可溶性表达.将人鼻病毒3C蛋白酶的DNA编码序列克隆到pRK792载体上,然后导入到大肠杆菌BL21(DE3),进行诱导表达,获得MBP-LEVLFQGP-6x His-人鼻病毒3C蛋白酶融合蛋白,随后通过Ni柱亲和纯化获得目的蛋白.用该产物切割纯化的MBP-LEVLFQGP-6x His-木聚糖酶融合蛋白,可获得木聚糖酶并用底物平板检测其活性.实验结果表明人鼻病毒3C蛋白酶能够在大肠杆菌中表达,并且能特异性地在其底物识别序列进行切割,从而移除MBP标签,获得仅带有6x His的人鼻病毒3C蛋白酶.该酶可以特异性地去除木聚糖酶融合蛋白中的MBP标签,获得有活性的木聚糖酶.利用MBP标签能够促进人鼻病毒3C蛋白酶可溶性表达,且人鼻病毒3C蛋白酶的活性不受影响,有利于一些不稳定的蛋白在低温下的标签去除和纯化.Human rhinovirus3C protease has high specificity to substrate,and it remains high activity under low temperature.This report was aim to heterologous expression of human rhinovirus3C protease in Escherichia coli expression system with MBP tag to improve its solubility.The coding sequence of human rhinovirus3C protease was cloned into vector pRK792and the recombinant plasmid was transformed into Escherichia coli BL21(DE3),followed by inducing with IPTG to gain MBP-LEVLFQGP-6xHis-human rhinovirus3C protease fusion protein.The target protein was purified with Ni column.The purified human rhinovirus3C protease was used to digest MBP-LEVLFQGP-6xHis-xylanase.Xylanase with N terminal6xHis tag was detected its activity.The results show that human rhinovirus3C protease was expressed in Escherichia coli successfully.It recognized and cleaved between MBP tag and6xHis tag,leading to human rhinovirus3C protease with only6xHis tag.Then human rhinovirus3C protease was used to remove MBP tag,obtained active xylanase.In conclusion,the MBP tag increased the soluble expression of human rhinovirus3C protease,and the tag didn’t affect the activity of human rhinovirus3C protease,which was an advantage during purification and removed tag in the low temperature of unstable proteins.

关 键 词：人鼻病毒3C蛋白酶 MBP标签 融合表达 木聚糖酶 

分 类 号：Q786[生物学—分子生物学]