酵母Elp3在毕赤酵母中的分泌表达及活性测定  

Secreted Expression of Recombinant Yeast Elp3 in Pichia Pastoris and Its HAT Activity Detection in Vitro

作　　者：白雪[1] 马瑜[1] 陈百宝[1] 胡岩岩[1] 李芬[1] 

机构地区：[1]河南师范大学生命科学学院,河南新乡453007

出　　处：《河南师范大学学报（自然科学版）》2012年第4期98-102,共5页Journal of Henan Normal University(Natural Science Edition)

基　　金：国家自然科学基金(30600343);河南省重点科技攻关(092102310026);河南省重点学科建设项目资助

摘　　要：酵母Elp3是Elongator复合物的催化亚基,可参与组蛋白乙酰化修饰与基因转录延伸.生物信息学分析及有关研究表明Elp3除组蛋白乙酰转移酶(Histone acetyltranferase,HAT)活性外,可能还拥有组蛋白去甲基化酶活性.本文以yElp3的pYES2yElp3质粒为模板,PCR获得yElp3基因全长,插入改造过的毕赤酵母表达载体pPIC9KH,转化巴斯德毕赤酵母GS115菌株.经表型鉴定、PCR分析及G418筛选获Mut+型多拷贝整合菌,经0.5%的甲醇诱导和Ni-NTA纯化,得到目的蛋白并对其进行体外酶活测定.SDS-PAGE分析表明yElp3在毕赤酵母中实现了高效分泌表达,Western印迹证实得到的蛋白为yElp3.OPA法测得纯化蛋白拥有较好的HAT活性,具有生物活性的Elp3蛋白的获得为体外测定其第二结构域是否有催化活性奠定了基础.Yeast Elongator protein 3 （yElp3）, possessing Histone Acetyltransferase activity （HAT）, is the catalytic suhunit of the multi-subunit Elongator complex and involvs in histone acetylation and transcription elongation. Sequence analysis and relative research have revealed the presence of a second putative domain, bearing homology to the catalytic domain of Radical SAM enzymes. Here we amplified the full length of hElp3 gene from pYES2-yelp3 template and inserted into the modi- fied Pichia Pastoris expression vector pPIC9KH to construct the recombinant plasmid pPIC9KH-yElp3. After inducing the His-tagged fusion protein expressing in GSl15 （ His4＋ , Mut＋ ） by methanol, a 66 kD yElp3 fusion protein was obtained. Then the protein was purified by Ni-NTA affinity column and we used the soluble purified protein to detect its HAT activity in vitro through OPA method. The results showed that the recombinant yElp3, successfully expressed in Pichia pastoris and has high HAT activity. The work has laid down the basis for further in vitro studies into enzyme activity of the second domain of Elp3.

关 键 词：酵母Elp3 毕赤酵母 分泌表达 活性测定 

分 类 号：Q291[生物学—细胞生物学]