黄曲霉尿酸氧化酶在毕赤酵母中的分泌表达及纯化  

Secretory Expression and Purification of Aspergillus Flavus Urate Oxidase in Pichia Pastoris

作　　者：曾雨雷[1,2] 杨波[1,2] 刘志刚[1,2] 邓贝[1,2] 成细瑶[1,2] 徐雪荣[2] 杨康[2] 胡征[1,2] 

机构地区：[1]发酵工程教育部重点实验室湖北工业大学,湖北武汉430068 [2]湖北工业大学生物工程学院,湖北武汉430068

出　　处：《食品与发酵科技》2012年第4期20-24,共5页Food and Fermentation Science & Technology

基　　金：国家863计划项目(项目编号2010AA101501)

摘　　要：从黄曲霉(Aspergillus flavus)中钓取尿酸氧化酶(UOX)基因,并将该基因以正确的阅读框插入到载体pPIC9K,构建成pPIC9K-UOX重组分泌表达载体。用Sal I线性化处理重组表达载体,电击法转化毕赤酵母(GS115)感受态细胞,G418浓度梯增法筛选多拷贝阳性转化子。0.5%甲醇诱导后的上清经SDS-PAGE电泳检测,证明重组尿酸氧化酶成功分泌到培养基中;由阴离子层析柱纯化产物,目的蛋白纯度可达90%;体外活性测定具有分解尿酸的能力。毕赤酵母(GS115)成功分泌表达了黄曲霉尿酸氧化酶。The urate oxidase （UOX） gene was isolated by RT-PCR from Aspergillus flavus genome and insterted in-to the vector pPIC9K. After being linearized by Sal I, the recombinant vector （pPIC9K-UOX） was transformed into competent cells of Pichia pastoris GSll5 by electroporation. The multi-copy positive transformants were screened by G418 concentration gradient. The recombinant urate oxidase was induced by adding 0.5% methyl alcohol into the me- dia, then purified by anion exchange chromatography and the purity of interest protein was 90%. The purified urate oxidase exhibited the enzymatic activity of oxidation of uric acid to allantoin in vitro. In conclusion, the Aspergillus flavus urate oxidase was successfully expressed in Pichia vastoris.

关 键 词：黄曲霉 尿酸氧化酶 毕赤酵母 表达 纯化 

分 类 号：TQ926.2[轻工技术与工程—发酵工程]