人Elp3在毕赤酵母中的分泌表达及HAT活性测定  

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

作　　者：胡岩岩[1] 李玉会 白雪[1] 陈百宝[1] 刘鸣宇[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 [2]河南省项城市公疗医院,河南项城466200

出　　处：《河南师范大学学报（自然科学版）》2012年第2期132-136,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：人Elp3蛋白(human Elongator protein 3,hElp3)是组蛋白乙酰转移酶(Histone acetyltranferase,HAT)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,其功能异常与人类多种疾病相关.目前对其羧基末端高度保守的第二功能域研究较少.以pYES2hElp3质粒为模板,PCR获hElp3基因全长,插入改造的毕赤酵母表达载体pPIC9KH,转化巴斯德毕赤酵母菌GS115菌株.经表型鉴定、PCR分析和G418筛选得到Mut+型多拷贝整合菌.0.5%的甲醇诱导hElp3高效分泌表达,Ni-NTA纯化及Western印迹证实获目的蛋白.OPA法测得其拥有良好的HAT活性,为体外测定其第二结构域是否拥有催化活性奠定了基础,对筛选抑制其HAT活性的小分子药物用于疾病治疗研究至关重要.Human Elongator protein 3 （hElp3） is the catalytic subunit of the multi-subunit Histone Acetyhransferase （HAT） complex Elongator and involvs in histone acetylation and transcription elongation. Variants of ELP3 gene are associated with many human diseases. Few works focus on its second conserved domain which is the homology to the catalytic domain of Radical SAM enzymes. The study has amplified the full length of hElp3 gene from pYES2-hElp3 template and inserted into the modified Pichia Pastoris expression vector pPICgKH to construct the recombinant plasmid pPICgKH-hElp3. After inducing the His-tagged fusion protein expressing in GSll5 （ His4＋, Mut＋ ） by SDS-PAGE,Western Blot and HAT activity detection methanol, a 66 kD help3 fusion protein has been obtained. The research has purified the protein by Ni-NTA affinity column and used the soluble purified protein to detect its HAT activity in vitro through OPA method. The results show that the recom- binant human EIp3 is successfully expressed in Piehia pastoris and has high HAT activity. The work has laid down the basis for further vitro studies into enzyme activity of the second domain of Elp3 and the selection of small-molecular inhibitor against its HAT activity can be used as the therapeutic agents.

关 键 词：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]