酿酒酵母环核苷酸磷酸二酯酶1的异源表达、分离纯化与活性检测  

Heterologous Expression,Purification and Enzymatic Activity of Cyclic Nucleotide Phosphodiesterase 1 from Saccharomyces cerevisiae

作　　者：陈滢 李赤霞 陈玉娟[1] 田元元[1] 张萌[1] 韩潆仪 王友升[1] CHEN Ying;LI Chixia;CHEN Yujuan;TIAN Yuanyuan;ZHANG Meng;HAN Yingyi;WANG Yousheng(Beijing Advanced Innovation Center for Food Nutrition and Human Health,School of Light Industry,Beijing Technology&Business University(BTBU),Beijing 100048,China)

机构地区：[1]北京食品营养与人类健康高精尖创新中心,北京工商大学轻工科学技术学院,北京100048

出　　处：《食品科学》2020年第18期71-76,共6页Food Science

基　　金：国家自然科学基金面上项目(31972127);北京市教委科技计划重点项目(KZ201910011013)。

摘　　要：制备获得高纯度酿酒酵母(Saccharomyces cerevisiae)环核苷酸磷酸二酯酶1(phosphodiesterase 1,PDE1)蛋白,以pGM-T-S.cerevisiae基因组为模板,聚合酶链式反应扩增目的基因,构建表达质粒,转化至大肠杆菌BL21中,并诱导表达。高压破碎菌体提取物先后通过亲和层析纯化系统(Ni-NAT柱)、离子交换纯化系统(Q-Sepharose柱)和分子筛纯化系统(Sephacryl S200柱)进行纯化,经酶活性测定显示,纯化后的蛋白活性达92%。研究结果可为将来该蛋白的晶体学分析和在酿酒酵母中的代谢调控提供前期基础。In order to prepare high-purity cyclic nucleotide phosphodiesterase 1(PDE1)from Saccharomyces cerevisiae,the pGM-T-S.cerevisiae was used as a template to amplify the target gene by PCR,and an expression plasmid carrying this gene was constructed,and then transformed into Escherichia coli BL21 cells for expression.High-pressure broken bacteria extract was purified consecutively by nickel affinity column chromatography,Q-Sepharose ion exchange column chromatography and Sephacryl S200 column chromatography.The purified enzyme activity remained 92%of the original activity.This study may provide a basis for future crystallographic analysis of the protein and its metabolic regulation in S.cerevisiae.

关 键 词：酿酒酵母 环核苷酸磷酸二酯酶 表达纯化 酶活性检测 

分 类 号：TS201.2[轻工技术与工程—食品科学]