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.
出处
《食品与发酵科技》
CAS
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)
关键词
黄曲霉
尿酸氧化酶
毕赤酵母
表达
纯化
Aspergillus flavus
urate oxidase
pichia pastoris
expression
purification
