菌丝霉素MP1106融合蛋白的复性及纯化方法  

Study on Refolding and Purification of Plectasin MP1106

作　　者：王鹏举[1] 谭焕波[1] 苏文成[1] 张文宇[1] 邹培建 WANG Peng-ju TAN Huan-bo SU Wen-cheng ZHANG Wen-yu ZOU Pei-jian(National Engineering Laboratory for Industrial Enzymes, Tianfin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianfin 30030)

机构地区：[1]中国科学院天津工业生物技术研究所工业酶国家工程实验室,天津300308

出　　处：《生物技术通报》2017年第9期94-100,共7页Biotechnology Bulletin

基　　金：天津市科技支撑计划重点项目(14ZC2DSY00057)

摘　　要：旨在建立高效、快捷的菌丝霉素衍生物MP1106大肠杆菌表达系统。通过基因融合的方式构建生物表面活性剂-菌丝霉素衍生物(DAMP_4-MP1106)融合蛋白表达载体,在大肠杆菌中进行表达;并对目的蛋白MP1106进行分离纯化和分子内二硫键鉴定。结果显示,融合蛋白DAMP_4-MP1106在大肠杆菌中以包涵体的形式成功表达,表达产物在变性条件下经Ni^(2+)-NTA亲和层析纯化;经检测分析,摇瓶中DAMP_4-MP1106的发酵产量为118 mg/L,纯度为94.7%;采用96孔板筛选并建立复性方法,获得水溶性融合蛋白DAMP_4-MP1106;并经TEV蛋白酶酶切以及二次Ni^(2+)-NTA亲和层析纯化,可获得纯度为99%的抗菌肽MP1106 18mg/L,回收率达到了38.4%。通过简捷方法快速鉴定分子内的二硫键,初步证实了抗菌肽MP1106完成了分子内结构正确折叠。建立了高效快捷的菌丝霉素大肠杆菌表达系统。This work aims to establish an efficient and simple Escherichia coli expression system for the antimicrobial peptide,plectasinMP1106. The recombinant MP1106 fusion protein was obtained by genetic fusion with the artificially designed bio-surfactant,DAMP4. Then,the recombinant DAMP4-MP1106 was expressed in E. coli and the protein was isolated,moreover the formation of the disulfide bonds within theprotein was identified. The results showed that the recombinant DAMP4-MP1106 was over-expressed in inclusion body. The protein was obtainedby the Ni^2＋-NTA chromatography under the denature condition. The total yield of the fusion protein after the first chromatography was 118 mg/L in flask and the purity was 94.7%. The refolding buffer condition was screened by 96-well plate and the soluble recombinant DAMP4-MP1106 was obtained by a refolding procedure. The soluble recombinant DAMP4-MP1106 was cleaved by TEV protease and further purified by anotherNi^2＋-NTA column. The purity of final MP1106 was 99% and the recovery of the protein was 38.4%. The comparison of the behaviour of theplectasin derivative,MP1106 with the standard plectasin on electrophoresis indicated that the disulfide bonds in MP1106 were folded correctly.In conclusion,the established E. coli expression system for the plectasin derivative MP1106 was efficient and successful.

关 键 词：抗菌肽 菌丝霉素 蛋白融合 复性 蛋白纯化 

分 类 号：Q51[生物学—生物化学]